Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure.

PubMed

Esmekaya, Meric Arda; Tuysuz, Mehmet Zahid; Tomruk, Arın; Canseven, Ayse G; Yücel, Engin; Aktuna, Zuhal; Keskil, Semih; Seyhan, Nesrin

2016-09-01

The objective of the this study was to evaluate the effects of cellular phone radiation on oxidative stress parameters and oxide levels in mouse brain during pentylenetetrazole (PTZ) induced epileptic seizure. Eight weeks old mice were used in the study. Animals were distributed in the following groups: Group I: Control group treated with PTZ, Group II: 15min cellular phone radiation+PTZ treatment+30min cellular phone radiation, Group III: 30min cellular phone radiation+PTZ treatment+30min cellular phone radiation. The RF radiation was produced by a 900MHz cellular phone. Lipid peroxidation, which is the indicator of oxidative stress was quantified by measuring the formation of thiobarbituric acid reactive substances (TBARS). The glutathione (GSH) levels were determined by the Ellman method. Tissue total nitric oxide (NOx) levels were obtained using the Griess assay. Lipid peroxidation and NOx levels of brain tissue increased significantly in group II and III compared to group I. On the contrary, GSH levels were significantly lower in group II and III than group I. However, no statistically significant alterations in any of the endpoints were noted between group II and Group III. Overall, the experimental findings demonstrated that cellular phone radiation may increase the oxidative damage and NOx level during epileptic activity in mouse brain. Copyright © 2016 Elsevier B.V. All rights reserved.

Nitric oxide-releasing prodrug triggers cancer cell death through deregulation of cellular redox balance☆

PubMed Central

Maciag, Anna E.; Holland, Ryan J.; Robert Cheng, Y.-S.; Rodriguez, Luis G.; Saavedra, Joseph E.; Anderson, Lucy M.; Keefer, Larry K.

2013-01-01

JS-K is a nitric oxide (NO)-releasing prodrug of the O2-arylated diazeniumdiolate family that has demonstrated pronounced cytotoxicity and antitumor properties in a variety of cancer models both in vitro and in vivo. The current study of the metabolic actions of JS-K was undertaken to investigate mechanisms of its cytotoxicity. Consistent with model chemical reactions, the activating step in the metabolism of JS-K in the cell is the dearylation of the diazeniumdiolate by glutathione (GSH) via a nucleophilic aromatic substitution reaction. The resulting product (CEP/NO anion) spontaneously hydrolyzes, releasing two equivalents of NO. The GSH/GSSG redox couple is considered to be the major redox buffer of the cell, helping maintain a reducing environment under basal conditions. We have quantified the effects of JS-K on cellular GSH content, and show that JS-K markedly depletes GSH, due to JS-K's rapid uptake and cascading release of NO and reactive nitrogen species. The depletion of GSH results in alterations in the redox potential of the cellular environment, initiating MAPK stress signaling pathways, and inducing apoptosis. Microarray analysis confirmed signaling gene changes at the transcriptional level and revealed alteration in the expression of several genes crucial for maintenance of cellular redox homeostasis, as well as cell proliferation and survival, including MYC. Pre-treating cells with the known GSH precursor and nucleophilic reducing agent N-acetylcysteine prevented the signaling events that lead to apoptosis. These data indicate that multiplicative depletion of the reduced glutathione pool and deregulation of intracellular redox balance are important initial steps in the mechanism of JS-K's cytotoxic action. PMID:24024144

Different effects of two cyclic chalcone analogues on redox status of Jurkat T cells.

PubMed

Rozmer, Zsuzsanna; Berki, Tímea; Maász, Gábor; Perjési, Pál

2014-12-01

Chalcones are intermediary compounds of the biosynthetic pathway of the naturally flavonoids. Previous studies have demonstrated that chalcones and their conformationally rigid cyclic analogues have tumour cell cytotoxic and chemopreventive effects. It has been shown that equitoxic doses of the two cyclic chalcone analogues (E)-2-(4'-methoxybenzylidene)-(2) and (E)-2-(4'-methylbenzylidene)-1-benzosuberone (3) have different effect on cell cycle progress of the investigated Jurkat cells. It was also found that the compounds affect the cellular thiol status of the treated cells and show intrinsic (non-enzyme-catalyzed) reactivity towards GSH under cell-free conditions. In order to gain new insights into the cytotoxic mechanism of the compounds, effects on the redox status and glutathione level of Jurkat cells were investigated. Detection of intracellular ROS level in Jurkat cells exposed to 2 and 3 was performed using the dichlorofluorescein-assay. Compound 2 did not influence ROS activity either on 1 or 4h exposure; in contrast, chalcone 3 showed to reduce ROS level at both timepoints. The two compounds had different effects on cellular glutathione status as well. Compound 2 significantly increased the oxidized glutathione (GSSG) level showing an interference with the cellular antioxidant defence. On the contrary, chalcone 3 enhanced the reduced glutathione level, indicating enhanced cellular antioxidant activity. To investigate the chalcone-GSH conjugation reactions under cellular conditions, a combination of a RP-HPLC method with electrospray ionization mass spectrometry (ESI-MS) was performed. Chalcone-GSH adducts could not be observed either in the cell supernatant or the cell sediment after deproteinization. The investigations provide further details of dual - cytotoxic and chemopreventive - effects of the cyclic chalcone analogues. Copyright © 2014 Elsevier Ltd. All rights reserved.

Protective effect of N-acetyl-L-cysteine against disulfiram-induced oxidative stress and apoptosis in V79 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grosicka-Maciag, Emilia; Kurpios-Piec, Dagmara; Grzela, Tomasz

2010-11-01

This work investigated the effect of N-acetyl-L-cysteine (NAC) on disulfiram (DSF) induced oxidative stress in Chinese hamster fibroblast cells (V79). An increase in oxidative stress induced by DSF was observed up to a 200 {mu}M concentration. It was evidenced by a statistically significant increase of both GSH{sub t} and GSSG levels, as well as elevated protein carbonyl (PC) content. There was no increase in lipid peroxidation (measured as TBARS). DSF increased CAT activity, but did not change SOD1 and SOD2 activities. Analysis of GSH related enzymes showed that DSF significantly increased GR activity, did not change Se-dependent GPx, but statisticallymore » significantly decreased non-Se-dependent GPx activity. DSF showed also pro-apoptotic activity. NAC alone did not produce any significant changes, besides an increase of GSH{sub t} level, in any of the variables measured. However, pre-treatment of cells with NAC ameliorated DSF-induced changes. NAC pre-treatment restored the viability of DSF-treated cells evaluated by Trypan blue exclusion assay and MTT test, GSSG level, and protein carbonyl content to the control values as well as it reduced pro-apoptotic activity of DSF. The increase of CAT and GR activity was not reversed. Activity of both GPx was significantly increased compared to their values after DSF treatment. In conclusion, oxidative properties are at least partially attributable to the cellular effects of disulfiram and mechanisms induced by NAC pre-treatment may lower or even abolish the observed effects. These observations illustrate the importance of the initial cellular redox state in terms of cell response to disulfiram exposure. -- Research Highlights: {yields}This report explores biological properties of disulfiram under a condition of modulated intra-cellular GSH level. It shows a protective role of N-acetyl-L-cysteine in V79 cells exposed to disulfiram (in GSH metabolism as well as in changes of antioxidant enzyme activity).« less

Protection of LLC-PK1 cells against hydrogen peroxide-induced cell death by modulation of ceramide level.

PubMed

Yoo, Jae-Myung; Lee, Youn-Sun; Choi, Heon-Kyo; Lee, Yong-Moon; Hong, Jin-Tae; Yun, Yeo-Pyo; Oh, Seikwan; Yoo, Hwan-Soo

2005-03-01

Oxidative stress has been reported to elevate ceramide level during cell death. The purpose of the present study was to modulate cell death in relation to cellular glutathione (GSH) level and GST (glutathione S-transferase) expression by regulating the sphingolipid metabolism. LLC-PK1 cells were treated with H2O2 in the absence of serum to induce cell death. Subsequent to exposure to H2O2, LLC-PK1 cells were treated with desipramine, sphingomyelinase inhibitor, and N-acetylcysteine (NAC), GSH substrate. Based on comparative visual observation with H2O2-treated control cells, it was observed that 0.5 microM of desipramine and 25 mM of NAC exhibited about 90 and 95% of cytoprotection, respectively, against H2O2-induced cell death. Desipramine and NAC lowered the release of LDH activity by 36 and 3%, respectively, when compared to 71% in H2O2-exposed cells. Cellular glutathione level in 500 microM H2O2-treated cells was reduced to 890 pmol as compared to control level of 1198 pmol per mg protein. GST P1-1 expression was decreased in H2O2-treated cells compared to healthy normal cells. In conclusion, it has been inferred that H2O2-induced cell death is closely related to cellular GSH level and GST P1-1 expression in LLC-PK1 cells and occurs via ceramide elevation by sphingomyelinase activation.

Preventive effects of fructose and N-acetyl-L-cysteine against cytotoxicity induced by the psychoactive compounds N-methyl-5-(2-aminopropyl)benzofuran and 3,4-methylenedioxy-N-methamphetamine in isolated rat hepatocytes.

PubMed

Nakagawa, Yoshio; Suzuki, Toshinari; Inomata, Akiko

2018-02-01

Psychoactive compounds, N-methyl-5-(2-aminopropyl)benzofuran (5-MAPB) and 3,4-methylenedioxy-N-methamphetamine (MDMA), are known to be hepatotoxic in humans and/or experimental animals. As previous studies suggested that these compounds elicited cytotoxicity via mitochondrial dysfunction and/or oxidative stress in rat hepatocytes, the protective effects of fructose and N-acetyl-l-cysteine (NAC) on 5-MAPB- and MDMA-induced toxicity were studied in rat hepatocytes. These drugs caused not only concentration-dependent (0-4 mm) and time-dependent (0-3 hours) cell death accompanied by the depletion of cellular levels of adenosine triphosphate (ATP) and glutathione (reduced form; GSH) but also an increase in the oxidized form of GSH. The toxic effects of 5-MAPB were greater than those of MDMA. Pretreatment of hepatocytes with either fructose at a concentration of 10 mm or NAC at a concentration of 2.5 mm prevented 5-MAPB-/MDMA-induced cytotoxicity. In addition, the exposure of hepatocytes to 5-MAPB/MDMA caused the loss of mitochondrial membrane potential, although the preventive effect of fructose was weaker than that of NAC. These results suggest that: (1) 5-MAPB-/MDMA-induced cytotoxicity is linked to mitochondrial failure and depletion of cellular GSH; (2) insufficient cellular ATP levels derived from mitochondrial dysfunction were ameliorated, at least in part, by the addition of fructose; and (3) GSH loss via oxidative stress was prevented by NAC. Taken collectively, these results indicate that the onset of toxic effects caused by 5-MAPB/MDMA may be partially attributable to cellular energy stress as well as oxidative stress. Copyright © 2017 John Wiley & Sons, Ltd.

Roles of the Yap1 Transcription Factor and Antioxidants in Saccharomyces cerevisiae's Tolerance to Furfural and 5-Hydroxymethylfurfural, Which Function as Thiol-Reactive Electrophiles Generating Oxidative Stress

PubMed Central

Kim, Daehee

2013-01-01

Development of the tolerance of Saccharomyces cerevisiae strains to furfural and 5-hydroxymethylfurfural (HMF) is an important issue for cellulosic ethanol production. Although furfural and HMF are known to induce oxidative stress, the underlying mechanisms are largely unknown. In this study, we show that both furfural and HMF act as thiol-reactive electrophiles, thus directly activating the Yap1 transcription factor via the H2O2-independent pathway, depleting cellular glutathione (GSH) levels, and accumulating reactive oxygen species in Saccharomyces cerevisiae. However, furfural showed higher reactivity than did HMF toward GSH in vitro and in vivo. In line with such toxic mechanisms, overexpression of YAP1C620F, a constitutively active mutant of YAP1, and Yap1 target genes encoding catalases (CTA1 and CTT1) increased tolerance to furfural and HMF. However, increasing GSH levels by overexpression of genes for GSH biosynthesis (GSH1 and GLR1) or by the exogenous addition of GSH to the culture medium enhanced tolerance to furfural but not to HMF. PMID:23793623

Roles of the Yap1 transcription factor and antioxidants in Saccharomyces cerevisiae's tolerance to furfural and 5-hydroxymethylfurfural, which function as thiol-reactive electrophiles generating oxidative stress.

PubMed

Kim, Daehee; Hahn, Ji-Sook

2013-08-01

Development of the tolerance of Saccharomyces cerevisiae strains to furfural and 5-hydroxymethylfurfural (HMF) is an important issue for cellulosic ethanol production. Although furfural and HMF are known to induce oxidative stress, the underlying mechanisms are largely unknown. In this study, we show that both furfural and HMF act as thiol-reactive electrophiles, thus directly activating the Yap1 transcription factor via the H2O2-independent pathway, depleting cellular glutathione (GSH) levels, and accumulating reactive oxygen species in Saccharomyces cerevisiae. However, furfural showed higher reactivity than did HMF toward GSH in vitro and in vivo. In line with such toxic mechanisms, overexpression of YAP1(C620F), a constitutively active mutant of YAP1, and Yap1 target genes encoding catalases (CTA1 and CTT1) increased tolerance to furfural and HMF. However, increasing GSH levels by overexpression of genes for GSH biosynthesis (GSH1 and GLR1) or by the exogenous addition of GSH to the culture medium enhanced tolerance to furfural but not to HMF.

Profiling cellular bioenergetics, glutathione levels, and caspase activities in stomach biopsies of patients with upper gastrointestinal symptoms

PubMed Central

Alfazari, Ali S; Al-Dabbagh, Bayan; Al-Dhaheri, Wafa; Taha, Mazen S; Chebli, Ahmad A; Fontagnier, Eva M; Koutoubi, Zaher; Kochiyi, Jose; Karam, Sherif M; Souid, Abdul-Kader

2015-01-01

AIM: To measure biochemical parameters in stomach biopsies and test their suitability as diagnostic biomarkers for gastritis and precancerous lesions. METHODS: Biopsies were obtained from the stomachs of two groups of patients (n = 40) undergoing fiber-optic endoscopy due to upper gastrointestinal symptoms. In the first group (n = 17), only the corpus region was examined. Biopsies were processed for microscopic examination and measurement of mitochondrial O2 consumption (cellular respiration), cellular adenosine triphosphate (ATP), glutathione (GSH), and caspase activity. In the second group of patients (n = 23), both corpus and antral regions were studied. Some biopsies were processed for microscopic examination, while the others were used for measurements of cellular respiration and GSH level. RESULTS: Microscopic examinations of gastric corpus biopsies from 17 patients revealed normal mucosae in 8 patients, superficial gastritis in 7 patients, and chronic atrophic gastritis in 1 patient. In patients with normal histology, the rate (mean ± SD) of cellular respiration was 0.17 ± 0.02 μmol/L O2 min-1 mg-1, ATP content was 487 ± 493 pmol/mg, and GSH was 469 ± 98 pmol/mg. Caspase activity was detected in 3 out of 8 specimens. The values of ATP and caspase activity were highly variable. The presence of superficial gastritis had insignificant effects on the measured biomarkers. In the patient with atrophic gastritis, cellular respiration was high and ATP was relatively low, suggesting uncoupling oxidative phosphorylation. In the second cohort of patients, the examined biopsies showed either normal or superficial gastritis. The rate of cellular respiration (O2. μmol/L min-1 mg-1) was slightly higher in the corpus than the antrum (0.18 ± 0.05 vs 0.15 ± 0.04, P = 0.019). The value of GSH was about the same in both tissues (310 ± 135 vs 322 ± 155, P = 0.692). CONCLUSION: The corpus mucosa was metabolically more active than the antrum tissue. The data in this study will help in understanding the pathophysiology of gastric mucosa. PMID:25593494

Haloperidol-induced changes in glutathione and energy metabolism: effect of nicergoline.

PubMed

Vairetti, M; Feletti, F; Battaglia, A; Pamparana, F; Canonico, P L; Richelmi, P; Bertè, F

1999-02-12

The aim of this study was to evaluate the possible effects of nicergoline, a semisynthetic ergot derivative, on the biochemical changes observed during chronic treatment with haloperidol in male Sprague-Dawley rats. Chronic treatment with haloperidol induced a significant decrease in the cellular glutathione (GSH) content in selected areas of the brain (cerebellum, striatum and cortex) and in the liver. Prolonged nicergoline administration was able to antagonize the haloperidol-induced GSH decrease, maintaining the GSH concentration at levels comparable to those observed in the control group. Analysis of the energy charge revealed changes similar to those observed for GSH: haloperidol induced a significant decrease in ATP and energy charge that was completely reversed by repeated nicergoline administration. In conclusion, chronic treatment with the classical antipsychotic haloperidol induces profound biochemical changes in the brain and in the liver. Nicergoline treatment is able to counteract the haloperidol-induced decrease in GSH levels and energy charge, suggesting a potential role of the drug in the treatment of neuroleptic-induced side effects.

CYP3A-mediated apoptosis of dauricine in cultured human bronchial epithelial cells and in lungs of CD-1 mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Hua; Shen, Shuijie; Chen, Xiaoyan

2012-06-15

Dauricine is the major bioactive component isolated from the root of Menispermum dauricum DC and has shown promising pharmacologic activities with a great potential for clinical use. Recently, we found that intraperitoneal exposure of dauricine produced selective pulmonary injury in mice. A quinone methide metabolite of dauricine was identified and is suggested to be associated with the pulmonary toxicity of dauricine. The present study evaluated the apoptotic effect of dauricine in cultured cells and mice, determined the change in cellular glutathione (GSH) contents after exposure to dauricine, investigated the role of GSH depletion in dauricine-induced cytotoxicity and apoptosis, and examinedmore » the role of CYP3A in dauricine-induced GSH depletion and apoptosis. Dauricine was found to induce apoptosis in NL-20 cells. Additionally, intraperitoneal administration of dauricine caused GSH depletion and apoptosis in lungs of mice. Treatment with ketoconazole, an inhibitor of CYP3A, reversed cellular GSH depletion in lungs of mice given dauricine and showed protective effect on dauricine-induced apoptosis in lungs of mice. This indicates that metabolic activation is involved in dauricine-induced GSH-depletion, cytotoxicity and apoptosis. The glutathione depletor L-buthionine sulfoximine showed potentiating effect on cytotoxicity and apoptosis induced by dauricine. We propose that dauricine is metabolized to a quinone methide intermediate which depletes cellular GSH, and the depletion of GSH may trigger and/or intensify the cytotoxicity and apoptosis induced by dauricine. -- Highlights: ► Dauricine induced apoptosis in lungs in mice and in cultured human pulmonary cells. ► Dauricine depleted cellular GSH in lungs of mice and in the human pulmonary cells. ► CYP3A subfamily mediated GSH depletion and apoptosis induced by dauricine. ► L-Buthionine sulfoximine potentiated dauricine-induced GSH depletion and apoptosis.« less

Increased Cysteine Biosynthesis Capacity of Transgenic Tobacco Overexpressing an O-Acetylserine(thiol) Lyase Modifies Plant Responses to Oxidative Stress1

PubMed Central

Youssefian, Shohab; Nakamura, Michimi; Orudgev, Emin; Kondo, Noriaki

2001-01-01

O-Acetylserine(thiol) lyase (OASTL), a key enzyme of plant sulfur metabolism, catalyzes the formation of Cys from sulfide and O-acetylserine. The biosynthesis of Cys is regarded as the exclusive function of sulfur reduction in plants, and a key limiting step in the production of glutathione (GSH), a thiol implicated in various cellular functions, including sulfur transport, gene expression, scavenging of reactive oxygen species (ROS), and resistance to biotic and abiotic stresses. To examine whether an increased capacity for cysteine (Cys) biosynthesis alters cellular responses to such stresses, we studied the differential changes in thiol levels and ROS scavenging of transgenic tobacco (Nicotiana tabacum) plants expressing the wheat (Triticum aestivum) OASTL gene, cys1, to SO2 and to the ROS generator, methyl viologen. Intracellular Cys and GSH contents were generally higher in cys1 transgenics than in controls under normal growth conditions, but became especially elevated in transgenic plants after SO2 exposure. An examination of differences in the ROS scavenging system of the transgenic plants also demonstrated the specific accumulation of Cu/Zn superoxide dismutase transcripts, known to be induced by Cys or GSH, and elevated cellular superoxide dismutase activities. The transgenic plants accordingly showed dramatic reductions in the extent of both foliar and photooxidative damage in response to acute SO2, as well as reduced levels of chlorosis and membrane damage following methyl viologen treatment. Overall, our results imply that OASTL plays a pivotal role in the synthesis of Cys and GSH that are required for regulation of plant responses to oxidative stress. PMID:11457951

An Expanding Range of Functions for the Copper Chaperone/Antioxidant Protein Atox1

PubMed Central

Hatori, Yuta

2013-01-01

Abstract Significance: Antioxidant protein 1 (Atox1 in human cells) is a copper chaperone for the copper export pathway with an essential role in cellular copper distribution. In vitro, Atox1 binds and transfers copper to the copper-transporting ATPases, stimulating their catalytic activity. Inactivation of Atox1 in cells inhibits maturation of secreted cuproenzymes as well as copper export from cells. Recent Advances: Accumulating data suggest that cellular functions of Atox1 are not limited to its copper-trafficking role and may include storage of labile copper, modulation of transcription, and antioxidant defense. The conserved metal binding site of Atox1, CxGC, differs from the metal-binding sites of copper-transporting ATPases and has a physiologically relevant redox potential that equilibrates with the GSH:GSSG pair. Critical Issues: Tight relationship appears to exist between intracellular copper levels and glutathione (GSH) homeostasis. The biochemical properties of Atox1 place it at the intersection of cellular networks that regulate copper distribution and cellular redox balance. Mechanisms through which Atox1 facilitates copper export and contributes to oxidative defense are not fully understood. Future Directions: The current picture of cellular redox homeostasis and copper physiology will be enhanced by further mechanistic studies of functional interactions between the GSH:GSSG pair and copper-trafficking machinery. Antioxid. Redox Signal. 19, 945–957. PMID:23249252

Role of cellular antioxidants (glutathione and ascorbic acid) in the growth and development of wild carrot suspension cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Earnshaw, B.A.

1986-01-01

Determinations of endogenous glutathione (GSH), glutathione disulfide (GSSG), ascorbic acid (AA) and dehydroascorbic acid (DHA) in proliferating and developing wild carrot cultures showed that lower levels of GSH and AA were associated with developing cultures. The GSSG and DHA levels did not account for the changes in the levels of antioxidants between proliferating and developing cultures. Studies were designed to test an observed auxin (2,4-Dichlorophenoxyacetic acid, 2,4-D)-antioxidant association. Two fractions (embryo and less developed) were obtained by screening developed cultures which were previously grown in the presence of /sup 14/C-2, 4-D. The embryo fraction had a lower concentration of /supmore » 14/C than the less developed fraction, supporting the association, since the two fractions showed this relationship with respect to GSH and AA concentrations. Determinations of GSH and AA levels of cells grown in various concentrations of 2,4-D showed the association, decreases in the 2,4-D concentration correlated with decreases in the GSH and AA concentrations. The existence of a respiratory pathway involving GSSG reductase, DHA reductase, and AA oxidase was investigated to test whether inhibition of AA oxidase by 2,4-D could explain the auxin-antioxidant association; however, AA oxidase activity was not detected.« less

Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

PubMed Central

Cruz-Gregorio, Alfredo; Manzo-Merino, Joaquín; Gonzaléz-García, María Cecilia; Pedraza-Chaverri, José; Medina-Campos, Omar Noel; Valverde, Mahara; Rojas, Emilio; Rodríguez-Sastre, María Alexandra; García-Cuellar, Claudia María; Lizano, Marcela

2018-01-01

Oxidative stress has been proposed as a risk factor for cervical cancer development. However, few studies have evaluated the redox state associated with human papillomavirus (HPV) infection. The aim of this work was to determine the role of the early expressed viral proteins E1, E2, E6 and E7 from HPV types 16 and 18 in the modulation of the redox state in an integral form. Therefore, generation of reactive oxygen species (ROS), concentration of reduced glutathione (GSH), levels and activity of the antioxidant enzymes catalase and superoxide dismutase (SOD) and deoxyribonucleic acid (DNA) damage, were analysed in epithelial cells ectopically expressing the viral proteins. Our research shows that E6 oncoproteins decreased GSH and catalase protein levels, as well as its enzymatic activity, which was associated with an increase in ROS production and DNA damage. In contrast, E7 oncoproteins increased GSH, as well as catalase protein levels and its activity, which correlated with a decrease in ROS without affecting DNA integrity. The co-expression of both E6 and E7 oncoproteins neutralized the effects that were independently observed for each of the viral proteins. Additionally, the combined expression of E1 and E2 proteins increased ROS levels with the subsequent increase in the marker for DNA damage phospho-histone 2AX (γH2AX). A decrease in GSH, as well as SOD2 levels and activity were also detected in the presence of E1 and E2, even though catalase activity increased. This study demonstrates that HPV early expressed proteins differentially modulate cellular redox state and DNA damage. PMID:29483822

The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells.

PubMed

Schaaf, G J; Nijmeijer, S M; Maas, R F M; Roestenberg, P; de Groene, E M; Fink-Gremmels, J

2002-11-20

Balkan endemic nephropathy (BEN), a disease characterized by progressive renal fibrosis in human patients, has been associated with exposure to ochratoxin A (OTA). This mycotoxin is a frequent contaminant of human and animal food products, and is toxic to all animal species tested. OTA predominantly affects the kidney and is known to accumulate in the proximal tubule (PT). The induction of oxidative stress is implicated in the toxicity of this mycotoxin. In the present study, primary rat PT cells and LLC-PK(1) cells, which express characteristics of the PT, were used to investigate the OTA-mediated oxidative stress response. OTA exposure of these cells resulted in a concentration-dependent elevation of reactive oxygen species (ROS) levels, depletion of cellular glutathione (GSH) levels and an increase in the formation of 8-oxoguanine. The OTA-induced ROS response was significantly reduced following treatment with alpha-tocopherol (TOCO). However, this chain-braking anti-oxidant did not reduce the cytotoxicity of OTA and was unable to prevent the depletion of total GSH levels in OTA-exposed cells. In contrast, pre-incubation of the cell with N-acetyl-L-cysteine (NAC) completely prevented the OTA-induced increase in ROS levels as well as the formation of 8-oxoguanine and completely protected against the cytotoxicity of OTA. In addition, NAC treatment also limited the GSH depletion in OTA-exposed PT- and LLC-PK(1) cells. From these data, we conclude that oxidative stress contributes to the tubular toxicity of OTA. Subsequently, cellular GSH levels play a pivotal role in limiting the short-term toxicity of this mycotoxin in renal tubular cells.

A novel mitochondria-targeted two-photon fluorescent probe for dynamic and reversible detection of the redox cycles between peroxynitrite and glutathione.

PubMed

Sun, Chunlong; Du, Wen; Wang, Peng; Wu, Yang; Wang, Baoqin; Wang, Jun; Xie, Wenjun

2017-12-16

Redox homeostasis is important for maintenance of normal physiological functions within cells. Redox state of cells is primarily a consequence of precise balance between levels of reducing equivalents and reactive oxygen species. Redox homeostasis between peroxynitrite (ONOO - ) and glutathione (GSH) is closely associated with physiological and pathological processes, such as prolonged relaxation in vascular tissues and smooth muscle preparations, attenuation of hepatic necrosis, and activation of matrix metalloproteinase-2. We report a two-photon fluorescent probe (TP-Se) based on water-soluble carbazole-based compound, which integrates with organic selenium, to monitor changes in ONOO - /GSH levels in cells. This probe can reversibly respond to ONOO - and GSH and exhibits high selectivity, sensitivity, and mitochondrial targeting. The probe was successfully applied to visualize changes in redox cycles during ONOO - outbreak and antioxidant GSH repair in cells. The probe will lead to significant development on redox events involved in cellular redox regulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential Action between Schisandrin A and Schisandrin B in Eliciting an Anti-Inflammatory Action: The Depletion of Reduced Glutathione and the Induction of an Antioxidant Response

PubMed Central

Leong, Pou Kuan; Wong, Hoi Shan; Chen, Jihang; Chan, Wing Man; Leung, Hoi Yan; Ko, Kam Ming

2016-01-01

Schisandrin A (Sch A) and schisandrin B (Sch B) are active components of Schisandrae Fructus. We compared the biochemical mechanism underlying the anti-inflammatory action of Sch A and Sch B, using cultured lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and concanavalin (ConA)-stimulated mouse splenocytes. Pre-incubation with Sch A or Sch B produced an anti-inflammatory action in LPS-stimulated RAW264.7 cells, as evidenced by the inhibition of the pro-inflammatory c-Jun N-terminal kinases/p38 kinase/nuclear factor-κB signaling pathway as well as the suppression of various pro-inflammatory cytokines and effectors, with the extent of inhibition by Sch A being more pronounced. The greater activity of Sch A in anti-inflammatory response was associated with a greater decrease in cellular reduced glutathione (GSH) level and a greater increase in glutathione S-transferase activity than corresponding changes produced by Sch B. However, upon incubation, only Sch B resulted in the activation of the nuclear factor (erythroid-derived 2)-like factor 2 and the induction of a significant increase in the expression of thioredoxin (TRX) in RAW264.7 cells. The Sch B-induced increase in TRX expression was associated with the suppression of pro-inflammatory cytokines and effectors in LPS-stimulated macrophages. Studies in a mouse model of inflammation (carrageenan-induced paw edema) indicated that while long-term treatment with either Sch A or Sch B suppressed the extent of paw edema, only acute treatment with Sch A produced a significant degree of inhibition on the inflammatory response. Although only Sch A decreased the cellular GSH level and suppressed the release of pro-inflammatory cytokines and cell proliferation in ConA-simulated splenocytes in vitro, both Sch A and Sch B treatments, while not altering cellular GSH levels, suppressed ConA-stimulated splenocyte proliferation ex vivo. These results suggest that Sch A and Sch B may act differentially on activating GST/ depleting cellular GSH and inducing an antioxidant response involved in their anti-inflammatory actions. PMID:27195753

Quercetin attenuates toosendanin-induced hepatotoxicity through inducing the Nrf2/GCL/GSH antioxidant signaling pathway.

PubMed

Jin, Yao; Huang, Zhen-Lin; Li, Li; Yang, Yang; Wang, Chang-Hong; Wang, Zheng-Tao; Ji, Li-Li

2018-06-19

Toosendanin (TSN) is the main active compound in Toosendan Fructus and Meliae Cortex, two commonly used traditional Chinese medicines. TSN has been reported to induce hepatotoxicity, but its mechanism remains unclear. In this study, we demonstrated the critical role of nuclear factor erythroid 2-related factor 2 (Nrf2) in protecting against TSN-induced hepatotoxicity in mice and human normal liver L-02 cells. In mice, administration of TSN (10 mg/kg)-induced acute liver injury evidenced by increased serum alanine/aspartate aminotransferase (ALT/AST) and alkaline phosphatase (ALP) activities, and total bilirubin (TBiL) content as well as the histological changes. Furthermore, TSN markedly increased liver reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and decreased liver glutathione (GSH) content and Nrf2 expression. In L-02 cells, TSN (2 μM) time-dependently reduced glutamate-cysteine ligase (GCL) activity and cellular expression of the catalytic/modify subunit of GCL (GCLC/GCLM). Moreover, TSN reduced cellular GSH content and the increased ROS formation, and time-dependently decreased Nrf2 expression and increased the expression of the Nrf2 inhibitor protein kelch-like ECH-associated protein-1 (Keap1). Pre-administration of quercetin (40, 80 mg/kg) effectively inhibited TSN-induced liver oxidative injury and reversed the decreased expression of Nrf2 and GCLC/GCLM in vivo and in vitro. In addition, the quercetin-provided protection against TSN-induced hepatotoxicity was diminished in Nrf2 knock-out mice. In conclusion, TSN decreases cellular GSH content by reducing Nrf2-mediated GCLC/GCLM expression via decreasing Nrf2 expression. Quercetin attenuates TSN-induced hepatotoxicity by inducing the Nrf2/GCL/GSH antioxidant signaling pathway. This study implies that inducing Nrf2 activation may be an effective strategy to prevent TSN-induced hepatotoxicity.

Laser cytometric analysis of FIV-induced injury in astroglia.

PubMed

Zenger, E; Collisson, E W; Barhoumi, R; Burghardt, R C; Danave, I R; Tiffany-Castiglioni, E

1995-02-01

Glia are the predominant brain cells infected by the lentiviruses human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV). The importance of astrocytes in maintenance of central nervous system homeostasis suggests that astrocytes are likely to play a strategic role in the progression of neurological disease in lentiviral-infected patients. In consideration of this postulate, the ability of FIV to cause injury by infection of cultured feline astroglia was examined via vital fluorescence assays. Intracellular Ca2+ homeostasis, plasma membrane permeability and fluidity, and cytosolic glutathione (GSH) levels were evaluated. Although basal intracellular Ca2+ was not significantly different between groups, FIV-infected astroglia displayed both a significant delay in development of peak Ca2+ levels following ionophore application and a decrease in the amount of Ca2+ released from intracellular stores. Plasma membrane lipid mobility was increased in FIV-infected cells within 24 h of infection. Glutathione levels were affected in a dose dependent fashion. With a standard viral inoculum there was a decrease in GSH which became significant after 8 days postinfection. With a high inoculum dose there was rapid loss of cell viability with an increase in GSH in surviving cells. We have identified several cellular processes altered in FIV-infected astroglia and our findings suggest that FIV-infection of feline astroglia affects cellular membranes, both structurally and functionally.

Physical Training Status Determines Oxidative Stress and Redox Changes in Response to an Acute Aerobic Exercise

PubMed Central

Damirchi, Arsalan; Farjaminezhad, Manoochehr

2016-01-01

Objective. To assess the influence of different physical training status on exercise-induced oxidative stress and changes in cellular redox state. Methods. Thirty male subjects participated in this study and were assigned as well-trained (WT), moderately trained (MT), and untrained (UT) groups. The levels of cortisol, creatine kinase, plasma reduced glutathione to oxidized glutathione (GSH/GSSG), cysteine/cystine (Cys/CySS), and GSH/GSSG ratio in red blood cells (RBCs) were measured immediately and 10 and 30 min after exercise. Results. Following the exercise, plasma GSH/GSSG (p = 0.001) and Cys/CySS (p = 0.005) were significantly reduced in all groups. Reduction in plasma GSH/GSSG ratio in all groups induced a transient shift in redox balance towards a more oxidizing environment without difference between groups (p = 0.860), while RBCs GSH/GSSG showed significant reduction (p = 0.003) and elevation (p = 0.007) in UT and MT groups, respectively. The highest level of RBCs GSH/GSSG ratio was recorded in MT group, and the lowest one was recorded in the WT group. Conclusion. Long term regular exercise training with moderate intensity shifts redox balance towards more reducing environment, versus intensive exercise training leads to more oxidizing environment and consequently development of related diseases. PMID:27064342

N-acetylcysteine stimulates protein synthesis in enterocytes independently of glutathione synthesis.

PubMed

Yi, Dan; Hou, Yongqing; Wang, Lei; Long, Minhui; Hu, Shengdi; Mei, Huimin; Yan, Liqiong; Hu, Chien-An Andy; Wu, Guoyao

2016-02-01

Dietary supplementation with N-acetylcysteine (NAC) has been reported to improve intestinal health and treat gastrointestinal diseases. However, the underlying mechanisms are not fully understood. According to previous reports, NAC was thought to exert its effect through glutathione synthesis. This study tested the hypothesis that NAC enhances enterocyte growth and protein synthesis independently of cellular glutathione synthesis. Intestinal porcine epithelial cells were cultured for 3 days in Dulbecco's modified Eagle medium containing 0 or 100 μM NAC. To determine a possible role for GSH (the reduced form of glutathione) in mediating the effect of NAC on cell growth and protein synthesis, additional experiments were conducted using culture medium containing 100 μM GSH, 100 μM GSH ethyl ester (GSHee), diethylmaleate (a GSH-depletion agent; 10 μM), or a GSH-synthesis inhibitor (buthionine sulfoximine, BSO; 20 μM). NAC increased cell proliferation, GSH concentration, and protein synthesis, while inhibiting proteolysis. GSHee enhanced cell proliferation and GSH concentration without affecting protein synthesis but inhibited proteolysis. Conversely, BSO or diethylmaleate reduced cell proliferation and GSH concentration without affecting protein synthesis, while promoting protein degradation. At the signaling level, NAC augmented the protein abundance of total mTOR, phosphorylated mTOR, and phosphorylated 70S6 kinase as well as mRNA levels for mTOR and p70S6 kinase in IPEC-1 cells. Collectively, these results indicate that NAC upregulates expression of mTOR signaling proteins to stimulate protein synthesis in enterocytes independently of GSH generation. Our findings provide a hitherto unrecognized biochemical mechanism for beneficial effects of NAC in intestinal cells.

Exogenous Glutathione Enhances Mercury Tolerance by Inhibiting Mercury Entry into Plant Cells

PubMed Central

Kim, Yeon-Ok; Bae, Hyeun-Jong; Cho, Eunjin; Kang, Hunseung

2017-01-01

Despite the increasing understanding of the crucial roles of glutathione (GSH) in cellular defense against heavy metal stress as well as oxidative stress, little is known about the functional role of exogenous GSH in mercury (Hg) tolerance in plants. Here, we provide compelling evidence that GSH contributes to Hg tolerance in diverse plants. Exogenous GSH did not mitigate the toxicity of cadmium (Cd), copper (Cu), or zinc (Zn), whereas application of exogenous GSH significantly promoted Hg tolerance during seed germination and seedling growth of Arabidopsis thaliana, tobacco, and pepper. By contrast, addition of buthionine sulfoximine, an inhibitor of GSH biosynthesis, severely retarded seed germination and seedling growth of the plants in the presence of Hg. The effect of exogenous GSH on Hg specific tolerance was also evident in the presence of other heavy metals, such as Cd, Cu, and Zn, together with Hg. GSH treatment significantly decreased H2O2 and O2- levels and lipid peroxidation, but increased chlorophyll content in the presence of Hg. Importantly, GSH treatment resulted in significantly less accumulation of Hg in Arabidopsis plants, and thin layer chromatography and nuclear magnetic resonance analysis revealed that GSH had much stronger binding affinity to Hg than to Cd, Cu, or Zn, suggesting that tight binding of GSH to Hg impedes Hg uptake, leading to low Hg accumulation in plant cells. Collectively, the present findings reveal that GSH is a potent molecule capable of conferring Hg tolerance by inhibiting Hg accumulation in plants. PMID:28507557

A Novel Defensive Mechanism against Acetaminophen Toxicity in the Mouse Lateral Nasal Gland: Role of CYP2A5-Mediated Regulation of Testosterone Homeostasis and Salivary Androgen-Binding Protein Expression

PubMed Central

Zhou, Xin; Wei, Yuan; Xie, Fang; Laukaitis, Christina M.; Karn, Robert C.; Kluetzman, Kerri; Gu, Jun; Zhang, Qing-Yu; Roberts, Dean W.

2011-01-01

To identify novel factors or mechanisms that are important for the resistance of tissues to chemical toxicity, we have determined the mechanisms underlying the previously observed increases in resistance to acetaminophen (APAP) toxicity in the lateral nasal gland (LNG) of the male Cyp2g1-null/Cyp2a5-low mouse. Initial studies established that Cyp2a5-null mice, but not a newly generated strain of Cyp2g1-null mice, were resistant to APAP toxicity in the LNG; therefore, subsequent studies were focused on the Cyp2a5-null mice. Compared with the wild-type (WT) male mouse, the Cyp2a5-null male mouse had intact capability to metabolize APAP to reactive intermediates in the LNG, as well as unaltered circulating levels of APAP, APAP-GSH, APAP-glucuronide, and APAP-sulfate. However, it displayed reduced tissue levels of APAP and APAP-GSH and increased tissue levels of testosterone and salivary androgen-binding protein (ABP) in the LNG. Furthermore, we found that ABP was able to compete with GSH and cellular proteins for adduction with reactive metabolites of APAP in vitro. The amounts of APAP-ABP adducts formed in vivo were greater, whereas the amounts of APAP adducts formed with other cellular proteins were substantially lower, in the LNG of APAP-treated male Cyp2a5-null mice compared with the LNG of APAP-treated male WT mice. We propose that through its critical role in testosterone metabolism, CYP2A5 regulates 1) the bioavailability of APAP and APAP-GSH (presumably through modulation of the rates of xenobiotic excretion from the LNG) and 2) the expression of ABP, which can quench reactive APAP metabolites and thereby spare critical cellular proteins from inactivation. PMID:21252290

Glutathione in Cancer Cell Death

PubMed Central

Ortega, Angel L.; Mena, Salvador; Estrela, Jose M.

2011-01-01

Glutathione (L-γ-glutamyl-L-cysteinyl-glycine; GSH) in cancer cells is particularly relevant in the regulation of carcinogenic mechanisms; sensitivity against cytotoxic drugs, ionizing radiations, and some cytokines; DNA synthesis; and cell proliferation and death. The intracellular thiol redox state (controlled by GSH) is one of the endogenous effectors involved in regulating the mitochondrial permeability transition pore complex and, in consequence, thiol oxidation can be a causal factor in the mitochondrion-based mechanism that leads to cell death. Nevertheless GSH depletion is a common feature not only of apoptosis but also of other types of cell death. Indeed rates of GSH synthesis and fluxes regulate its levels in cellular compartments, and potentially influence switches among different mechanisms of death. How changes in gene expression, post-translational modifications of proteins, and signaling cascades are implicated will be discussed. Furthermore, this review will finally analyze whether GSH depletion may facilitate cancer cell death under in vivo conditions, and how this can be applied to cancer therapy. PMID:24212662

Role of glutathione biosynthesis in endothelial dysfunction and fibrosis.

PubMed

Espinosa-Díez, Cristina; Miguel, Verónica; Vallejo, Susana; Sánchez, Francisco J; Sandoval, Elena; Blanco, Eva; Cannata, Pablo; Peiró, Concepción; Sánchez-Ferrer, Carlos F; Lamas, Santiago

2018-04-01

Glutathione (GSH) biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL), which is composed of the catalytic (GCLc) and the modulatory (GCLm) subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice). In murine lung endothelial cells (MLEC) derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177) and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT) mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+) male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH 4 . To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+) mice. We observed that obstructed kidneys from Gclc(e/+) mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Effects of Glycyrrhetinic Acid on GSH Synthesis Induced by Realgar in the Mouse Hippocampus: Involvement of System [Formula: see text], System [Formula: see text], MRP-1, and Nrf2.

PubMed

Wang, Yan-Lei; Chen, Mo; Huo, Tao-Guang; Zhang, Ying-Hua; Fang, Ying; Feng, Cong; Wang, Shou-Yun; Jiang, Hong

2017-05-01

Realgar, a type of mineral drug-containing arsenic, exhibits neurotoxicity. Brain glutathione (GSH) is crucial to protect the nervous system and to resist arsenic toxicity. Therefore, the main aim of this study was to explore the neurotoxic mechanisms of realgar and the protective effects of glycyrrhetinic acid (GA) by observing the effects of GA on the hippocampal GSH biosynthetic pathway after exposure to realgar. Institute of Cancer Research (ICR) mice were randomly divided into five groups: a control group, a GA control group, a realgar alone group, a low-dose GA intervention group, and a high-dose GA intervention group. Cognitive ability was tested using an object recognition task (ORT). The ultrastructures of the hippocampal neurons and synapses were observed. mRNA and protein levels of EAAT1, EAAT2, EAAT3, xCT, Nrf2, HO-1, γ-GCS (GCLC, GCLM), and MRP-1 were measured, as was the cellular localization of EAAT3, xCT, MRP-1, and Nrf2. The levels of GSH in the hippocampus, the levels of glutamate (Glu) and cysteine (Cys) in the extracellular fluid of hippocampal CA1 region, and the levels of active sulfur in the brain were also investigated. The results indicate that realgar lowered hippocampal GSH levels, resulting in ultrastructural changes in hippocampal neurons and synapses and deficiencies in cognitive ability, ultimately inducing neurotoxicity. GA could trigger the expression of Nrf2, HO-1, EAAT1, EAAT2, EAAT3, xCT, MRP-1, GCLC, and GCLM. Additionally, the expression of γ-GT and the supply levels of Glu and Cys increased, ultimately causing a significant increase in hippocampal GSH to alleviate realgar-induced neurotoxicity. In conclusion, the findings from our study indicate that GA can antagonize decreased brain GSH levels induced by realgar and can lessen the neurotoxicity of realgar.

The partial pressure of oxygen affects biomarkers of oxidative stress in cultured rainbow trout (Oncorhynchus mykiss) hepatocytes.

PubMed

Finne, E F; Olsvik, P A; Berntssen, M H G; Hylland, K; Tollefsen, K E

2008-09-01

Oxidative stress, the imbalance between production of reactive oxygen species and the cellular detoxification of these reactive compounds, is believed to be involved in the pathology of various diseases. Several biomarkers for oxidative stress have been proposed to serve as tools in toxicological and ecotoxicological research. Not only may exposure to various pro-oxidants create conditions of cellular oxidative stress, but hyperoxic conditions may also increase the production of reactive oxygen species. The objective of the current study was to determine the extent to which differences in oxygen partial pressure would affect biomarkers of oxidative stress in a primary culture of hepatocytes from rainbow trout (Oncorhynchus mykiss). Membrane integrity, metabolic activity, levels of total and oxidized glutathione (tGSH/GSSG) was determined, as well as mRNA expression levels of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), gamma-glutamyl-cystein synthetase (GCS) and thioredoxin (TRX). The results show that different biomarkers of oxidative stress are affected when the cell culture is exposed to atmospheric oxygen, and that changes such as increased GSSG content and induction of GSSG-R and GSH-Px can be reduced by culturing the cells under lower oxygen tension. Oxygen tension may thus influence results of in vitro based cell research and is particularly important when assessing parameters in the antioxidant defence system. Further research is needed to establish the magnitude of this effect in different cellular systems.

Impaired cross-talk between the thioredoxin and glutathione systems is related to ASK-1 mediated apoptosis in neuronal cells exposed to mercury.

PubMed

Branco, Vasco; Coppo, Lucia; Solá, Susana; Lu, Jun; Rodrigues, Cecília M P; Holmgren, Arne; Carvalho, Cristina

2017-10-01

Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems - glutathione (GSH) and thioredoxin (Trx) systems - are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events. To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5μM of inorganic mercury (Hg 2+ ), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function. Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Ferroptosis is Involved in Acetaminophen Induced Cell Death.

PubMed

Lőrincz, Tamás; Jemnitz, Katalin; Kardon, Tamás; Mandl, József; Szarka, András

2015-09-01

The recently described form of programmed cell death, ferroptosis can be induced by agents causing GSH depletion or the inhibition of GPX4. Ferroptosis clearly shows distinct morphologic, biochemical and genetic features from apoptosis, necrosis and autophagy. Since NAPQI the highly reactive metabolite of the widely applied analgesic and antipyretic, acetaminophen induces a cell death which can be characterized by GSH depletion, GPX inhibition and caspase independency the involvement of ferroptosis in acetaminophen induced cell death has been investigated. The specific ferroptosis inhibitor ferrostatin-1 failed to elevate the viability of acetaminophen treated HepG2 cells. It should be noticed that these cells do not form NAPQI due to the lack of phase I enzyme expression therefore GSH depletion cannot be observed. However in the case of acetaminophen treated primary mouse hepatocytes the significant elevation of cell viability could be observed upon ferrostatin-1 treatment. Similar to ferrostatin-1 treatment, the addition of the RIP1 kinase inhibitor necrostatin-1 could also elevate the viability of acetaminophen treated primary hepatocytes. Ferrostatin-1 has no influence on the expression of CYP2E1 or on the cellular GSH level which suggest that the protective effect of ferrostatin-1 in APAP induced cell death is not based on the reduced metabolism of APAP to NAPQI or on altered NAPQI conjugation by cellular GSH. Our results suggest that beyond necroptosis and apoptosis a third programmed cell death, ferroptosis is also involved in acetaminophen induced cell death in primary hepatocytes.

The influence of microwave radiation from cellular phone on fetal rat brain.

PubMed

Jing, Ji; Yuhua, Zhang; Xiao-qian, Yang; Rongping, Jiang; Dong-mei, Guo; Xi, Cui

2012-03-01

The increasing use of cellular phones in our society has brought focus on the potential detrimental effects to human health by microwave radiation. The aim of our study was to evaluate the intensity of oxidative stress and the level of neurotransmitters in the brains of fetal rats chronically exposed to cellular phones. The experiment was performed on pregnant rats exposed to different intensities of microwave radiation from cellular phones. Thirty-two pregnant rats were randomly divided into four groups: CG, GL, GM, and GH. CG accepted no microwave radiation, GL group radiated 10 min each time, GM group radiated 30 min, and GH group radiated 60 min. The 3 experimental groups were radiated 3 times a day from the first pregnant day for consecutively 20 days, and on the 21st day, the fetal rats were taken and then the contents of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), noradrenaline (NE), dopamine (DA), and 5-hydroxyindole acetic acid (5-HT) in the brain were assayed. Compared with CG, there were significant differences (P<0.05) found in the contents of SOD, GSH-Px, and MDA in GM and GH; the contents of SOD and GSH-Px decreased and the content of MDA increased. The significant content differences of NE and DA were found in fetal rat brains in GL and GH groups, with the GL group increased and the GH group decreased. Through this study, we concluded that receiving a certain period of microwave radiation from cellular phones during pregnancy has certain harm on fetal rat brains.

Modulation of GSH with exogenous agents leads to changes in glyoxalase 1 enzyme activity in VL-17A cells exposed to chronic alcohol plus high glucose.

PubMed

Kumar, S Mathan; Swaminathan, Kavitha; Clemens, Dahn L; Dey, Aparajita

2014-02-01

Gluthathione (GSH) is a major cellular antioxidant. The present study utilizing VL-17A cells exposed to chronic alcohol plus high glucose investigated the changes in oxidative stress, toxicity, and glyoxalase 1 activity as a detoxification pathway due to changes in GSH level through GSH supplementation with N-acetyl cysteine (NAC) or ursodeoxycholic acid (UDCA) and its depletion through buthionine sulfoximine (BSO) or diethyl maleate (DEM). Glyoxalase 1 plays an important role in detoxification of methylglyoxal which is formed as a precursor of advanced glycated end products formed due to high glucose mediated oxidative stress. Significant changes in glyoxalase 1 activity utilizing methylglyoxal or glyoxal as substrates occurred with NAC or UDCA or BSO or DEM supplementation in chronic alcohol plus high glucose treated VL-17A cells. NAC or UDCA administration in chronic alcohol plus high glucose treated VL-17A cells increased viability and decreased ROS levels, lipid peroxidation and 3-nitrotyrosine adduct formation. Similarly, GSH depletion with BSO or DEM had an opposite effect on the parameters in chronic alcohol plus high glucose treated VL-17A cells. In conclusion, modulation of GSH with NAC or UDCA or BSO or DEM leads to significant changes in oxidative stress, glyoxalase 1 enzyme activity and toxicity in chronic alcohol plus high glucose treated VL-17A cells.

Lipid peroxidation, antioxidant enzymes and glutathione levels in human erythrocytes exposed to colloidal iron hydroxide in vitro.

PubMed

Ferreira, A L; Machado, P E; Matsubara, L S

1999-06-01

The free form of the iron ion is one of the strongest oxidizing agents in the cellular environment. The effect of iron at different concentrations (0, 1, 5, 10, 50, and 100 microM Fe3+) on the normal human red blood cell (RBC) antioxidant system was evaluated in vitro by measuring total (GSH) and oxidized (GSSG) glutathione levels, and superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and reductase (GSH-Rd) activities. Membrane lipid peroxidation was assessed by measuring thiobarbituric acid reactive substance (TBARS). The RBC were incubated with colloidal iron hydroxide and phosphate-buffered saline, pH 7.45, at 37 degrees C, for 60 min. For each assay, the results for the control group were: a) GSH = 3.52 +/- 0.27 microM/g Hb; b) GSSG = 0.17 +/- 0.03 microM/g Hb; c) GSH-Px = 19.60 +/- 1.96 IU/g Hb; d) GSH-Rd = 3.13 +/- 0.17 IU/g Hb; e) catalase = 394.9 +/- 22.8 IU/g Hb; f) SOD = 5981 +/- 375 IU/g Hb. The addition of 1 to 100 microM Fe3+ had no effect on the parameters analyzed. No change in TBARS levels was detected at any of the iron concentrations studied. Oxidative stress, measured by GSH kinetics over time, occurs when the RBC are incubated with colloidal iron hydroxide at concentrations higher than 10 microM of Fe3+. Overall, these results show that the intact human RBC is prone to oxidative stress when exposed to Fe3+ and that the RBC has a potent antioxidant system that can minimize the potential damage caused by acute exposure to a colloidal iron hydroxide in vitro.

Potentiation of lead-induced cell death in PC12 cells by glutamate: Protection by N-acetylcysteine amide (NACA), a novel thiol antioxidant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Penugonda, Suman; Mare, Suneetha; Lutz, P.

2006-10-15

Oxidative stress has been implicated as an important factor in many neurological diseases. Oxidative toxicity in a number of these conditions is induced by excessive glutamate release and subsequent glutamatergic neuronal stimulation. This, in turn, causes increased generation of reactive oxygen species (ROS), oxidative stress, excitotoxicity, and neuronal damage. Recent studies indicate that the glutamatergic neurotransmitter system is involved in lead-induced neurotoxicity. Therefore, this study aimed to (1) investigate the potential effects of glutamate on lead-induced PC12 cell death and (2) elucidate whether the novel thiol antioxidant N-acetylcysteine amide (NACA) had any protective abilities against such cytotoxicity. Our results suggestmore » that glutamate (1 mM) potentiates lead-induced cytotoxicity by increased generation of ROS, decreased proliferation (MTS), decreased glutathione (GSH) levels, and depletion of cellular adenosine-triphosphate (ATP). Consistent with its ability to decrease ATP levels and induce cell death, lead also increased caspase-3 activity, an effect potentiated by glutamate. Exposure to glutamate and lead elevated the cellular malondialdehyde (MDA) levels and phospholipase-A{sub 2} (PLA{sub 2}) activity and diminished the glutamine synthetase (GS) activity. NACA protected PC12 cells from the cytotoxic effects of glutamate plus lead, as evaluated by MTS assay. NACA reduced the decrease in the cellular ATP levels and restored the intracellular GSH levels. The increased levels of ROS and MDA in glutamate-lead treated cells were significantly decreased by NACA. In conclusion, our data showed that glutamate potentiated the effects of lead-induced PC12 cell death by a mechanism involving mitochondrial dysfunction (ATP depletion) and oxidative stress. NACA had a protective role against the combined toxic effects of glutamate and lead by inhibiting lipid peroxidation and scavenging ROS, thus preserving intracellular GSH.« less

Depletion of intracellular GSH and NPSH by buthionine sulfoximine and diethyl maleate: factors that influence enhancement of aerobic radiation response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varnes, M.E.; Biaglow, J.E.; Roizin-Towle, L.

1984-08-01

Many investigators have observed aerobic sensitization of V79, CHO and A549 (human lung carcinoma) cells upon depletion of GSH using buthionine sulfoximine (BSO). Recently the authors discovered that this aerobic sensitization can be reversed if WR-2721 or N-acetylcysteine is added to the cells just prior to irradiation. Reversal requires that the exogenous thiols be present during the time of irradiation. One possible explanation was that these thiols entered the cells and either increased the pool of cellular nonprotein thiols or reversed the thiol-depleted state by stimulation of GSH synthesis. Cells treated with BSO do not readily regenerate intracellular GSH becausemore » this agent irreversibly inhibits ..gamma..-glutamyl synthetase. They found that addition of WR-2721 or N-acetylcysteine to BSO-treated cells did not affect the rate of regeneration of intracellular GSH. Thus, reversal of the aerobic sensitization of A549 cells by BSO cannot be explained on the basis of intracellular thiol levels alone, or by rapid reversal of BSO inhibition. In addition, diethylmaleate (DEM)-treated cells are considerably different from BSO-treated cells with respect to the ability to regenerate GSH.« less

Vitamin K3 suppressed inflammatory and immune responses in a redox-dependent manner.

PubMed

Checker, Rahul; Sharma, Deepak; Sandur, Santosh K; Khan, Nazir M; Patwardhan, Raghavendra S; Kohli, Vineet; Sainis, Krishna B

2011-08-01

Recent investigations suggest that cellular redox status may play a key role in the regulation of several immune functions. Treatment of lymphocytes with vitamin K3 (menadione) resulted in a significant decrease in cellular GSH/GSSG ratio and concomitant increase in the ROS levels. It also suppressed Concanavalin A (Con A)-induced proliferation and cytokine production in lymphocytes and CD4 + T cells in vitro. Immunosuppressive effects of menadione were abrogated only by thiol containing antioxidants. Mass spectrometric analysis showed that menadione directly interacted with thiol antioxidant GSH. Menadione completely suppressed Con A-induced activation of ERK, JNK and NF-κB in lymphocytes. It also significantly decreased the homeostasis driven proliferation of syngeneic CD4 + T cells. Further, menadione significantly delayed graft-vs-host disease morbidity and mortality in mice. Menadione suppressed phytohemagglutinin-induced cytokine production in human peripheral blood mononuclear cells. These results reveal that cellular redox perturbation by menadione is responsible for significant suppression of lymphocyte responses.

Chloro-Functionalized Photo-crosslinking BODIPY for Glutathione Sensing and Subcellular Trafficking.

PubMed

Murale, Dhiraj P; Hong, Seong Cheol; Haque, Md Mamunul; Lee, Jun-Seok

2018-05-18

Glutathione (GSH) is one of major antioxidants inside cells that regulates oxidoreduction homeostasis. Recently, there have been extensive efforts to visualize GSH in live cells, but most of the probes available today are simple detection sensors and do not provide details of cellular localization. A new fluorescent probe (pcBD2-Cl), which is cell permeable and selectively reacts with GSH in situ, has been developed. The in situ GSH-labeled probe (pcBD2-GSH) exhibited quenches fluorescence, but subsequent binding to cellular abundant glutathione S-transferase (GST) recovers the fluorescence intensity, which makes it possible to image the GSH-GST complex in live cells. Interactions between probe and GST were confirmed by means of photo-crosslinking under intact live-cell conditions. Interestingly, isomers of chloro-functionalized 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) compounds behaved very distinctively inside the cells. Following co-staining imaging with MitoTracker and mitochondria fractionation upon lipopolysaccharide-mediated reactive oxygen species induction experiments showed that pcBD2-GSH accumulated in mitochondria. This is the first example of a live-cell imaging probe to visualize translocation of GSH from the cytosol to mitochondria. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Timing of developmental reduction in epithelial glutathione redox potential is associated with increased epithelial proliferation in the immature murine intestine.

PubMed

Reid, Graham K; Berardinelli, Andrew J; Ray, Laurie; Jackson, Arena R; Neish, Andrew S; Hansen, Jason M; Denning, Patricia W

2017-08-01

BackgroundThe intracellular redox potential of the glutathione (GSH)/glutathione disulfide (GSSG) couple regulates cellular processes. In vitro studies indicate that a reduced GSH/GSSG redox potential favors proliferation, whereas a more oxidized redox potential favors differentiation. Intestinal growth depends upon an appropriate balance between the two. However, how the ontogeny of intestinal epithelial cellular (IEC) GSH/GSSG redox regulates these processes in the developing intestine has not been fully characterized in vivo.MethodsOntogeny of intestinal GSH redox potential and growth were measured in neonatal mice.ResultsWe show that IEC GSH/GSSG redox potential becomes increasingly reduced (primarily driven by increased GSH concentration) over the first 3 weeks of life. Increased intracellular GSH has been shown to drive proliferation through increased poly-ADP-ribose polymerase (PARP) activity. We show that increasing IEC poly-ADP-ribose chains can be measured over the first 3 weeks of life, indicating an increase in IEC PARP activity. These changes are accompanied by increased intestinal growth and IEC proliferation as assessed by villus height/crypt depth, intestinal length, and Ki67 staining.ConclusionUnderstanding how IEC GSH/GSSG redox potential is developmentally regulated may provide insight into how premature human intestinal redox states can be manipulated to optimize intestinal growth and adaptation.

Radiation-induced cyclooxygenase 2 up-regulation is dependent on redox status in prostate cancer cells.

PubMed

Li, Lingyun; Steinauer, Kirsten K; Dirks, Amie J; Husbeck, Bryan; Gibbs, Iris; Knox, Susan J

2003-12-01

Cyclooxygenase 2 (COX2) is the inducible isozyme of COX, a key enzyme in arachidonate metabolism and the conversion of arachidonic acid (AA) to prostaglandins (PGs) and other eicosanoids. Previous studies have demonstrated that the COX2 protein is up-regulated in prostate cancer cells after irradiation and that this results in elevated levels of PGE(2). In the present study, we further investigated whether radiation-induced COX2 up-regulation is dependent on the redox status of cells from the prostate cancer cell line PC-3. l-Buthionine sulfoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), and the antioxidants alpha-lipoic acid and N-acetyl-l-cysteine (NAC) were used to modulate the cellular redox status. BSO decreased the cellular GSH level and increased cellular reactive oxygen species (ROS) in PC-3 cells, whereas alpha-lipoic acid and NAC increased the GSH level and decreased cellular ROS. Both radiation and the oxidant H(2)O(2) had similar effects on COX2 up-regulation and PGE(2) production in PC-3 cells, suggesting that radiation-induced COX2 up-regulation is secondary to the production of ROS. The relative increases in COX2 expression and PGE(2) production induced by radiation and H(2)O(2) were even greater when PC-3 cells were pretreated with BSO. When the cells were pretreated with alpha-lipoic acid or NAC for 24 h, both radiation- and H(2)O(2)-induced COX2 up-regulation and PGE(2) production were markedly inhibited. These results demonstrate that radiation-induced COX2 up-regulation in prostate cancer cells is modulated by the cellular redox status. Radiation-induced increases in ROS levels contribute to the adaptive response of PC-3 cells, resulting in elevated levels of COX2.

Multidrug Resistance-Associated Protein 1 (MRP1) mediated vincristine resistance: effects of N-acetylcysteine and Buthionine Sulfoximine

PubMed Central

Akan, Ilhan; Akan, Selma; Akca, Hakan; Savas, Burhan; Ozben, Tomris

2005-01-01

Background Multidrug resistance mediated by the multidrug resistance-associated protein 1 (MRP1) decreases cellular drug accumulation. The exact mechanism of MRP1 involved multidrug resistance has not been clarified yet, though glutathione (GSH) is likely to have a role for the resistance to occur. N-acetylcysteine (NAC) is a pro-glutathione drug. DL-Buthionine (S,R)-sulfoximine (BSO) is an inhibitor of GSH synthesis. The aim of our study was to investigate the effect of NAC and BSO on MRP1-mediated vincristine resistance in Human Embryonic Kidney (HEK293) and its MRP1 transfected 293MRP cells. Human Embryonic Kidney (HEK293) cells were transfected with a plasmid encoding whole MRP1 gene. Both cells were incubated with vincristine in the presence or absence of NAC and/or BSO. The viability of both cells was determined under different incubation conditions. GSH, Glutathione S-Transferase (GST) and glutathione peroxidase (GPx) levels were measured in the cell extracts obtained from both cells incubated with different drugs. Results N-acetylcysteine increased the resistance of both cells against vincristine and BSO decreased NAC-enhanced MRP1-mediated vincristine resistance, indicating that induction of MRP1-mediated vincristine resistance depends on GSH. Vincristine decreased cellular GSH concentration and increased GPx activity. Glutathione S-Transferase activity was decreased by NAC. Conclusion Our results demonstrate that NAC and BSO have opposite effects in MRP1 mediated vincristine resistance and BSO seems a promising chemotherapy improving agent in MRP1 overexpressing tumor cells. PMID:16042792

Nitric-oxide Synthase Forms N-NO-pterin and S-NO-Cys

PubMed Central

Rosenfeld, Robin J.; Bonaventura, Joseph; Szymczyna, Blair R.; MacCoss, Michael J.; Arvai, Andrew S.; Yates, John R.; Tainer, John A.; Getzoff, Elizabeth D.

2010-01-01

Inducible nitric-oxide synthase (iNOS) produces biologically stressful levels of nitric oxide (NO) as a potent mediator of cellular cytotoxicity or signaling. Yet, how this nitrosative stress affects iNOS function in vivo is poorly understood. Here we define two specific non-heme iNOS nitrosation sites discovered by combining UV-visible spectroscopy, chemiluminescence, mass spectrometry, and x-ray crystallography. We detected auto-S-nitrosylation during enzymatic turnover by using chemiluminescence. Selective S-nitrosylation of the ZnS4 site, which bridges the dimer interface, promoted a dimer-destabilizing order-to-disorder transition. The nitrosated iNOS crystal structure revealed an unexpected N-NO modification on the pterin cofactor. Furthermore, the structurally defined N-NO moiety is solvent-exposed and available to transfer NO to a partner. We investigated glutathione (GSH) as a potential transnitrosation partner because the intracellular GSH concentration is high and NOS can form S-nitrosoglutathione. Our computational results predicted a GSH binding site adjacent to the N-NO-pterin. Moreover, we detected GSH binding to iNOS with saturation transfer difference NMR spectroscopy. Collectively, these observations resolve previous paradoxes regarding this uncommon pterin cofactor in NOS and suggest means for regulating iNOS activity via N-NO-pterin and S-NO-Cys modifications. The iNOS self-nitrosation characterized here appears appropriate to help control NO production in response to cellular conditions. PMID:20659888

Xc- inhibitor sulfasalazine sensitizes colorectal cancer to cisplatin by a GSH-dependent mechanism.

PubMed

Ma, Ming-zhe; Chen, Gang; Wang, Peng; Lu, Wen-hua; Zhu, Chao-feng; Song, Ming; Yang, Jing; Wen, Shijun; Xu, Rui-hua; Hu, Yumin; Huang, Peng

2015-11-01

Sulfasalazine (SSZ) is an anti-inflammatory drug that has been demonstrated to induce apoptosis and tumor regression through inhibition of plasma membrane cystine transporter xc(-). Cysteine is a rate-limiting precursor for intracellular glutathione (GSH) synthesis, which is vital for compound detoxification and maintaining redox balance. Platinum-based chemotherapy is an important regimen used in clinics for various cancers including colorectal cancer (CRC). We hypothesized that targeting xc(-) transporter by SSZ may annihilate cellular detoxification through interruption of GSH synthesis and may enhance the anti-cancer activity of cisplatin (CDDP) by increasing drug transport. In the present study, we revealed that xCT, the active subunit of xc(-), is highly expressed in CRC cell lines and human colorectal carcinoma tissues compared with their normal counterparts. SSZ effectively depleted cellular GSH, leading to significant accumulation of reactive oxygen species and growth inhibition in CRC cells. In contrast, the normal epithelial cells of colon origin were less sensitive to SSZ, showing a moderate ROS elevation. Importantly, SSZ effectively enhanced the intracellular platinum level and cytotoxicity of CDDP in CRC cells. The synergistic effect of SSZ and CDDP was reversed by antioxidant N-acetyl-L-cysteine (NAC). Together, these results suggest that SSZ, a relatively non-toxic drug that targets cystine transporter, may, in combination with CDDP, have effective therapy for colorectal cancer. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Determining Glutathione Levels in Plants.

PubMed

Sahoo, Smita; Awasthi, Jay Prakash; Sunkar, Ramanjulu; Panda, Sanjib Kumar

2017-01-01

Upon exposure to abiotic stresses, plants tend to accumulate excessive amounts of reactive oxygen species (ROS) that inturn react with cellular lipids, proteins, and DNA. Therefore, decreasing ROS accumulation is indispensible to survive under stress, which is accomplished by inducing enzymatic and nonenzymatic antioxidant defense pathways. Glutathione, particularly reduced glutathione (GSH), represents a principal anitioxidant that could decrease ROS through scavenging them directly or indirectly through ascorbate-glutathione cycle or GSH peroxidases. Glutathione content can be determined using HPLC or spectrophotometric assays. In this chapter, we provided detailed assays to determine total, reduced, and oxidized gluathione using spectrophotometric method.

Insights into the Distinguishing Stress-induced Cytotoxicity of Chiral Gold Nanoclusters and the Relationship with GSTP1

PubMed Central

Zhang, Chunlei; Zhou, Zhijun; Zhi, Xiao; Ma, Yue; Wang, Kan; Wang, Yuxia; Zhang, Yingge; Fu, Hualin; Jin, Weilin; Pan, Fei; Cui, Daxiang

2015-01-01

Chiral gold nanoclusters (Au NCs) exhibit attracting properties owing to their unique physical and chemical properties. Herein we report for the first time chiral gold nanoclusters' cytotoxicity and potential molecular mechanism. The L-glutathione (i.e. L-GSH) and D-glutathione (i.e. D-GSH)-capped Au NCs were prepared and characterized by HRTEM, UV-vis, photoluminescence and circular dichroism (CD) spectroscopy. Results showed that the CD spectra of L-glutathione (i.e. L-GSH) and D-glutathione (i.e. D-GSH)-capped Au NCs exhibited multiple bands which were identically mirror-imaged, demonstrating that the chirality of GSH-capped NCs had contributions from both the metal core and the ligand. The effects of AuNCs@L-GSH and AuNCs@D-GSH on cells were similar based on the cell physiology related cytotoxicity, although the effects became more prominent in AuNCs@D-GSH treated cells, including ROS generation, mitochondrial membrane depolarization, cell cycle arrest and apoptosis. Global gene expression and pathway analysis displayed that both AuNCs@L-GSH and AuNCs@D-GSH caused the up-regulation of genes involved in cellular rescue and stress response, while AuNCs@D-GSH individually induced up-regulation of transcripts involved in some metabolic- and biosynthetic-related response. MGC-803 cells were more sensitive to the oxidative stress damage induced by chiral Au NCs than GES-1 cells, which was associated with GSTP1 hypermethylation. In conclusion, chiral gold nanoclusters exhibit this chirality-associated regulation of cytotoxicity, different gene expression profiling and epigenetic changes should be responsible for observed phenomena. Our study highlights the importance of the interplays between chiral materials and biological system at sub-nano level. PMID:25553104

Kinetic study of the interaction of glutathione with four antitumor disulfides: possible mechanism for cellular glutathione depletion.

PubMed

Kirkpatrick, D L

1989-01-01

The reactions between the cellular tripeptide, glutathione (GSH) and four disulfide derivatives of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) (compounds 1-4) were studied kinetically. The decyl and phenyl derivatives of 6-MP and 6-TG were reacted with GSH in phosphate buffer (pH 7.4 or 6.0) at 25.0 degrees C and were monitored spectrophotometrically by observing the release of 6-MP and 6-TG. Second order kinetics were observed, with rate constants of 142, 564, 4174 and 429 M-1 s-1 being measured for compounds 1-4, respectively. When the reactions were carried out in the presence of GSH-S-transferase the rates were enhanced 1.3-5.4 times those observed in the absence of enzyme. Products of the reactions were isolated by chromatography and tentatively identified by TLC or fast atom bombardment mass spectrometry. It was observed that GSH reacted with each disulfide in a 1:1 manner, forming a mixed disulfide between GSH and decanethiol or thiophenol while releasing 6-MP or 6-TG. It was concluded that the reported depletion of GSH from EMT6 cells after exposure to these disulfides could be due to their reaction with GSH, and the formation of the mixed disulfides.

Generic method for the absolute quantification of glutathione S-conjugates: Application to the conjugates of acetaminophen, clozapine and diclofenac.

PubMed

den Braver, Michiel W; Vermeulen, Nico P E; Commandeur, Jan N M

2017-03-01

Modification of cellular macromolecules by reactive drug metabolites is considered to play an important role in the initiation of tissue injury by many drugs. Detection and identification of reactive intermediates is often performed by analyzing the conjugates formed after trapping by glutathione (GSH). Although sensitivity of modern mass spectrometrical methods is extremely high, absolute quantification of GSH-conjugates is critically dependent on the availability of authentic references. Although 1 H NMR is currently the method of choice for quantification of metabolites formed biosynthetically, its intrinsically low sensitivity can be a limiting factor in quantification of GSH-conjugates which generally are formed at low levels. In the present study, a simple but sensitive and generic method for absolute quantification of GSH-conjugates is presented. The method is based on quantitative alkaline hydrolysis of GSH-conjugates and subsequent quantification of glutamic acid and glycine by HPLC after precolumn derivatization with o-phthaldialdehyde/N-acetylcysteine (OPA/NAC). Because of the lower stability of the glycine OPA/NAC-derivate, quantification of the glutamic acid OPA/NAC-derivate appeared most suitable for quantification of GSH-conjugates. The novel method was used to quantify the concentrations of GSH-conjugates of diclofenac, clozapine and acetaminophen and quantification was consistent with 1 H NMR, but with a more than 100-fold lower detection limit for absolute quantification. Copyright © 2017. Published by Elsevier B.V.

Arsenite-induced stress granule formation is inhibited by elevated levels of reduced glutathione in West Nile virus-infected cells

PubMed Central

Basu, Mausumi; Courtney, Sean C.

2017-01-01

Oxidative stress activates the cellular kinase HRI, which then phosphorylates eIF2α, resulting in stalled translation initiation and the formation of stress granules (SGs). SG assembly redirects cellular translation to stress response mRNAs and inhibits cap-dependent viral RNA translation. Flavivirus infections were previously reported to induce oxidative stress in infected cells but flavivirus-infected cells paradoxically develop resistance to arsenite (Ars)-induced SG formation with time after infection. This resistance was previously postulated to be due to sequestration of the SG protein Caprin1 by Japanese encephalitis virus capsid protein. However, Caprin1 did not co-localize with West Nile virus (WNV) capsid protein in infected cells. Other stressors induced SGs with equal efficiency in mock- and WNV-infected cells indicating the intrinsic ability of cells to assemble SGs was not disabled. Induction of both reactive oxygen species (ROS) and the antioxidant response was detected at early times after WNV-infection. The transcription factors, Nrf2 and ATF4, which activate antioxidant genes, were upregulated and translocated to the nucleus. Knockdown of Nrf2, ATF4 or apoptosis-inducing factor (AIF), a mitochondrial protein involved in regenerating intracellular reduced glutathione (GSH) levels, with siRNA or treatment of cells with buthionine sulphoximine, which induces oxidative stress by inhibiting GSH synthesis, decreased intracellular GSH levels and increased the number of SG-positive, infected cells. Mitochondria were protected from Ars-induced damage by WNV infection until late times in the infection cycle. The results indicate that the increase in virus-induced ROS levels is counterbalanced by a virus-induced antioxidant response that is sufficient to also overcome the increase in ROS induced by Ars treatment and prevent Ars-induced SG assembly and mitochondrial damage. The virus-induced alterations in the cellular redox status appear to provide benefits for the virus during its lifecycle. PMID:28241074

Cellular Antioxidant and Anti-Inflammatory Effects of Coffee Extracts with Different Roasting Levels.

PubMed

Jung, Soohan; Kim, Min Hyung; Park, Jae Hee; Jeong, Yoonhwa; Ko, Kwang Suk

2017-06-01

During roasting, major changes occur in the composition and physiological effects of coffee beans. In this study, in vitro antioxidant effects and anti-inflammatory effects of Coffea arabica green coffee extracts were investigated at different roasting levels corresponding to Light, Medium, City, and French roast. Total caffeine did not show huge difference according to roasting level, but total chlorogenic acid contents were higher in light roasted coffee extract than other roasted groups. In addition, light roasted coffee extract had the highest antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. To determine the in vitro antioxidant property, coffee extracts were used to treat AML-12 cells. Intracellular glutathione (GSH) concentration and mRNA expression levels of genes related to GSH synthesis were negatively related to roasting levels. The anti-inflammatory effects of coffee extracts were investigated in lipopolysaccharide-treated RAW 264.7 macrophage cells. The cellular antioxidant activity of coffee extracts exhibited similar patterns as the AML-12 cells. The expression of mRNA for tumor necrosis factor-alpha and interleukin-6 was decreased in cells treated with the coffee extracts and the expression decreased with increasing roasting levels. These data suggest that coffee has physiological antioxidant and anti-inflammatory activities and these effects are negatively correlated with roasting levels in the cell models.

Milk thistle seed extract protects rat C6 astroglial cells from acute cocaine toxicity.

PubMed

Badisa, Ramesh B; Fitch-Pye, Cheryl A; Agharahimi, Maryam; Palm, Donald E; Latinwo, Lekan M; Goodman, Carl B

2014-11-01

Cocaine is a powerful addictive drug, widely abused in most Western countries. It easily reaches various domains within and outside of the central nervous system (CNS), and triggers varying levels of cellular toxicity. No pharmacological treatment is available to alleviate cocaine-induced toxicity in the cells without side-effects. Here, we discerned the role of milk thistle (MT) seed extract against cocaine toxicity. First, we investigated acute cytotoxicity induced by treatment with 2, 3 and 4 mM cocaine for 1 h in astroglial, liver and kidney cells in vitro, and then in living shrimp larvae in vivo. We showed that astroglial cells are more sensitive to cocaine than liver, kidney cells or larvae. Cocaine exposure disrupted the general architecture of astroglial cells, induced vacuolation, decreased cell viability, and depleted the glutathione (GSH) level. These changes may represent the underlying pathology of cocaine in the astrocytes. By contrast, MT pretreatment (200 µg/ml) for 30 min sustained the cell morphological features and increased both cell viability and the GSH level. Besides its protective effects, the MT extract was revealed to be non-toxic to astroglial cells, and displayed high free-radical scavenging activity. The results from this study suggest that enhanced GSH level underlies cell protection, and indicate that compounds that promote GSH synthesis in the cells may be beneficial against cocaine toxicity.

A γ-glutamyl cyclotransferase protects Arabidopsis plants from heavy metal toxicity by recycling glutamate to maintain glutathione homeostasis.

PubMed

Paulose, Bibin; Chhikara, Sudesh; Coomey, Joshua; Jung, Ha-Il; Vatamaniuk, Olena; Dhankher, Om Parkash

2013-11-01

Plants detoxify toxic metals through a GSH-dependent pathway. GSH homeostasis is maintained by the γ-glutamyl cycle, which involves GSH synthesis and degradation and the recycling of component amino acids. The enzyme γ-glutamyl cyclotransferase (GGCT) is involved in Glu recycling, but the gene(s) encoding GGCT has not been identified in plants. Here, we report that an Arabidopsis thaliana protein with a cation transport regulator-like domain, hereafter referred to as GGCT2;1, functions as γ-glutamyl cyclotransferase. Heterologous expression of GGCT2;1 in Saccharomyces cerevisiae produced phenotypes that were consistent with decreased GSH content attributable to either GSH degradation or the diversion of γ-glutamyl peptides to produce 5-oxoproline (5-OP). 5-OP levels were further increased by the addition of arsenite and GSH to the medium, indicating that GGCT2;1 participates in the cellular response to arsenic (As) via GSH degradation. Recombinant GGCT2;1 converted both GSH and γ-glutamyl Ala to 5-OP in vitro. GGCT2;1 transcripts were upregulated in As-treated Arabidopsis, and ggct2;1 knockout mutants were more tolerant to As and cadmium than the wild type. Overexpression of GGCT2;1 in Arabidopsis resulted in the accumulation of 5-OP. Under As toxicity, the overexpression lines showed minimal changes in de novo Glu synthesis, while the ggct2;1 mutant increased nitrogen assimilation by severalfold, resulting in a very low As/N ratio in tissue. Thus, our results suggest that GGCT2;1 ensures sufficient GSH turnover during abiotic stress by recycling Glu.

Reversible near-infrared fluorescent probe introducing tellurium to mimetic glutathione peroxidase for monitoring the redox cycles between peroxynitrite and glutathione in vivo.

PubMed

Yu, Fabiao; Li, Peng; Wang, Bingshuai; Han, Keli

2013-05-22

The redox homeostasis between peroxynitrite and glutathione is closely associated with the physiological and pathological processes, e.g. vascular tissue prolonged relaxation and smooth muscle preparations, attenuation hepatic necrosis, and activation matrix metalloproteinase-2. We report a near-infrared fluorescent probe based on heptamethine cyanine, which integrates with telluroenzyme mimics for monitoring the changes of ONOO(-)/GSH levels in cells and in vivo. The probe can reversibly respond to ONOO(-) and GSH and exhibits high selectivity, sensitivity, and mitochondrial target. It is successfully applied to visualize the changes of redox cycles during the outbreak of ONOO(-) and the antioxidant GSH repair in cells and animal. The probe would provide a significant advance on the redox events involved in the cellular redox regulation.

Redox activation of DUSP4 by N-acetylcysteine protects endothelial cells from Cd²⁺-induced apoptosis.

PubMed

Barajas-Espinosa, Alma; Basye, Ariel; Jesse, Erin; Yan, Haixu; Quan, David; Chen, Chun-An

2014-09-01

Redox imbalance is a primary cause of endothelial dysfunction (ED). Under oxidant stress, many critical proteins regulating endothelial function undergo oxidative modifications that lead to ED. Cellular levels of glutathione (GSH), the primary reducing source in cells, can significantly regulate cell function via reversible protein thiol modification. N-acetylcysteine (NAC), a precursor for GSH biosynthesis, is beneficial for many vascular diseases; however, the detailed mechanism of these benefits is still not clear. From HPLC analysis, NAC significantly increases both cellular GSH and tetrahydrobiopterin levels. Immunoblotting of endothelial NO synthase (eNOS) and DUSP4, a dual-specificity phosphatase with a cysteine as its active residue, revealed that both enzymes are upregulated by NAC. EPR spin trapping further demonstrated that NAC enhances NO generation from cells. Long-term exposure to Cd(2+) contributes to DUSP4 degradation and the uncontrolled activation of p38 and ERK1/2, leading to apoptosis. Treatment with NAC prevents DUSP4 degradation and protects cells against Cd(2+)-induced apoptosis. Moreover, the increased DUSP4 expression can redox-regulate the p38 and ERK1/2 pathways from hyperactivation, providing a survival mechanism against the toxicity of Cd(2+). DUSP4 gene knockdown further supports the hypothesis that DUSP4 is an antioxidant gene, critical in the modulation of eNOS expression, and thus protects against Cd(2+)-induced stress. Depletion of intracellular GSH by buthionine sulfoximine makes cells more susceptible to Cd(2+)-induced apoptosis. Pretreatment with NAC prevents p38 overactivation and thus protects the endothelium from this oxidative stress. Therefore, the identification of DUSP4 activation by NAC provides a novel target for future drug design. Copyright © 2014 Elsevier Inc. All rights reserved.

Progesterone amplifies oxidative stress signal and promotes NO production via H2O2 in mouse kidney arterial endothelial cells.

PubMed

Yuan, Xiao-Hua; Fan, Yang-Yang; Yang, Chun-Rong; Gao, Xiao-Rui; Zhang, Li-Li; Hu, Ying; Wang, Ya-Qin; Jun, Hu

2016-01-01

The role of progesterone on the cardiovascular system is controversial. Our present research is to specify the effect of progesterone on arterial endothelial cells in response to oxidative stress. Our result showed that H2O2 (150 μM and 300 μM) induced cellular antioxidant response. Glutathione (GSH) production and the activity of Glutathione peroxidase (GPx) were increased in H2O2-treated group. The expression of glutamate cysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) was induced in response to H2O2. However, progesterone absolutely abolished the antioxidant response through increasing ROS level, inhibiting the activity of Glutathione peroxidase (GPx), decreasing GSH level and reducing expression of GClC and GCLM. In our study, H2O2 induced nitrogen monoxide (NO) production and endothelial nitric oxide synthase (eNOS) expression, and progesterone promoted H2O2-induced NO production. Progesterone increased H2O2-induced expression of hypoxia inducible factor-α (HIFα) which in turn regulated eNOS expression and NO synthesis. Further study demonstrated that progesterone increased H2O2 concentration of culture medium which may contribute to NO synthesis. Exogenous GSH decreased the content of H2O2 of culture medium pretreated by progesterone combined with H2O2 or progesterone alone. GSH also inhibited expression of HIFα and eNOS, and abolished NO synthesis. Collectively, our study demonstrated for the first time that progesterone inhibited cellular antioxidant effect and increased oxidative stress, promoted NO production of arterial endothelial cells, which may be due to the increasing H2O2 concentration and amplified oxidative stress signal. Copyright © 2015. Published by Elsevier Ltd.

Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death.

PubMed

Dunning, Sandra; Ur Rehman, Atta; Tiebosch, Marjolein H; Hannivoort, Rebekka A; Haijer, Floris W; Woudenberg, Jannes; van den Heuvel, Fiona A J; Buist-Homan, Manon; Faber, Klaas Nico; Moshage, Han

2013-12-01

In chronic liver disease, hepatic stellate cells (HSCs) are activated, highly proliferative and produce excessive amounts of extracellular matrix, leading to liver fibrosis. Elevated levels of toxic reactive oxygen species (ROS) produced during chronic liver injury have been implicated in this activation process. Therefore, activated hepatic stellate cells need to harbor highly effective anti-oxidants to protect against the toxic effects of ROS. To investigate the protective mechanisms of activated HSCs against ROS-induced toxicity. Culture-activated rat HSCs were exposed to hydrogen peroxide. Necrosis and apoptosis were determined by Sytox Green or acridine orange staining, respectively. The hydrogen peroxide detoxifying enzymes catalase and glutathione-peroxidase (GPx) were inhibited using 3-amino-1,2,4-triazole and mercaptosuccinic acid, respectively. The anti-oxidant glutathione was depleted by L-buthionine-sulfoximine and repleted with the GSH-analogue GSH-monoethylester (GSH-MEE). Upon activation, HSCs increase their cellular glutathione content and GPx expression, while MnSOD (both at mRNA and protein level) and catalase (at the protein level, but not at the mRNA level) decreased. Hydrogen peroxide did not induce cell death in activated HSCs. Glutathione depletion increased the sensitivity of HSCs to hydrogen peroxide, resulting in 35% and 75% necrotic cells at 0.2 and 1mmol/L hydrogen peroxide, respectively. The sensitizing effect was abolished by GSH-MEE. Inhibition of catalase or GPx significantly increased hydrogen peroxide-induced apoptosis, which was not reversed by GSH-MEE. Activated HSCs have increased ROS-detoxifying capacity compared to quiescent HSCs. Glutathione levels increase during HSC activation and protect against ROS-induced necrosis, whereas hydrogen peroxide-detoxifying enzymes protect against apoptotic cell death. © 2013.

Modulation of O-GlcNAc Levels in the Liver Impacts Acetaminophen-Induced Liver Injury by Affecting Protein Adduct Formation and Glutathione Synthesis.

PubMed

McGreal, Steven R; Bhushan, Bharat; Walesky, Chad; McGill, Mitchell R; Lebofsky, Margitta; Kandel, Sylvie E; Winefield, Robert D; Jaeschke, Hartmut; Zachara, Natasha E; Zhang, Zhen; Tan, Ee Phie; Slawson, Chad; Apte, Udayan

2018-04-01

Overdose of acetaminophen (APAP) results in acute liver failure. We have investigated the role of a posttranslational modification of proteins called O-GlcNAcylation, where the O-GlcNAc transferase (OGT) adds and O-GlcNAcase (OGA) removes a single β-D-N-acetylglucosamine (O-GlcNAc) moiety, in the pathogenesis of APAP-induced liver injury. Hepatocyte-specific OGT knockout mice (OGT KO), which have reduced O-GlcNAcylation, and wild-type (WT) controls were treated with 300 mg/kg APAP and the development of injury was studied over a time course from 0 to 24 h. OGT KO mice developed significantly lower liver injury as compared with WT mice. Hepatic CYP2E1 activity and glutathione (GSH) depletion following APAP treatment were not different between WT and OGT KO mice. However, replenishment of GSH and induction of GSH biosynthesis genes were significantly faster in the OGT KO mice. Next, male C57BL/6 J mice were treated Thiamet-G (TMG), a specific inhibitor of OGA to induce O-GlcNAcylation, 1.5 h after APAP administration and the development of liver injury was studied over a time course of 0-24 h. TMG-treated mice exhibited significantly higher APAP-induced liver injury. Treatment with TMG did not affect hepatic CYP2E1 levels, GSH depletion, APAP-protein adducts, and APAP-induced mitochondrial damage. However, GSH replenishment and GSH biosynthesis genes were lower in TMG-treated mice after APAP overdose. Taken together, these data indicate that induction in cellular O-GlcNAcylation exacerbates APAP-induced liver injury via dysregulation of hepatic GSH replenishment response.

3-bromopyruvate inhibits glycolysis, depletes cellular glutathione, and compromises the viability of cultured primary rat astrocytes.

PubMed

Ehrke, Eric; Arend, Christian; Dringen, Ralf

2015-07-01

The pyruvate analogue 3-bromopyruvate (3-BP) is an electrophilic alkylator that is considered a promising anticancer drug because it has been shown to kill cancer cells efficiently while having little toxic effect on nontumor cells. To test for potential adverse effects of 3-BP on brain cells, we exposed cultured primary rat astrocytes to 3-BP and investigated the effects of this compound on cell viability, glucose metabolism, and glutathione (GSH) content. The presence of 3-BP severely compromised cell viability and slowed cellular glucose consumption and lactate production in a time- and concentration-dependent manner, with half-maximal effects observed at about 100 µM 3-BP after 4 hr of incubation. The cellular hexokinase activity was not affected in 3-BP-treated astrocytes, whereas within 30 min after application of 3-BP the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inhibited, and cellular GSH content was depleted in a concentration-dependent manner, with half-maximal effects observed at about 30 µM 3-BP. The depletion of cellular GSH after exposure to 100 µM 3-BP was not prevented by the presence of 10 mM of the monocarboxylates lactate or pyruvate, suggesting that 3-BP is not taken up into astrocytes predominantly by monocarboxylate transporters. The data suggest that inhibition of glycolysis by inactivation of GAPDH and GSH depletion contributes to the toxicity that was observed for 3-BP-treated cultured astrocytes. © 2014 Wiley Periodicals, Inc.

Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism

PubMed Central

James, S. Jill; Rose, Shannon; Melnyk, Stepan; Jernigan, Stefanie; Blossom, Sarah; Pavliv, Oleksandra; Gaylor, David W.

2009-01-01

Research into the metabolic phenotype of autism has been relatively unexplored despite the fact that metabolic abnormalities have been implicated in the pathophysiology of several other neurobehavioral disorders. Plasma biomarkers of oxidative stress have been reported in autistic children; however, intracellular redox status has not yet been evaluated. Lymphoblastoid cells (LCLs) derived from autistic children and unaffected controls were used to assess relative concentrations of reduced glutathione (GSH) and oxidized disulfide glutathione (GSSG) in cell extracts and isolated mitochondria as a measure of intracellular redox capacity. The results indicated that the GSH/GSSG redox ratio was decreased and percentage oxidized glutathione increased in both cytosol and mitochondria in the autism LCLs. Exposure to oxidative stress via the sulfhydryl reagent thimerosal resulted in a greater decrease in the GSH/GSSG ratio and increase in free radical generation in autism compared to control cells. Acute exposure to physiological levels of nitric oxide decreased mitochondrial membrane potential to a greater extent in the autism LCLs, although GSH/GSSG and ATP concentrations were similarly decreased in both cell lines. These results suggest that the autism LCLs exhibit a reduced glutathione reserve capacity in both cytosol and mitochondria that may compromise antioxidant defense and detoxification capacity under prooxidant conditions.—James, S. J., Rose, S., Melnyk, S., Jernigan, S., Blossom, S., Pavliv, O., Gaylor, D. W. Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism. PMID:19307255

Enhanced cytostatic activity of the sesquiterpene lactone eupatoriopicrin by glutathione depletion.

PubMed Central

Woerdenbag, H. J.; Lemstra, W.; Malingré, T. M.; Konings, A. W.

1989-01-01

Eupatoriopicrin (EUP), a sesquiterpene lactone from Eupatorium cannabinum L., possesses cytostatic activity. This was demonstrated for FIO 26 cells in vitro with the aid of a clonogenic assay and in vivo by tumour growth delay in FIO 26 and Lewis lung tumour-bearing mice. In vitro the IC50 for 1 h exposure to EUP was 1.5 microgram ml-1 (4.1 nmol ml-1). This concentration depleted about 25% of its cellular GSH concentration. Pretreatment of FIO 26 cells with BSO, resulting in greater than 99%. GSH depletion, enhanced the cytotoxic effect of EUP. The dose-enhancement factor at the level of 10% cell survival was 2.3. Growth inhibition of the Lewis lung carcinoma and the FIO 26 fibrosarcoma, solidly growing in C57Bl mice, was found after i.v. injection of 20 or 40 mg kg-1 EUP, at a tumour volume of about 500 microliters. Pretreatment with BSO at a dose of 4 mmol kg-1 i.p., 6 h before EUP administration, resulted in a significantly stronger growth delay of both tumours compared with EUP only. At the time of EUP treatment, cellular GSH in the tumours was reduced by BSO treatment to about 60%. It is concluded that EUP possesses antitumour activity in vivo and that chemosensitisation of EUP may be accomplished by pretreatment with BSO, indicating that endogenous GSH protects against the cytostatic action of EUP. PMID:2757925

Glutathione depletion by valproic acid in sandwich-cultured rat hepatocytes: Role of biotransformation and temporal relationship with onset of toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kiang, Tony K.L.; Teng Xiaowei; Surendradoss, Jayakumar

2011-05-01

The present study was conducted in sandwich-cultured rat hepatocytes to investigate the chemical basis of glutathione (GSH) depletion by valproic acid (VPA) and evaluate the role of GSH depletion in VPA toxicity. Among the synthetic metabolites of VPA investigated, 4-ene-VPA and (E)-2,4-diene-VPA decreased cellular levels of total GSH, but only (E)-2,4-diene-VPA was more effective and more potent than the parent drug. The in situ generated, cytochrome P450-dependent 4-ene-VPA did not contribute to GSH depletion by VPA, as suggested by the experiment with a cytochrome P450 inhibitor, 1-aminobenzotriazole, to decrease the formation of this metabolite. In support of a role formore » metabolites, alpha-F-VPA and octanoic acid, which do not undergo biotransformation to form a 2,4-diene metabolite, CoA ester, or glucuronide, did not deplete GSH. A time course experiment showed that GSH depletion did not occur prior to the increase in 2',7'-dichlorofluorescein (a marker of oxidative stress), the decrease in [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) product formation (a marker of cell viability), or the increase in lactate dehydrogenase (LDH) release (a marker of necrosis) in VPA-treated hepatocytes. In conclusion, the cytochrome P450-mediated 4-ene-VPA pathway does not play a role in the in situ depletion of GSH by VPA, and GSH depletion is not an initiating event in VPA toxicity in sandwich-cultured rat hepatocytes.« less

Alterations of energy metabolism and glutathione levels of HL-60 cells induced by methacrylates present in composite resins.

PubMed

Nocca, G; De Palma, F; Minucci, A; De Sole, P; Martorana, G E; Callà, C; Morlacchi, C; Gozzo, M L; Gambarini, G; Chimenti, C; Giardina, B; Lupi, A

2007-03-01

Methacrylic compounds such as 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA) and bisphenol A glycerolate (1 glycerol/phenol) dimethacrylate (Bis-GMA) are largely present in auto- or photopolymerizable composite resins. Since the polymerization reaction is never complete, these molecules are released into the oral cavity tissues and biological fluids where they could cause local adverse effects. The aim of this work was to verify the hypothesis that the biological effects of HEMA, TEGDMA and Bis-GMA - at a non-cytotoxic concentration - depend on the interaction with mitochondria and exert consequent alterations of energy metabolism, GSH levels and the related pathways in human promyelocytic cell line (HL-60). The biological effects of methacrylic monomers were determined by analyzing the following parameters: GSH concentration, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activity, oxygen and glucose consumption and lactate production along with cell differentiation and proliferation. All monomers induced both cellular differentiation and decrease in oxygen consumption. Cells treated with TEGDMA and Bis-GMA showed a significant enhancement of glucose consumption and lactate production. TEGDMA and HEMA induced GSH depletion stimulating G6PDH and GR activity. All the monomers under study affect the metabolism of HL-60 cells and show differentiating activity. Since alterations in cellular metabolism occurred at compound concentrations well below cytotoxic levels, the changes in energy metabolism and glutathione redox balance could be considered as potential mechanisms for inducing clinical and sub-clinical adverse effects and thus providing useful parameters when testing biocompatibility of dental materials.

Resveratrol protects against arsenic trioxide-induced oxidative damage through maintenance of glutathione homeostasis and inhibition of apoptotic progression

PubMed Central

Chen, Chengzhi; Jiang, Xuejun; Lai, Yanhao; Liu, Yuan; Zhang, Zunzhen

2014-01-01

Arsenic trioxide (As2O3) is commonly used to treat acute promyelocytic leukemia and solid tumors. However, the clinical application of the agent is limited by its cyto- and genotoxic effects on normal cells. Thus, relief of As2O3 toxicity in normal cells is essentially necessary for improvement of As2O3-mediated chemotherapy. In this study, we have identified a series of protective effects of resveratrol against As2O3-induced oxidative damage in normal human bronchial epithelial (HBE) cells. We showed that treatment of HBE cells with resveratrol significantly reduced cellular levels of DNA damage, chromosomal breakage and apoptosis induced by As2O3. The effect of resveratrol against DNA damage was associated with a decreased level of reactive oxygen species and lipid peroxidation in cells treated by As2O3, suggesting that resveratrol protects against As2O3 toxicity via a cellular anti-oxidative stress pathway. Further analysis of the roles of resveratrol demonstrated that it modulated biosynthesis, recycling and consumption of glutathione (GSH), thereby promoting GSH homeostasis in HBE cells treated by As2O3. This was further supported by results showing that resveratrol prevented an increase in the activities and levels of caspases, Fas, Fas-L and cytochrome c proteins induced by As2O3. Our study indicates that resveratrol relieves As2O3-induced oxidative damage in normal human lung cells via maintenance of GSH homeostasis and suppression of apoptosis. PMID:25339131

2',5'-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways.

PubMed

Kachadourian, Remy; Pugazhenthi, Subbiah; Velmurugan, Kalpana; Backos, Donald S; Franklin, Christopher C; McCord, Joe M; Day, Brian J

2011-09-15

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest because of their anti-inflammatory and antiangiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide synthase and to induce the expression of heme oxygenase-1. This study examines the mechanisms by which 2',5'-dihydroxychalcone (2',5'-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant-response elements (MCF-7/AREc32). The 2',5'-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP(5+), suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2',5'-DHC treatment induced phosphorylation of the c-Jun N-terminal kinase (JNK) pathway, which was also inhibited by MnTDE-1,3-IP(5+). These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, whereas 2',5'-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, cotreatment with MnTDE-1,3-IP(5+) did not decrease 2',5'-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein kinase (MAPK) pathways showed a role for JNK and p38MAPK in mediating the 2',5'-DHC-induced Nrf2 response. These findings suggest that the 2',5'-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways. Copyright © 2011 Elsevier Inc. All rights reserved.

Sulfur amino acid metabolism in doxorubicin-resistant breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryu, Chang Seon; Kwak, Hui Chan; Lee, Kye Sook

2011-08-15

Although methionine dependency is a phenotypic characteristic of tumor cells, it remains to be determined whether changes in sulfur amino acid metabolism occur in cancer cells resistant to chemotherapeutic medications. We compared expression/activity of sulfur amino acid metabolizing enzymes and cellular levels of sulfur amino acids and their metabolites between normal MCF-7 cells and doxorubicin-resistant MCF-7 (MCF-7/Adr) cells. The S-adenosylmethionine/S-adenosylhomocysteine ratio, an index of transmethylation potential, in MCF-7/Adr cells decreased to {approx} 10% relative to that in MCF-7 cells, which may have resulted from down-regulation of S-adenosylhomocysteine hydrolase. Expression of homocysteine-clearing enzymes, such as cystathionine beta-synthase, methionine synthase/methylene tetrahydrofolate reductase,more » and betaine homocysteine methyltransferase, was up-regulated in MCF-7/Adr cells, suggesting that acquiring doxorubicin resistance attenuated methionine-dependence and activated transsulfuration from methionine to cysteine. Homocysteine was similar, which is associated with a balance between the increased expressions of homocysteine-clearing enzymes and decreased extracellular homocysteine. Despite an elevation in cysteine, cellular GSH decreased in MCF-7/Adr cells, which was attributed to over-efflux of GSH into the medium and down-regulation of the GSH synthesis enzyme. Consequently, MCF-7/Adr cells were more sensitive to the oxidative stress induced by bleomycin and menadione than MCF-7 cells. In conclusion, our results suggest that regulating sulfur amino acid metabolism may be a possible therapeutic target for chemoresistant cancer cells. These results warrant further investigations to determine the role of sulfur amino acid metabolism in acquiring anticancer drug resistance in cancer cells using chemical and biological regulators involved in sulfur amino acid metabolism. - Research Highlights: > MCF-7/Adr cells showed decreases in cellular GSH, which were attributed to increase efflux of GSH. > MCF-7/Adr was more sensitive to oxidative stress induced by bleomycin and menadione. > Hcy-clearing enzymes involved in were up-regulated in MCF-7/Adr cells. > Doxorubicin-resistance attenuated Met-dependence and activated transsulfuration. > Regulating sulfur amino acid metabolism may be a possible therapeutic target.« less

A γ-Glutamyl Cyclotransferase Protects Arabidopsis Plants from Heavy Metal Toxicity by Recycling Glutamate to Maintain Glutathione Homeostasis[C][W

PubMed Central

Paulose, Bibin; Chhikara, Sudesh; Coomey, Joshua; Jung, Ha-il; Vatamaniuk, Olena; Dhankher, Om Parkash

2013-01-01

Plants detoxify toxic metals through a GSH-dependent pathway. GSH homeostasis is maintained by the γ-glutamyl cycle, which involves GSH synthesis and degradation and the recycling of component amino acids. The enzyme γ-glutamyl cyclotransferase (GGCT) is involved in Glu recycling, but the gene(s) encoding GGCT has not been identified in plants. Here, we report that an Arabidopsis thaliana protein with a cation transport regulator-like domain, hereafter referred to as GGCT2;1, functions as γ-glutamyl cyclotransferase. Heterologous expression of GGCT2;1 in Saccharomyces cerevisiae produced phenotypes that were consistent with decreased GSH content attributable to either GSH degradation or the diversion of γ-glutamyl peptides to produce 5-oxoproline (5-OP). 5-OP levels were further increased by the addition of arsenite and GSH to the medium, indicating that GGCT2;1 participates in the cellular response to arsenic (As) via GSH degradation. Recombinant GGCT2;1 converted both GSH and γ-glutamyl Ala to 5-OP in vitro. GGCT2;1 transcripts were upregulated in As-treated Arabidopsis, and ggct2;1 knockout mutants were more tolerant to As and cadmium than the wild type. Overexpression of GGCT2;1 in Arabidopsis resulted in the accumulation of 5-OP. Under As toxicity, the overexpression lines showed minimal changes in de novo Glu synthesis, while the ggct2;1 mutant increased nitrogen assimilation by severalfold, resulting in a very low As/N ratio in tissue. Thus, our results suggest that GGCT2;1 ensures sufficient GSH turnover during abiotic stress by recycling Glu. PMID:24214398

Selective Targeting of the Cysteine Proteome by Thioredoxin and Glutathione Redox Systems

PubMed Central

Go, Young-Mi; Roede, James R.; Walker, Douglas I.; Duong, Duc M.; Seyfried, Nicholas T.; Orr, Michael; Liang, Yongliang; Pennell, Kurt D.; Jones, Dean P.

2013-01-01

Thioredoxin (Trx) and GSH are the major thiol antioxidants protecting cells from oxidative stress-induced cytotoxicity. Redox states of Trx and GSH have been used as indicators of oxidative stress. Accumulating studies suggest that Trx and GSH redox systems regulate cell signaling and metabolic pathways differently and independently during diverse stressful conditions. In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx- and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized more than 1.3-fold by ARF, whereas BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than the GSH system, support distinct function of Trx and GSH in cellular redox control, and show for the first time in mammalian cells selective targeting peptidyl Cys and biological pathways due to deficient function of the Trx system. PMID:23946468

Differential response of DU145 and PC3 prostate cancer cells to ionizing radiation: role of reactive oxygen species, GSH and Nrf2 in radiosensitivity.

PubMed

Jayakumar, Sundarraj; Kunwar, Amit; Sandur, Santosh K; Pandey, Badri N; Chaubey, Ramesh C

2014-01-01

Radioresistance is the major impediment in radiotherapy of many cancers including prostate cancer, necessitating the need to understand the factors contributing to radioresistance in tumor cells. In the present study, the role of cellular redox and redox sensitive transcription factor, Nrf2 in the radiosensitivity of prostate cancer cell lines PC3 and DU145, has been investigated. Differential radiosensitivity of PC3 and DU145 cells was assessed using clonogenic assay, flow cytometry, and comet assay. Their redox status was measured using DCFDA and DHR probes. Expression of Nrf2 and its dependent genes was measured by EMSA and real time PCR. Knockdown studies were done using shRNA transfection. PC3 and DU145 cells differed significantly in their radiosensitivity as observed by clonogenic survival, apoptosis and neutral comet assays. Both basal and inducible levels of ROS were higher in PC3 cells than that of DU145 cells. DU145 cells showed higher level of basal GSH content and GSH/GSSG ratio than that of PC3 cells. Further, significant increase in both basal and induced levels of Nrf2 and its dependent genes was observed in DU145 cells. Knock-down experiments and pharmacological intervention studies revealed the involvement of Nrf2 in differential radio-resistance of these cells. Cellular redox status and Nrf2 levels play a causal role in radio-resistance of prostate cancer cells. The pivotal role Nrf2 has been shown in the radioresistance of tumor cells and this study will further help in exploiting this factor in radiosensitization of other tumor cell types. © 2013.

Polyamines are traps for reactive intermediates in furan metabolism

PubMed Central

Peterson, Lisa A.; Phillips, Martin B.; Lu, Ding; Sullivan, Mathilde M.

2011-01-01

Furan is toxic and carcinogenic in rodents. Because of the large potential for human exposure, furan is classified as a possible human carcinogen. The detailed mechanism by which furan causes toxicity and cancer is not yet known. Since furan toxicity requires cytochrome P450-catalyzed oxidation of furan, we have characterized the urinary and hepatocyte metabolites of furan to gain insight into the chemical nature of the reactive intermediate. Previous studies in hepatocytes indicated that furan is oxidized to the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA), which reacts with glutathione (GSH) to form 2-(S-glutathionyl)-succinaldehyde (GSH-BDA). This intermediate forms pyrrole cross-links with cellular amines such as lysine and glutamine. In this report, we demonstrate that GSH-BDA also forms cross-links with ornithine, putrescine and spermidine when furan is incubated with rat hepatocytes. The relative levels of these metabolites are not completely explained by hepatocellular levels of the amines or by their reactivity with GSH-BDA. Mercapturic acid derivatives of the spermidine cross-links were detected in the urine of furan-treated rats, which indicates that this metabolic pathway occurs in vivo. Their detection in furan-treated hepatocytes and in urine from furan-treated rats indicates that polyamines may play an important role in the toxicity of furan PMID:21842885

Fifty hertz extremely low-frequency magnetic field exposure elicits redox and trophic response in rat-cortical neurons.

PubMed

Di Loreto, Silvia; Falone, Stefano; Caracciolo, Valentina; Sebastiani, Pierluigi; D'Alessandro, Antonella; Mirabilio, Alessandro; Zimmitti, Vincenzo; Amicarelli, Fernanda

2009-05-01

Large research activity has raised around the mechanisms of interaction between extremely low-frequency magnetic fields (ELF-MFs) and biological systems. ELF-MFs may interfere with chemical reactions involving reactive oxygen species (ROS), thus facilitating oxidative damages in living cells. Cortical neurons are particularly susceptible to oxidative stressors and are also highly dependent on the specific factors and proteins governing neuronal development, activity and survival. The aim of the present work was to investigate the effects of exposures to two different 50 Hz sinusoidal ELF-MFs intensities (0.1 and 1 mT) in maturing rat cortical neurons' major anti-oxidative enzymatic and non-enzymatic cellular protection systems, membrane peroxidative damage, as well as growth factor, and cytokine expression pattern. Briefly, our results showed that ELF-MFs affected positively the cell viability and concomitantly reduced the levels of apoptotic death in rat neuronal primary cultures, with no significant effects on the main anti-oxidative defences. Interestingly, linear regression analysis suggested a positive correlation between reduced glutathione (GSH) and ROS levels in 1 mT MF-exposed cells. On this basis, our hypothesis is that GSH could play an important role in the antioxidant defence towards the ELF-MF-induced redox challenge. Moreover, the GSH-based cellular response was achieved together with a brain-derived neurotrophic factor over-expression as well as with the interleukin 1beta-dependent regulation of pro-survival signaling pathways after ELF-MF exposure.

Neurotoxicity of a polybrominated diphenyl ether mixture (DE-71) in mouse neurons and astrocytes is modulated by intracellular glutathione levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giordano, Gennaro; Kavanagh, Terrance J.; Costa, Lucio G.

2008-10-15

Polybrominated diphenyl ether (PBDE) flame retardants have become widespread environmental contaminants. Body burden in the U.S. population has been shown to be higher than in other countries, and infants and toddlers have highest exposure through maternal breast milk and household dust. The primary concern for adverse health effects of PBDEs relates to their potential developmental neurotoxicity, which has been found in a number of animal studies. Information on the possible mechanisms of PBDE neurotoxicity is limited, though some studies have suggested that PBDEs may elicit oxidative stress. The present study examined the in vitro neurotoxicity of DE-71, a penta-BDE mixture,more » in primary neurons and astrocytes obtained from wild-type and Gclm knockout mice, which lack the modifier subunit of glutamate-cysteine ligase and, as a consequence, have very low levels of glutathione (GSH). These experiments show that neurotoxicity of DE-71 in these cells is modulated by cellular GSH levels. Cerebellar granule neurons (CGNs) from Gclm (-/-) mice displayed a higher sensitivity to DE-71 toxicity compared to CGNs from wild-type animals. DE-71 neurotoxicity in CGNs from Gclm (+/+) mice was exacerbated by GSH depletion, and in CGNs from both genotypes it was antagonized by increasing GSH levels and by antioxidants. DE-71 caused an increase in reactive oxygen species and in lipid peroxidation in CGNs, that was more pronounced in Gclm (-/-) mice. Toxicity of DE-71 was mostly due to the induction of apoptotic cell death. An analysis of DE-71-induced cytotoxicity and apoptosis in neurons and astrocytes from different brain areas (cerebellum, hippocampus, cerebral cortex) in both mouse genotypes showed a significant correlation with intracellular GSH levels. As an example, DE-71 caused cytotoxicity in hippocampal neurons with IC50s of 2.2 and 0.3 {mu}M, depending on genotype, and apoptosis with IC50s of 2.3 and 0.4 {mu}M, respectively. These findings suggest that the developmental neurotoxicity of PBDE may involve oxidative stress, and that individual with genetic polymorphisms leading to lower GSH levels may be more susceptible to their adverse effects.« less

Selected flavonoids potentiate the toxicity of cisplatin in human lung adenocarcinoma cells: a role for glutathione depletion.

PubMed

Kachadourian, Remy; Leitner, Heather M; Day, Brian J

2007-07-01

Adjuvant therapies that enhance the anti-tumor effects of cis-diammineplatinum(II) dichloride (cisplatin, CDDP) are actively being pursued. Growing evidence supports the involvement of mitochondrial dysfunction in the anti-cancer effect of cisplatin. We examined the potential of using selective flavonoids that are effective in depleting tumor cells of glutathione (GSH) to potentiate cisplatin-mediated cytotoxicity in human lung adenocarcinoma (A549) cells. We found that cisplatin (40 microM, 48-h treatment) disrupts the steady-state levels of mitochondrial respiratory complex I, which correlates with elevated mitochondrial reactive oxygen species (ROS) production and cytochrome c release. The flavonoids, 2',5'-dihydroxychalcone (2',5'-DHC, 20 microM) and chrysin (20 microM) potentiated the cytotoxicity of cisplatin (20 microM), which could be blocked by supplementation of the media with exogenous GSH (500 microM). Both 2',5'-DHC and chrysin were more effective than the specific inhibitor of GSH synthesis, L-buthionine sulfoximine (BSO, 20 microM), in inducing GSH depletion and potentiating the cytotoxic effect of cisplatin. These data suggest that the flavonoid-induced potentiation of cisplatin's toxicity is due, in part, to synergetic pro-oxidant effects of cisplatin by inducing mitochondrial dysfunction, and the flavonoids by depleting cellular GSH, an important antioxidant defense.

Quantitative optical imaging of paracetamol-induced metabolism changes in the liver

NASA Astrophysics Data System (ADS)

Liang, Xiaowen; Wang, Haolu; Liu, Xin; Roberts, Michael

2016-12-01

Paracetamol is the most readily available and widely used painkiller. However, its toxicity remains the most common cause of liver injury. The toxicity of paracetamol has been attributing to its toxic metabolite, which depletes cellular glutathione (GSH) stores and reacts within cells to increase oxidative stress, leading to mitochondrial dysfunction and cell necrosis. Multiphoton microscopy (MPM) and fluorescence lifetime imaging (FLIM) can provide quantitative imaging of biological tissues and organs in vivo and allow direct visualization of cellular events, which were used to monitor cellular metabolism in paracetamol-induced toxicity in this study. To better understand mechanisms of paracetamol induced liver injury, the redox ratio of NADH/FAD in liver cells were detected and quantified by MPM imaging to represent the relative rates of glycolysis and oxidative phosphorylation within cells. Compared to normal liver, average fluorescence lifetime of NADH and redox ratio of NADH/FAD in hepatocytes was significantly decreased after paracetamol overdose for 12 and 24 hrs, reflecting impaired metabolic activity. GSH levels of treatment groups were significantly lower than those of normal livers, with gradually decreasing from periportal to centrilobular zonation. This imaging technique has significant implications for investigating metabolic mechanisms of paracetamol toxicity.

Pummelo Protects Doxorubicin-Induced Cardiac Cell Death by Reducing Oxidative Stress, Modifying Glutathione Transferase Expression, and Preventing Cellular Senescence

PubMed Central

Chularojmontri, L.; Gerdprasert, O.; Wattanapitayakul, S. K.

2013-01-01

Citrus flavonoids have been shown to reduce cardiovascular disease (CVD) risks prominently due to their antioxidant effects. Here we investigated the protective effect of pummelo (Citrus maxima, CM) fruit juice in rat cardiac H9c2 cells against doxorubicin (DOX-) induced cytotoxicity. Four antioxidant compositions (ascorbic acid, hesperidin, naringin, and gallic acid) were determined by HPLC. CM significantly increased cardiac cell survival from DOX toxicity as evaluated by MTT assay. Reduction of cellular oxidative stress was monitored by the formation of DCF fluorescent product and total glutathione (GSH) levels. The changes in glutathione-S-transferase (GST) activity and expression were determined by enzyme activity assay and Western blot analysis, respectively. Influence of CM on senescence-associated β-galactosidase activity (SA-β-gal) was also determined. The mechanisms of cytoprotection involved reduction of intracellular oxidative stress, maintaining GSH availability, and enhanced GST enzyme activity and expression. DOX-induced cellular senescence was also attenuated by long-term CM treatment. Thus, CM fruit juice can be promoted as functional fruit to protect cells from oxidative cell death, enhance the phase II GSTP enzyme activity, and decrease senescence phenotype population induced by cardiotoxic agent such as DOX. PMID:23401708

Vitamin B12 deficiency results in severe oxidative stress, leading to memory retention impairment in Caenorhabditis elegans.

PubMed

Bito, Tomohiro; Misaki, Taihei; Yabuta, Yukinori; Ishikawa, Takahiro; Kawano, Tsuyoshi; Watanabe, Fumio

2017-04-01

Oxidative stress is implicated in various human diseases and conditions, such as a neurodegeneration, which is the major symptom of vitamin B 12 deficiency, although the underlying disease mechanisms associated with vitamin B 12 deficiency are poorly understood. Vitamin B 12 deficiency was found to significantly increase cellular H 2 O 2 and NO content in Caenorhabditis elegans and significantly decrease low molecular antioxidant [reduced glutathione (GSH) and L-ascorbic acid] levels and antioxidant enzyme (superoxide dismutase and catalase) activities, indicating that vitamin B 12 deficiency induces severe oxidative stress leading to oxidative damage of various cellular components in worms. An NaCl chemotaxis associative learning assay indicated that vitamin B 12 deficiency did not affect learning ability but impaired memory retention ability, which decreased to approximately 58% of the control value. When worms were treated with 1mmol/L GSH, L-ascorbic acid, or vitamin E for three generations during vitamin B 12 deficiency, cellular malondialdehyde content as an index of oxidative stress decreased to the control level, but the impairment of memory retention ability was not completely reversed (up to approximately 50%). These results suggest that memory retention impairment formed during vitamin B 12 deficiency is partially attributable to oxidative stress. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

2′,5′-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways

PubMed Central

Kachadourian, Remy; Pugazhenthi, Subbiah; Velmurugan, Kalpana; Backos, Donald S.; Franklin, Christopher C.; McCord, Joe M.; Day, Brian J.

2011-01-01

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest due to their anti-inflammatory and anti-angiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide (NO) synthase and to induce the expression of heme oxygenase-1 (HO-1). This study examines the mechanisms by which 2′,5′-dihydroxychalcone (2′,5′-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant response elements (MCF-7/AREc32). 2′,5′-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP5+, suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2′,5′-DHC treatment induced the phosphorylation of c-Jun N-terminal kinase (JNK) pathway that was also inhibited by MnTDE-1,3-IP5+. These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, while 2′,5′-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, co-treatment with MnTDE-1,3-IP5+ did not decrease 2′,5′-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein (MAP) kinase pathways showed a role for JNK and p38MAPK in mediating the 2′,5′-DHC-induced Nrf2 response. These findings suggest that the 2′,5′-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways. PMID:21712085

Regulation of Cigarette Smoke (CS)-Induced Autophagy by Nrf2.

PubMed

Zhu, Lingxiang; Barrett, Erika C; Barret, Erika C; Xu, Yuxue; Liu, Zuguo; Manoharan, Aditya; Chen, Yin

2013-01-01

Cigarette smoke (CS) has been reported to induce autophagy in airway epithelial cells. The subsequent autophagic cell death has been proposed to play an important pathogenic role in chronic obstructive pulmonary disease (COPD); however, the underlying molecular mechanism is not entirely clear. Using CS extract (CSE) as a surrogate for CS, we found that it markedly increased the expressions of both LC3B-I and LC3B-II as well as autophagosomes in airway epithelial cells. This is in contrast to the common autophagy inducer (i.e., starvation) that increases LC3B-II but reduces LC3B-I. Further studies indicate that CSE regulated LC3B at transcriptional and post-translational levels. In addition, CSE, but not starvation, activated Nrf2-mediated adaptive response. Increase of cellular Nrf2 by either Nrf2 overexpression or the knockdown of Keap1 (an Nrf2 inhibitor) significantly repressed CSE-induced LC3B-I and II as well as autophagosomes. Supplement of NAC (a GSH precursor) or GSH recapitulated the effect of Nrf2, suggesting the increase of cellular GSH level is responsible for Nrf2 effect on LC3B and autophagosome. Interestingly, neither Nrf2 activation nor GSH supplement could restore the repressed activities of mTOR or its downstream effctor-S6K. Thus, the Nrf2-dependent autophagy-suppression was not due to the re-activation of mTOR-the master repressor of autophagy. To search for the downstream effector of Nrf2 on LC3B and autophagosome, we tested Nrf2-dependent genes (i.e., NQO1 and P62) that are also increased by CSE treatment. We found that P62, but not NQO1, could mimic the effect of Nrf2 activation by repressing LC3B expression. Thus, Nrf2->P62 appears to play an important role in the regulation of CSE-induced LC3B and autophagosome.

Direct structural evidence of protein redox regulation obtained by in-cell NMR.

PubMed

Mercatelli, Eleonora; Barbieri, Letizia; Luchinat, Enrico; Banci, Lucia

2016-02-01

The redox properties of cellular environments are critical to many functional processes, and are strictly controlled in all living organisms. The glutathione-glutathione disulfide (GSH-GSSG) couple is the most abundant intracellular redox couple. A GSH redox potential can be calculated for each cellular compartment, which reflects the redox properties of that environment. This redox potential is often used to predict the redox state of a disulfide-containing protein, based on thermodynamic considerations. However, thiol-disulfide exchange reactions are often catalyzed by specific partners, and the distribution of the redox states of a protein may not correspond to the thermodynamic equilibrium with the GSH pool. Ideally, the protein redox state should be measured directly, bypassing the need to extrapolate from the GSH. Here, by in-cell NMR, we directly observe the redox state of three human proteins, Cox17, Mia40 and SOD1, in the cytoplasm of human and bacterial cells. We compare the observed distributions of redox states with those predicted by the GSH redox potential, and our results partially agree with the predictions. Discrepancies likely arise from the fact that the redox state of SOD1 is controlled by a specific partner, its copper chaperone (CCS), in a pathway which is not linked to the GSH redox potential. In principle, in-cell NMR allows determining whether redox proteins are at the equilibrium with GSH, or they are kinetically regulated. Such approach does not need assumptions on the redox potential of the environment, and provides a way to characterize each redox-regulating pathway separately. Copyright © 2015 Elsevier B.V. All rights reserved.

Glutathione Efflux and Cell Death

PubMed Central

2012-01-01

Abstract Significance: Glutathione (GSH) depletion is a central signaling event that regulates the activation of cell death pathways. GSH depletion is often taken as a marker of oxidative stress and thus, as a consequence of its antioxidant properties scavenging reactive species of both oxygen and nitrogen (ROS/RNS). Recent Advances: There is increasing evidence demonstrating that GSH loss is an active phenomenon regulating the redox signaling events modulating cell death activation and progression. Critical Issues: In this work, we review the role of GSH depletion by its efflux, as an important event regulating alterations in the cellular redox balance during cell death independent from oxidative stress and ROS/RNS formation. We discuss the mechanisms involved in GSH efflux during cell death progression and the redox signaling events by which GSH depletion regulates the activation of the cell death machinery. Future Directions: The evidence summarized here clearly places GSH transport as a central mechanism mediating redox signaling during cell death progression. Future studies should be directed toward identifying the molecular identity of GSH transporters mediating GSH extrusion during cell death, and addressing the lack of sensitive approaches to quantify GSH efflux. Antioxid. Redox Signal. 17, 1694–1713. PMID:22656858

Outer membrane vesicles enhance the carcinogenic potential of Helicobacter pylori.

PubMed

Chitcholtan, Kenny; Hampton, Mark B; Keenan, Jacqueline I

2008-12-01

Chronic Helicobacter pylori infection is associated with an increased risk of gastric carcinogenesis. These non-invasive bacteria colonize the gastric mucosa and constitutively shed small outer membrane vesicles (OMV). In this study, we investigated the direct effect of H.pylori OMV on cellular events associated with carcinogenesis. We observed increased micronuclei formation in AGS human gastric epithelial cells treated with OMV isolated from a toxigenic H.pylori strain (60190). This effect was absent in OMV from strain 60190v:1 that has a mutant vacA, indicating VacA-dependent micronuclei formation. VacA induces intracellular vacuolation, and reduced acridine orange staining indicated disruption in the integrity of these vacuoles. This was accompanied by an alteration in iron metabolism and glutathione (GSH) loss, suggesting a role for oxidative stress in genomic damage. Increasing intracellular GSH levels with a GSH ester abrogated the VacA-mediated increase in micronuclei formation. In conclusion, OMV-mediated delivery of VacA to the gastric epithelium may constitute a new mechanism for H.pylori-induced gastric carcinogenesis.

Selected flavonoids potentiate the toxicity of cisplatin in human lung adenocarcinoma cells: A role for glutathione depletion

PubMed Central

KACHADOURIAN, REMY; LEITNER, VHEATHER M.; DAY, BRIAN J.

2014-01-01

Adjuvant therapies that enhance the anti-tumor effects of cisplatin are actively being pursued. Growing evidence supports the involvement of mitochondrial dysfunction in the anti-cancer effect of cis-diammineplatinum(II) dichloride (cisplatin, CDDP). We examined the potential of using selective flavonoids that are effective in depleting tumor cells of glu-tathione (GSH) to potentiate cisplatin-mediated cytotoxicity in human lung adenocarcinoma (A549) cells. We found that cisplatin (40 μM, 48-h treatment) disrupts the steady-state levels of mitochondrial respiratory complex I, which correlates with elevated mitochondrial reactive oxygen species (ROS) production and cytochrome c release. The flavonoids, 2′,5′-dihydroxychalcone (2′,5′-DHC, 20 μM) and chrysin (20 μM) potentiated the cytotoxicity of cisplatin (20 μM), which could be blocked by supplementation of the media with exogenous GSH (500 μM). Both 2′,5′-DHC and chrysin were more effective than the specific inhibitor of GSH synthesis, L-buthionine sulfoximine (BSO, 20 μM), in inducing GSH depletion and potentiating the cytotoxic effect of cisplatin. These data suggest that the flavonoid-induced potentiation of cisplatin’s toxicity is due, in part, to synergetic pro-oxidant effects of cisplatin by inducing mitochondrial dysfunction, and the flavonoids by depleting cellular GSH, an important antioxidant defense. PMID:17549417

Puerarin protects against lead-induced cytotoxicity in cultured primary rat proximal tubular cells.

PubMed

Liu, Gang; Li, Zifa; Wang, Jinqiu; Wang, Hong; Wang, Zhenyong; Wang, Lin

2014-10-01

Puerarin, a potent free radicals scavenger, has been demonstrated to have protective efficacy in oxidative damage induced by nephrotoxins. In the present study, the attenuating effect of puerarin (PU) on lead (Pb)-induced apoptosis and oxidative stress was investigated in cultured primary rat proximal tubular (rPT) cells. Results showed that exposure to 0.5 µM Pb induced a decrease in cell viability accompanied with obvious cellular morphological alterations and caused an increase in apoptotic rate and apoptotic morphological changes. Simultaneously, depletion of mitochondrial membrane potential (ΔΨ) and intracellular glutathione (GSH); elevation of caspase-3 activity, intracellular reactive oxygen species, and malondialdehyde levels; and inhibition of GSH peroxidase (GSH-Px) activity were revealed in the cells exposed to Pb alone. However, simultaneous supplementation with PU (50 and 100 µM) protected rPT cells from Pb-induced cytotoxicity through inhibiting apoptosis, attenuating lipid peroxidation, renewing mitochondrial function, and elevating the intracellular antioxidants (nonenzymatic and enzymic) levels. In conclusion, these findings suggested that PU, as a widely distributed dietary antioxidant, contributes potentially to inhibition of Pb-induced cytotoxicity in rPT cells. © The Author(s) 2014.

The fairytale of the GSSG/GSH redox potential.

PubMed

Flohé, Leopold

2013-05-01

The term GSSG/GSH redox potential is frequently used to explain redox regulation and other biological processes. The relevance of the GSSG/GSH redox potential as driving force of biological processes is critically discussed. It is recalled that the concentration ratio of GSSG and GSH reflects little else than a steady state, which overwhelmingly results from fast enzymatic processes utilizing, degrading or regenerating GSH. A biological GSSG/GSH redox potential, as calculated by the Nernst equation, is a deduced electrochemical parameter based on direct measurements of GSH and GSSG that are often complicated by poorly substantiated assumptions. It is considered irrelevant to the steering of any biological process. GSH-utilizing enzymes depend on the concentration of GSH, not on [GSH](2), as is predicted by the Nernst equation, and are typically not affected by GSSG. Regulatory processes involving oxidants and GSH are considered to make use of mechanistic principles known for thiol peroxidases which catalyze the oxidation of hydroperoxides by GSH by means of an enzyme substitution mechanism involving only bimolecular reaction steps. The negligibly small rate constants of related spontaneous reactions as compared with enzyme-catalyzed ones underscore the superiority of kinetic parameters over electrochemical or thermodynamic ones for an in-depth understanding of GSH-dependent biological phenomena. At best, the GSSG/GSH potential might be useful as an analytical tool to disclose disturbances in redox metabolism. This article is part of a Special Issue entitled Cellular Functions of Glutathione. Copyright © 2012 Elsevier B.V. All rights reserved.

Changes in the Antioxidant Systems as Part of the Signaling Pathway Responsible for the Programmed Cell Death Activated by Nitric Oxide and Reactive Oxygen Species in Tobacco Bright-Yellow 2 Cells1

PubMed Central

de Pinto, Maria Concetta; Tommasi, Franca; De Gara, Laura

2002-01-01

Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS), for the activation of the hypersensitive reaction, a defense response induced in the noncompatible plant-pathogen interaction. However, its involvement in activating programmed cell death (PCD) in plant cells has been questioned. In this paper, the involvement of the cellular antioxidant metabolism in the signal transduction triggered by these bioactive molecules has been investigated. NO and ROS levels were singularly or simultaneously increased in tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells by the addition to the culture medium of NO and/or ROS generators. The individual increase in NO or ROS had different effects on the studied parameters than the simultaneous increase in the two reactive species. NO generation did not cause an increase in phenylalanine ammonia-lyase (PAL) activity or induction of cellular death. It only induced minor changes in ascorbate (ASC) and glutathione (GSH) metabolisms. An increase in ROS induced oxidative stress in the cells, causing an oxidation of the ASC and GSH redox pairs; however, it had no effect on PAL activity and did not induce cell death when it was generated at low concentrations. In contrast, the simultaneous increase of NO and ROS activated a process of death with the typical cytological and biochemical features of hypersensitive PCD and a remarkable rise in PAL activity. Under the simultaneous generation of NO and ROS, the cellular antioxidant capabilities were also suppressed. The involvement of ASC and GSH as part of the transduction pathway leading to PCD is discussed. PMID:12376637

Distinct Redox Regulation in Sub-Cellular Compartments in Response to Various Stress Conditions in Saccharomyces cerevisiae

PubMed Central

Ayer, Anita; Sanwald, Julia; Pillay, Bethany A.; Meyer, Andreas J.; Perrone, Gabriel G.; Dawes, Ian W.

2013-01-01

Responses to many growth and stress conditions are assumed to act via changes to the cellular redox status. However, direct measurement of pH-adjusted redox state during growth and stress has never been carried out. Organellar redox state (E GSH) was measured using the fluorescent probes roGFP2 and pHluorin in Saccharomyces cerevisiae. In particular, we investigated changes in organellar redox state in response to various growth and stress conditions to better understand the relationship between redox-, oxidative- and environmental stress response systems. E GSH values of the cytosol, mitochondrial matrix and peroxisome were determined in exponential and stationary phase in various media. These values (−340 to −350 mV) were more reducing than previously reported. Interestingly, sub-cellular redox state remained unchanged when cells were challenged with stresses previously reported to affect redox homeostasis. Only hydrogen peroxide and heat stress significantly altered organellar redox state. Hydrogen peroxide stress altered the redox state of the glutathione disulfide/glutathione couple (GSSG, 2H+/2GSH) and pH. Recovery from moderate hydrogen peroxide stress was most rapid in the cytosol, followed by the mitochondrial matrix, with the peroxisome the least able to recover. Conversely, the bulk of the redox shift observed during heat stress resulted from alterations in pH and not the GSSG, 2H+/2GSH couple. This study presents the first direct measurement of pH-adjusted redox state in sub-cellular compartments during growth and stress conditions. Redox state is distinctly regulated in organelles and data presented challenge the notion that perturbation of redox state is central in the response to many stress conditions. PMID:23762325

IDH1 R132H Mutation Enhances Cell Migration by Activating AKT-mTOR Signaling Pathway, but Sensitizes Cells to 5-FU Treatment as NADPH and GSH Are Reduced.

PubMed

Zhu, Huixia; Zhang, Ye; Chen, Jianfeng; Qiu, Jiangdong; Huang, Keting; Wu, Mindan; Xia, Chunlin

2017-01-01

Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation. Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH. We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment. Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment.

Antioxidant potential properties of mushroom extract (Agaricus bisporus) against aluminum-induced neurotoxicity in rat brain.

PubMed

Waly, Mostafa I; Guizani, Nejib

2014-09-01

Aluminum (Al) is an environmental toxin that induces oxidative stress in neuronal cells. Mushroom cultivar extract (MCE) acted as a potent antioxidant agent and protects against cellular oxidative stress in human cultured neuronal cells. This study aimed to investigate the neuroprotective effect of MCE against Al-induced neurotoxicity in rat brain. Forty Sprague-Dawley rats were divided into 4 groups (10 rats per group), control group, MCE-fed group, Al-administered group and MCE/Al-treated group. Animals were continuously fed ad-libitum their specific diets for 4 weeks. At the end of the experiment, all rats were sacrificed and the brain tissues were homogenized and examined for biochemical measurements of neurocellular oxidative stress indices [glutathione (GSH), Total Antioxidant Capacity (TAC), antioxidant enzymes and oxidized dichlorofluorescein (DCF)]. Al-administration caused inhibition of antioxidant enzymes and a significant decrease in GSH and TAC levels, meanwhile it positively increased cellular oxidized DCF level, as well as Al concentration in brain tissues. Feeding animals with MCE had completely offset the Al-induced oxidative stress and significantly restrict the Al accumulation in brain tissues of Al-administered rats. The results obtained suggest that MCE acted as a potent dietary antioxidant and protects against Al-mediated neurotoxicity, by abrogating neuronal oxidative stress.

Glutathione peroxidase: fact and fiction.

PubMed

Flohé, L

The present knowledge of glutathione (GSH) peroxidase is briefly reviewed: GSH peroxidase has a molecular weight of about 85,000, consists of four apparently-identical subunits and contains four g atom of selenium/mol. The enzyme-bound selenium can undergo a substrate-induced redox change and is obviously essential for activity. In accordance with the assumption that a selenol group is reversibly oxidized during catalysis, ping-pong kinetics are observed. Limiting maximum velocities and Michaelis constants, indicating the formation of an enzyme-substrate complex, are not detectable. The enzyme is highly specific for GSH but reacts with many hydroperoxides. It can be deduced from the kinetic analysis of GSH peroxidase that in physiological conditions removal of hydroperoxide is largely independent of fluctuations in the cellular concentration of GSH. However, the system will abruptly collapse if the rate of hydroperoxide formation exceeds that of regeneration of GSH. By these considerations, the pathophysiological manifestation of disorders in GSH metabolism and pentose-phosphate shunt may be explained. With regard to its low specificity for hydroperoxides, GSH peroxidase could be involved in various metabolic events such as H2O2 removal in compartments low in catalase, hydroperoxide-mediated mutagenesis, protection of unsaturated lipids in biomembranes, prostaglandin biosynthesis, and regulation of prostacyclin formation.

Increased Activity of [gamma]-Glutamylcysteine Synthetase in Tomato Cells Selected for Cadmium Tolerance.

PubMed

Chen, J.; Goldsbrough, P. B.

1994-09-01

Two cell lines of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) were systematically compared for their capacity to tolerate cadmium. Unselected CdS cells died in the presence of 0.3 mM CdCl2. CdR6-0 cells, which were selected from CdS, survived and grew in medium supplemented with 0.3 mM CdCl2. Growth of CdR6-0 cells under this condition was accompanied by synthesis of cadmium-binding phytochelatins and maintenance of cellular glutathione (GSH) levels. CdR6-0 cells also exhibited increased tolerance to buthionine sulfoximine, in both the presence and absence of 0.1 mM CdCl2. The specific activity of [gamma]-glutamylcysteine synthetase (EC 6.3.2.2) was approximately 2-fold higher in CdR6-0 cells than in CdS cells, whereas there was no difference between cell lines in specific activity of GSH synthetase (EC 6.3.2.3). Increased activity of the first enzyme of GSH biosynthesis in CdR6-0 cells, presumably a result of selection for increased cadmium tolerance, provides an enhanced capacity to synthesize GSH and to maintain the production of phytochelatins in response to cadmium. This adaptation may contribute to the enhanced cadmium tolerance of CdR6-0 cells.

Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics.

PubMed

Umezawa, Keitaro; Yoshida, Masafumi; Kamiya, Mako; Yamasoba, Tatsuya; Urano, Yasuteru

2017-03-01

Alterations in glutathione (GSH) homeostasis are associated with a variety of diseases and cellular functions, and therefore, real-time live-cell imaging and quantification of GSH dynamics are important for understanding pathophysiological processes. However, existing fluorescent probes are unsuitable for these purposes due to their irreversible fluorogenic mechanisms or slow reaction rates. In this work, we have successfully overcome these problems by establishing a design strategy inspired by Mayr's work on nucleophilic reaction kinetics. The synthesized probes exhibit concentration-dependent, reversible and rapid absorption/fluorescence changes (t 1/2  = 620 ms at [GSH] = 1 mM), as well as appropriate K d values (1-10 mM: within the range of intracellular GSH concentrations). We also developed FRET-based ratiometric probes, and demonstrated that they are useful for quantifying GSH concentration in various cell types and also for real-time live-cell imaging of GSH dynamics with temporal resolution of seconds.

Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics

NASA Astrophysics Data System (ADS)

Umezawa, Keitaro; Yoshida, Masafumi; Kamiya, Mako; Yamasoba, Tatsuya; Urano, Yasuteru

2017-03-01

Alterations in glutathione (GSH) homeostasis are associated with a variety of diseases and cellular functions, and therefore, real-time live-cell imaging and quantification of GSH dynamics are important for understanding pathophysiological processes. However, existing fluorescent probes are unsuitable for these purposes due to their irreversible fluorogenic mechanisms or slow reaction rates. In this work, we have successfully overcome these problems by establishing a design strategy inspired by Mayr's work on nucleophilic reaction kinetics. The synthesized probes exhibit concentration-dependent, reversible and rapid absorption/fluorescence changes (t1/2 = 620 ms at [GSH] = 1 mM), as well as appropriate Kd values (1-10 mM: within the range of intracellular GSH concentrations). We also developed FRET-based ratiometric probes, and demonstrated that they are useful for quantifying GSH concentration in various cell types and also for real-time live-cell imaging of GSH dynamics with temporal resolution of seconds.

A role for CFTR in the elevation of glutathione levels in the lung by oral glutathione administration

PubMed Central

Kariya, Chirag; Leitner, Heather; Min, Elysia; van Heeckeren, Christiaan; van Heeckeren, Anna; Day, Brian J.

2014-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is the only known apical glutathione (GSH) transporter in the lung. The purpose of these studies was to determine whether oral GSH or glutathione disulfide (GSSG) treatment could increase lung epithelial lining fluid (ELF) GSH levels and whether CFTR plays a role in this process. The pharmacokinetic profile of an oral bolus dose of GSH (300 mg/kg) was determined in mice. Plasma, ELF, bronchoalveolar lavage (BAL) cells, and lung tissue were analyzed for GSH content. There was a rapid elevation in the GSH levels that peaked at 30 min in the plasma and 60 min in the lung, ELF, and BAL cells after oral GSH dosing. Oral GSH treatment produced a selective increase in the reduced and active form of GSH in all lung compartments examined. Oral GSSG treatment (300 mg/kg) resulted in a smaller increase of GSH levels. To evaluate the role of CFTR in this process, Cftr knockout (KO) mice and gut-corrected Cftr KO-transgenic (Tg) mice were given an oral bolus dose of GSH (300 mg/kg) and compared with wild-type mice for changes in GSH levels in plasma, lung, ELF, and BAL cells. There was a twofold increase in plasma, a twofold increase in lung, a fivefold increase in ELF, and a threefold increase in BAL cell GSH levels at 60 min in wild-type mice; however, GSH levels only increased by 40% in the plasma, 60% in the lung, 50% in the ELF, and twofold in the BAL cells within the gut-corrected Cftr KO-Tg mice. No change in GSH levels was observed in the uncorrected Cftr KO mice. These studies suggest that CFTR plays an important role in GSH uptake from the diet and transport processes in the lung. PMID:17369290

The toxicity of N-methyl-alpha-methyldopamine to freshly isolated rat hepatocytes is prevented by ascorbic acid and N-acetylcysteine.

PubMed

Carvalho, Márcia; Remião, Fernando; Milhazes, Nuno; Borges, Fernanda; Fernandes, Eduarda; Carvalho, Félix; Bastos, Maria Lourdes

2004-08-05

In the past decade, clinical evidence has increasingly shown that the liver is a target organ for 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") toxicity. The aims of the present in vitro study were: (1) to evaluate and compare the hepatotoxic effects of MDMA and one of its main metabolites, N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and (2) to investigate the ability of antioxidants, namely ascorbic acid and N-acetyl-L-cysteine (NAC), to prevent N-Me-alpha-MeDA-induced toxic injury, using freshly isolated rat hepatocytes. Cell suspensions were incubated with MDMA or N-Me-alpha-MeDA in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 3 h. To evaluate the potential protective effects of antioxidants, cells were preincubated with ascorbic acid in the final concentrations of 0.1 and 0.5 mM, or NAC in the final concentrations of 0.1 and 1 mM for 15 min before treatment with 1.6 mM N-Me-alpha-MeDA for 3 h (throughout this incubation period the cells were exposed to both compounds). The toxic effects were evaluated by measuring the cell viability, glutathione (GSH) and glutathione disulfide (GSSG), ATP, and the cellular activities of GSH peroxidase (GPX), GSSG reductase (GR), and GSH S-transferase (GST). MDMA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on cell viability, ATP levels, or on the activities of GR, GPX, and GST. In contrast, N-Me-alpha-MeDA was shown to induce not only a concentration- and time-dependent depletion of GSH, but also a depletion of ATP levels accompanied by a loss in cell viability, and decreases in the antioxidant enzyme activities. For both compounds, GSH depletion was not accompanied by increases in GSSG levels, which seems to indicate GSH depletion by adduct formation. Importantly, the presence of ascorbic acid (0.5 mM) or NAC (1 mM) prevented cell death and GSH depletion induced by N-Me-alpha-MeDA. The results provide evidence that MDMA and its metabolite N-Me-alpha-MeDA induce toxicity to freshly isolated rat hepatocytes. Oxidative stress may play a major role in N-Me-alpha-MeDA-induced hepatic toxicity since antioxidant defense systems are impaired and administration of antioxidants prevented N-Me-alpha-MeDA toxicity.

Effects of JP-8 Jet Fuel on Homeostasis of Clone 9 Rat Liver Cells

NASA Technical Reports Server (NTRS)

Wilson, C. L.; Barhoumi, R.; Burghardt, R.; Miladi, A.; Jung, A.

2000-01-01

Chronic exposure to JP-8 and other kerosene-based petroleum distillates has been associated with hepatic, renal, neurologic, pulmonary, and immune toxicity. However, the effects of kerosene-type jet fuels on cellular homeostasis hitherto have not been reported. Fluorescence imaging using a Meridian Ultima laser scanning fluorescence microscope was used to evaluate the effect of JP-8 jet fuel on a communication competent rat liver cell line. Several endpoints of cellular function were measured including gap junctional intercellular communication (GJIC), mitochondrial and plasma membrane potential (MMP and PMP, respectively), intracellular glutathione (GSH) concentration, glutathione-S-transferase (GST) activity, and reactive oxygen species (ROS) generation. Cells were treated with JP-8 (0.01 to 2% in ethanol (EtOH)) for the following time points: 1 h, 24 h, 48 h, and analysis immediately after addition of jet fuel. GJIC analyzed directly after addition of 1% JP-8 was reduced 4.9-fold relative to EtOH-dosed control groups and further reduction (12.6-fold) was observed in cells treated for 1 h. Moreover, GJIC was not recoverable in cells treated with 1% JP-8 for 1 h and subsequently washed and incubated in fresh medium for 1 h. Significant changes in GSH content and GST activity were observed in cells analyzed directly after addition of 1% JP-8. GSH content increased in cells treated for 1 h with less than 2% JP-8 whereas treatment with 2% JP-8 for 1 h resulted in a 50% reduction in intracellular GSH relative to EtOH-dosed controls. Cells treated with 1% JP-8 for 48 h exhibited changes in GSH levels. However, higher JP-8 concentrations exhibited more pronounced changes in GSH and GST, which led to suppression of GSH synthesis. ROS increased in a dose-responsive fashion at JP-8 concentrations up to 1%, but decreased to 80% of control values at 2% and 3% JP-8. A 25% reduction in PMP was observed in cells treated for 1 h with 1% JP-8. In contrast, cells treated for 48 h with 2% JP-8 exhibited a 25% increase when compared to control. No significant changes were noted in the 0.01 and 1% treatment groups. Moreover, no significant changes were observed in MMP or intracellular calcium concentrations in cells treated with 0.01 to 2% JP-8 for up to 48 h. In summary, the most significant effects observed in the present study which may contribute to the toxicity of JP-8 jet fuel in cultured rat liver cells include effects on GJIC, ROS production, and GSH depletion at high (i.e., greater than 2%) JP-8 concentrations.

Correlation of α-Lipoic Acid and S. Glutathione Level with Free Radical Excess in Tobacco Consumers.

PubMed

Sharma, Suman; Kaur, Manjinder; Suhalka, M L; Shrivastav, Chanchal

2016-04-01

Tobacco consumption is a serious health hazard and most important avoidable cause of death worldwide. Tobacco is recognized as lethal toxin, ripping off 7-11 minutes of human life with each cigarette through harmful compounds and inducing free radical synthesis and a high rate of lipid peroxidation. These free radicals are scavenged by the endogenous antioxidants viz. S. Glutathione (S.GSH) and S. α-Lipoic acid (S. α-LA), thus preventing the endothelial damage. The present study was designed with an aim to find out the lipid peroxidative stress through S. Malondialdehyde (S.MDA) and its correlation with antioxidant levels like S. Glutathione (S. GSH) and S. α- Lipoic acid (S. α- LA) among tobacco users (in both smokers and chewers). A case control cross-sectional study was carried out in the Department of Physiology among 200 subjects; aged 18-50 years of both sexes which were chosen randomly from institutional campus and healthy volunteers. The subjects were broadly divided into two groups (A & B); group A comprised of tobacco users (n=150) with history of smoking cigarette/biddies and chewing tobacco daily, for at least one year and group B had controls (non tobacco users) (n=50). S. MDA, S.GSH and S. α-LA levels were estimated by standardized methods. The data was analysed by unpaired student t-test and Pearson's correlation coefficient (r) for finding the correlation between antioxidants and S.MDA in group-A and group-B. The present study reports the significantly higher (p<0.0001) levels of S.MDA and lower (p<0.0001) levels of S.GSH and S. α-LA in tobacco users as compared to nontobacco users. The observed value of S.MDA was (2.72±0.87, 1.39±0.47) nmol/ml, S. α-LA was (9.94±5.96, 14.24 ± 4.34) μg/ml and S.GSH was (23.24±7.04, 32.82±2.95) mg/dl respectively in group-A and group-B. A significant (p<0.01) strong negative correlation was observed between S. MDA and antioxidants (S.GSH and S. α-LA) with a Pearson co-efficient of r=-0.619, r= -0.625 respectively, in group A. The decreased level of S. α-LA and S. GSH, in our study clearly indicates potential risk of cellular damage in tobacco users due to lipid peroxidation. Hence, the present study recommends supplementation of S. α-LA and Vitamin C in tobacco users to prevent this damage whereas quitting this evil habit will be the best available option.

Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione S-transferase Pi

PubMed Central

Henderson, Colin J.; Wolf, C. Roland; Kitteringham, Neil; Powell, Helen; Otto, Diana; Park, B. Kevin

2000-01-01

Overdose of acetaminophen, a widely used analgesic drug, can result in severe hepatotoxicity and is often fatal. This toxic reaction is associated with metabolic activation by the P450 system to form a quinoneimine metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), which covalently binds to proteins and other macromolecules to cause cellular damage. At low doses, NAPQI is efficiently detoxified, principally by conjugation with glutathione, a reaction catalyzed in part by the glutathione S-transferases (GST), such as GST Pi. To assess the role of GST in acetaminophen hepatotoxicity, we examined acetaminophen metabolism and liver damage in mice nulled for GstP (GstP1/P2(−/−)). Contrary to our expectations, instead of being more sensitive, GstP null mice were highly resistant to the hepatotoxic effects of this compound. No significant differences between wild-type (GstP1/P2(+/+)) mice and GstP1/P2(−/−) nulls in either the rate or route of metabolism, particularly to glutathione conjugates, or in the levels of covalent binding of acetaminophen-reactive metabolites to cellular protein were observed. However, although a similar rapid depletion of hepatic reduced glutathione (GSH) was found in both GstP1/P2(+/+) and GstP1/P2(−/−) mice, GSH levels only recovered in the GstP1/P2(−/−) mice. These data demonstrate that GstP does not contribute in vivo to the formation of glutathione conjugates of acetaminophen but plays a novel and unexpected role in the toxicity of this compound. This study identifies new ways in which GST can modulate cellular sensitivity to toxic effects and suggests that the level of GST Pi may be an important and contributing factor in the sensitivity of patients with acetaminophen-induced hepatotoxicity. PMID:11058152

Glycine facilitates gamma-glutamylcysteinylethyl ester-mediated increase in liver glutathione level.

PubMed

Nishida, K; Ohta, Y; Ishiguro, I

1997-08-27

gamma-Glutamylcysteinylethyl ester (gamma-GCE) increases reduced glutathione (GSH) levels in GSH-depleted rat hepatocytes. Because glycine, a constituent of GSH, exists at 0.3 to 0.4 mM in rat plasma, we examined the influence of glycine added to the medium on the action of gamma-GCE to increase GSH levels in the rat hepatocytes. Glycine (0.2-0.8 mM) dose-dependently enhanced gamma-GCE-mediated increase in intracellular GSH levels with an increase in intracellular gamma-GCE levels. These results indicate that exogenous glycine facilitates gamma-GCE-mediated increase in intracellular GSH levels in rat hepatocytes possibly by enhancing the uptake of gamma-GCE into the cells.

Toxicity of nano- and micro-sized silver particles in human hepatocyte cell line L02

NASA Astrophysics Data System (ADS)

Liu, Pengpeng; Guan, Rongfa; Ye, Xingqian; Jiang, Jiaxin; Liu, Mingqi; Huang, Guangrong; Chen, Xiaoting

2011-07-01

Silver nanoparticles (Ag NPs) previously classified as antimicrobial agents have been widely used in consumers and industrial products, especially food storage material. Ag NPs used as antimicrobial agents may be found in liver. Thus, examination of the ability of Ag NPs to penetrate the liver is warranted. The aim of the study was to determine the optimal viability assay for using with Ag NPs in order to assess their toxicity to liver cells. For toxicity evaluations, cellular morphology, mitochondrial function (3-(4, 5-dimethylazol-2-yl)-2, 5-diphenyl-tetrazolium bromide, MTT assay), membrane leakage of lactate dehydrogenase (lactate dehydrogenase, LDH release assay), Oxidative stress markers (malonaldehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD)), DNA damage (single cell gel eletrophoresis, SCGE assay), and protein damage were assessed under control and exposed conditions (24 h of exposure). The results showed that mitochondrial function decreased significantly in cells exposed to Ag NPs at 25 μg·mL-1. LDH leakage significantly increased in cells exposed to Ag NPs (>= 25 μg mL-1) while micro-sized silver particles tested displayed LDH leakage only at higher doses (100 μg·mL-1). The microscopic studies demonstrated that nanoparticle-exposed cells at higher doses became abnormal in size, displaying cellular shrinkage, and an acquisition of an irregular shape. Due to toxicity of silver, further study conducted with reference to its oxidative stress. The results exhibited significant depletion of GSH level, increase in SOD levels and lead to lipid peroxidation, which suggested that cytotoxicity of Ag NPs in liver cells might be mediated through oxidative stress. The results demonstrates that Ag NPs lead to cellular morphological modifications, LDH leakage, mitochondrial dysfunction, and cause increased generation of ROS, depletion of GSH, lipid peroxidation, oxidative DNA damage and protein damage. Though the exact mechanism behind Ag NPs toxicity is suggested oxidative stress and lipid peroxidation playing an important role in Ag NPs elicited cell membrane disruption, DNA damage, protein damage and subsequent cell death. Our preliminary data suggest that oxidative stress might contribute to Ag NPs cytotoxicity. To reveal whether apoptosis involved in Ag NPs toxicity, further studies are underway.

Modulation of Hepatic and Renal Metabolism and Toxicity of Trichloroethylene and Perchloroethylene by Alterations in Status of Cytochrome P450 and Glutathione

PubMed Central

Lash, Lawrence H.; Putt, David A.; Huang, Paul; Hueni, Sarah E.; Parker, Jean C.

2007-01-01

The relative importance of metabolism of trichloroethylene (Tri) and perchloroethylene (Perc) by the cytochrome P450 (P450) and glutathione (GSH) conjugation pathways in their acute renal and hepatic toxicity was studied in isolated cells and microsomes from rat kidney and liver after various treatments to modulate P450 activity/expression or GSH status. Inhibitors of P450 stimulated GSH conjugation of Tri and, to a lesser extent, Perc, in both kidney cells and hepatocytes. Perc was a more potent, acute cytotoxic agent in isolated kidney cells than Tri but Perc-induced toxicity was less responsive than Tri-induced toxicity to modulation of P450 status. These observations are consistent with P450-dependent bioactivation being more important for Tri than for Perc. Incubation of isolated rat hepatocytes with Tri produced no acute cytotoxicity in isolated hepatocytes while Perc produced comparable cytotoxicity as in kidney cells. Modulation of P450 status in hepatocytes produced larger changes in Tri- and Perc-induced cytotoxicity than in kidney cells, with non-selective P450 inhibitors increasing toxicity. Induction of CYP2E1 with pyridine also markedly increased sensitivity of hepatocytes to Tri but had little effect on Perc-induced cytotoxicity. Increases in cellular GSH concentrations increased Tri- and Perc-induced cytotoxicity in kidney cells but not in hepatocytes, consistent with the role of GSH conjugation in Tri- and Perc-induced nephrotoxicity. In contrast, depletion of cellular GSH concentrations moderately decreased Tri- and Perc-induced cytotoxicity in kidney cells but increased cytotoxicity in hepatocytes, again pointing to the importance of different bioactivation pathways and modes of action in kidney and liver. PMID:17433522

Glutathione Fine-Tunes the Innate Immune Response toward Antiviral Pathways in a Macrophage Cell Line Independently of Its Antioxidant Properties

PubMed Central

Diotallevi, Marina; Checconi, Paola; Palamara, Anna Teresa; Celestino, Ignacio; Coppo, Lucia; Holmgren, Arne; Abbas, Kahina; Peyrot, Fabienne; Mengozzi, Manuela; Ghezzi, Pietro

2017-01-01

Glutathione (GSH), a major cellular antioxidant, is considered an inhibitor of the inflammatory response involving reactive oxygen species (ROS). However, evidence is largely based on experiments with exogenously added antioxidants/reducing agents or pro-oxidants. We show that depleting macrophages of 99% of GSH does not exacerbate the inflammatory gene expression profile in the RAW264 macrophage cell line or increase expression of inflammatory cytokines in response to the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS); only two small patterns of LPS-induced genes were sensitive to GSH depletion. One group, mapping to innate immunity and antiviral responses (Oas2, Oas3, Mx2, Irf7, Irf9, STAT1, il1b), required GSH for optimal induction. Consequently, GSH depletion prevented the LPS-induced activation of antiviral response and its inhibition of influenza virus infection. LPS induction of a second group of genes (Prdx1, Srxn1, Hmox1, GSH synthase, cysteine transporters), mapping to nrf2 and the oxidative stress response, was increased by GSH depletion. We conclude that the main function of endogenous GSH is not to limit inflammation but to fine-tune the innate immune response to infection. PMID:29033950

Reversible and Dynamic Fluorescence Imaging of Cellular Redox Self-Regulation Using Fast-Responsive Near-Infrared Ge-Pyronines.

PubMed

Nie, Hailiang; Jing, Jing; Tian, Yong; Yang, Wen; Zhang, Rubo; Zhang, Xiaoling

2016-04-13

Cellular self-regulation of reactive oxygen species (ROS) stress via glutathione (GSH) antioxidant repair plays a crucial role in maintaining redox balance, which affects various physiological and pathological pathways. In this work, we developed a simple yet effective strategy for reversible, dynamic, and real-time fluorescence imaging of ROS stress and GSH repair, based on novel Ge-pyronine dyes (GePs). Unlike the current O-pyronine (OP) dye, the fluorescence of GePs can be quenched in GSH reduction and then greatly restored by ROS (e.g., ClO(-), ONOO(-), and HO(•)) oxidation because of their unique affinity toward thiols. The "on-off" and "off-on" fluorescence switch can complete in 10 and 20 s, respectively, and exhibit excellent reversibility in vitro and in cells. GePs also show excitation in the long wavelength from the deep-red to near-infrared (NIR) (621-662 nm) region, high fluorescence quantum yield (Φ(fl) = 0.32-0.44) in aqueous media, and excellent cell permeability. Our results demonstrated that GePs can be used for real-time monitoring of the reversible and dynamic interconversion between ROS oxidation and GSH reduction in living cells. GePs might be a useful tool for investigating various redox-related physiological and pathological pathways.

Analysis of the effects of iron and vitamin C co-supplementation on oxidative damage, antioxidant response and inflammation in THP-1 macrophages.

PubMed

Marcil, V; Lavoie, J C; Emonnot, L; Seidman, E; Levy, E

2011-07-01

The aims of the study were to test the susceptibility of THP-1 macrophages to develop oxidative stress and to deploy antioxidant defense mechanisms that insure the balance between the pro- and antioxidant molecules. Differentiated THP-1 were incubated in the presence or absence of iron-ascorbate (Fe/As) (100/1000μM) and the antioxidants Trolox, BHT, α-Tocopherol and NAC. Fe/As promoted the production of lipid peroxidation as reflected by the formation of malondialdehyde and H(2)O(2) along with reduced PUFA levels and elevated glutathione disulfide/total glutathione ratio, a reliable index of cellular redox status. THP-1 macrophages developed an increase in cytoplasmic SOD activity due in part to high cytoplasmic SOD1. On the other hand, a decline was noted in mRNA and protein of extra-cellular SOD3, as well as the activity of GSH-peroxidase, GSH-transferase and ATOX-1 expression. Macrophages activated under conditions of oxidative stress do not adequately deploy a powerful endogenous antioxidant response, a situation that can lead to an enhanced inflammatory response. Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Non-invasive imaging of the levels and effects of glutathione on the redox status of mouse brain using electron paramagnetic resonance imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Emoto, Miho C.; Department of Neurology, Sapporo Medical University School of Medicine, Sapporo, Hokkaido 060-8556; Matsuoka, Yuta

Glutathione (GSH) is the most abundant non-protein thiol that buffers reactive oxygen species in the brain. GSH does not reduce nitroxides directly, but in the presence of ascorbates, addition of GSH increases ascorbate-induced reduction of nitroxides. In this study, we used electron paramagnetic resonance (EPR) imaging and the nitroxide imaging probe, 3-methoxycarbonyl-2,2,5,5-tetramethyl-piperidine-1-oxyl (MCP), to non-invasively obtain spatially resolved redox data from mouse brains depleted of GSH with diethyl maleate compared to control. Based on the pharmacokinetics of the reduction reaction of MCP in the mouse heads, the pixel-based rate constant of its reduction reaction was calculated as an index ofmore » the redox status in vivo and mapped as a “redox map”. The obtained redox maps from control and GSH-depleted mouse brains showed a clear change in the brain redox status, which was due to the decreased levels of GSH in brains as measured by a biochemical assay. We observed a linear relationship between the reduction rate constant of MCP and the level of GSH for both control and GSH-depleted mouse brains. Using this relationship, the GSH level in the brain can be estimated from the redox map obtained with EPR imaging. - Highlights: • Redox status of glutathione-depleted mouse brain was examined with EPR imaging. • Redox status of mouse brain changed depending on glutathione (GSH) levels in brains. • Linear relationship between GSH levels and redox status in brains was found. • Using this relation, estimation of GSH levels in brains is possible from EPR images.« less

Differential correlations between changes to glutathione redox state, protein ubiquitination, and stress-inducible HSPA chaperone expression after different types of oxidative stress.

PubMed

Girard, Pierre-Marie; Peynot, Nathalie; Lelièvre, Jean-Marc

2018-05-12

In primary bovine fibroblasts with an hspa1b/luciferase transgene, we examined the intensity of heat-shock response (HSR) following four types of oxidative stress or heat stress (HS), and its putative relationship with changes to different cell parameters, including reactive oxygen species (ROS), the redox status of the key molecules glutathione (GSH), NADP(H) NAD(H), and the post-translational protein modifications carbonylation, S-glutathionylation, and ubiquitination. We determined the sub-lethal condition generating the maximal luciferase activity and inducible HSPA protein level for treatments with hydrogen peroxide (H 2 O 2 ), UVA-induced oxygen photo-activation, the superoxide-generating agent menadione (MN), and diamide (DA), an electrophilic and sulfhydryl reagent. The level of HSR induced by oxidative stress was the highest after DA and MN, followed by UVA and H 2 O 2 treatments, and was not correlated to the level of ROS production nor to the extent of protein S-glutathionylation or carbonylation observed immediately after stress. We found a correlation following oxidative treatments between HSR and the level of GSH/GSSG immediately after stress, and the increase in protein ubiquitination during the recovery period. Conversely, HS treatment, which led to the highest HSR level, did not generate ROS nor modified or depended on GSH redox state. Furthermore, the level of protein ubiquitination was maximum immediately after HS and lower than after MN and DA treatments thereafter. In these cells, heat-induced HSR was therefore clearly different from oxidative stress-induced HSR, in which conversely early redox changes of the major cellular thiol predicted the level of HSR and polyubiquinated proteins.

IDH1 R132H Mutation Enhances Cell Migration by Activating AKT-mTOR Signaling Pathway, but Sensitizes Cells to 5-FU Treatment as NADPH and GSH Are Reduced

PubMed Central

Qiu, Jiangdong; Huang, Keting; Wu, Mindan; Xia, Chunlin

2017-01-01

Aim of study Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation. Materials and methods Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH. Results We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment. Conclusion Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment. PMID:28052098

Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singhal, Sharad S., E-mail: ssinghal@coh.org; Singh, Sharda P.; Singhal, Preeti

2015-12-15

4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxidesmore » and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes — higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article. - Highlights: • GSTs are the major determinants of the intracellular concentration of 4HNE. • Higher concentrations of 4HNE promote apoptosis whereas lower promote proliferation. • Stress-mediated signaling can be modulated by the α-class glutathione S-transferase. • Genotoxic effect of 4HNE may be ameliorated by modulating the cellular GSH levels. • RLIP76 (RalBP1) mediates ATP-dependent transport of GSH-conjugate of 4HNE (GSHNE).« less

Effects of exogenous glutathione and cysteine on growth, lead accumulation, and tolerance of Iris lactea var. chinensis.

PubMed

Yuan, Haiyan; Zhang, Yongxia; Huang, Suzhen; Yang, Yongheng; Gu, Chunsun

2015-02-01

Effects of exogenous reduced glutathione (GSH) and cysteine (Cys) on growth, lead (Pb) accumulation, and nonprotein thiol (NPT) contents of Iris lactea var. chinensis under 100 and 500 mg L(-1) Pb stress were studied. Our results showed that 500 mg L(-1) Pb stress caused a dramatical decline in fresh weights, while the reduction of aboveground biomass was alleviated by exogenous GSH and Cys even though keeping higher Pb contents in roots and shoots. Exogenous GSH and Cys could enhance Pb accumulation in the shoots and roots compared with single Pb treatment. The promoting effect of GSH to Pb accumulation was larger than the effect of Cys, and the Pb contents in the shoots and roots treated with 500 mg L(-1) Pb + GSH reached 1,712 and 14,603 mg kg(-1), about 4.19 and 2.78 times of single 500 mg L(-1) Pb treatment, respectively. Microscopic imaging of Pb in roots and leaves showed that higher intensive fluorescence was observed in cell wall of root epidermis, stele, vascular tissues of the roots, and sclerenchyma cells of leaves treated with 500 mg L(-1) Pb + GSH and treated with 500 mg L(-1) Pb + Cys. Exogenous GSH had an apparent promoting effect on root and shoot GSH synthesis, while exogenous Cys reduced the synthesis of cellular GSH in shoot and increased Cys contents. Pb only induced the synthesis of phytochelatin (PC)2 in roots, and the PC2 content declined in GSH- and Cys-treated plant roots. These results suggested that GSH synthesis was a more effective approach to improve Pb accumulation and translocation of I. lactea var. chinensis. Further analysis of protein expression in plants by exogenous GSH and buthionine sulfoximine (BSO) application showed that the proteins regulated by GSH and BSO may constitute various enzymes involved in GSH biosynthesis and play certain roles in Pb accumulation and tolerance of I. lactea var. chinensis.

Targeting of the Glutathione, Thioredoxin, and Nrf2 Antioxidant Systems in Head and Neck Cancer.

PubMed

Roh, Jong-Lyel; Jang, Hyejin; Kim, Eun Hye; Shin, Daiha

2017-07-10

The glutathione (GSH), thioredoxin (Trx), and Nrf2 systems represent a major defense against reactive oxygen species (ROS), the cellular imbalance of which in cancer promotes growth and therapeutic resistance. This study investigated whether targeting the GSH, Trx, and Nrf2 antioxidant systems effectively eliminated head and neck cancer (HNC). At high concentrations, auranofin, but not buthionine sulfoximine (BSO) alone, decreased the viability of HNC, whereas even at low concentrations, auranofin plus BSO synergized to kill HNC cells. Dual silencing of the genes for GCLM and TrxR1 induced GSH depletion, Trx activity inhibition, and ROS accumulation, synergistically killing HNC cells. Inhibition of the GSH and Trx systems resulted in activation of the Nrf2-antioxidant response element (ARE) pathway, which may result in suboptimal GSH and Trx inhibition where HNC is resistant. Genetic inhibition of Nrf2 and/or HO-1 or trigonelline enhanced growth suppression, ROS accumulation, and cell death from GSH and Trx inhibition. The in vivo effects of GSH, Trx, and Nrf2 system inhibition were confirmed in a mouse HNC xenograft model by achieving growth inhibition >60% compared with those of control. Innovations: This study is the first to show that triple inhibition of GSH, Trx, and Nrf2 pathways could be an effective method to overcome the resistance of HNC. Inhibition of the Nrf2-ARE pathway in addition to dual inhibition of the GSH and Trx antioxidant systems can effectively eliminate resistant HNC. Antioxid. Redox Signal. 27, 106-114.

Paclitaxel-resistant HeLa cells have up-regulated levels of reactive oxygen species and increased expression of taxol resistance gene 1.

PubMed

Bi, Wenxiang; Wang, Yuxia; Sun, Gaoying; Zhang, Xiaojin; Wei, Yongqing; Li, Lu; Wang, Xiaoyuan

2014-07-01

This study is to establish a paclitaxel (PTX)-resistant human cervical carcinoma HeLa cell line (HeLa/PTX) and to investigate its redox characteristics and the expression of taxol resistance gene 1 (Txr1). HeLa cells were treated with PTX and effects of PTX on cell proliferation were detected through cell counting and the MTT assay. Levels of cellular reactive oxygen species (ROS), reduced glutathione (GSH), and oxidized glutathione (GSSG) as well as the ratio of GSH to GSSG were measured by the 2,7-difluorescein diacetate (DCFH-DA) method and the 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) method. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined by the nitrite formation method, the molybdate colorimetric method, and the DTNB colorimetric method, respectively. The level of Txr1 mRNA was determined by real-time PCR. Compared with the regular HeLa cells, HeLa/PTX cells were larger in size and had more cytoplasmic granules. The population doubling time for HeLa/PTX cells was 1.32 times of that of HeLa cells (P<0.01). HeLa/PTX cells showed stronger resistance to PTX than HeLa cells with a resistance index of 122.69. HeLa/PTX cells had higher levels of ROS (P<0.01) and Txr1 mRNA (P<0.01), lower level of GSH (P < 0.05), and lower activities of SOD (P<0.01) and GPx (P < 0.05) than HeLa cells. HeLa/PTX cells, with higher levels of ROS and Txr1 mRNA expression, are more resistant to PTX than HeLa cells.

Mechanisms of sulfur mustard analog 2-chloroethyl ethyl sulfide-induced DNA damage in skin epidermal cells and fibroblasts.

PubMed

Inturi, Swetha; Tewari-Singh, Neera; Gu, Mallikarjuna; Shrotriya, Sangeeta; Gomez, Joe; Agarwal, Chapla; White, Carl W; Agarwal, Rajesh

2011-12-15

Employing mouse skin epidermal JB6 cells and dermal fibroblasts, here we examined the mechanisms of DNA damage by 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of sulfur mustard (SM). CEES exposure caused H2A.X and p53 phosphorylation as well as p53 accumulation in both cell types, starting at 1h, that was sustained for 24h, indicating a DNA-damaging effect of CEES, which was also confirmed and quantified by alkaline comet assay. CEES exposure also induced oxidative stress and oxidative DNA damage in both cell types, measured by an increase in mitochondrial and cellular reactive oxygen species and 8-hydroxydeoxyguanosine levels, respectively. In the studies distinguishing between oxidative and direct DNA damage, 1h pretreatment with glutathione (GSH) or the antioxidant Trolox showed a decrease in CEES-induced oxidative stress and oxidative DNA damage. However, only GSH pretreatment decreased CEES-induced total DNA damage measured by comet assay, H2A.X and p53 phosphorylation, and total p53 levels. This was possibly due to the formation of GSH-CEES conjugates detected by LC-MS analysis. Together, our results show that CEES causes both direct and oxidative DNA damage, suggesting that to rescue SM-caused skin injuries, pleiotropic agents (or cocktails) are needed that could target multiple pathways of mustard skin toxicities. Copyright © 2011 Elsevier Inc. All rights reserved.

The protective effect of diosmin on hepatic ischemia reperfusion injury: an experimental study

PubMed Central

Tanrikulu, Yusuf; Şahin, Mefaret; Kismet, Kemal; Kilicoglu, Sibel Serin; Devrim, Erdinc; Tanrikulu, Ceren Sen; Erdemli, Esra; Erel, Serap; Bayraktar, Kenan; Akkus, Mehmet Ali

2013-01-01

Liver ischemia reperfusion injury (IRI) is an important pathologic process leading to bodily systemic effects and liver injury. Our study aimed to investigate the protective effects of diosmin, a phlebotrophic drug with antioxidant and anti-inflammatory effects, in a liver IRI model. Forty rats were divided into 4 groups. Sham group, control group (ischemia-reperfusion), intraoperative treatment group, and preoperative treatment group. Ischemia reperfusion model was formed by clamping hepatic pedicle for a 60 minute of ischemia followed by liver reperfusion for another 90 minutes. Superoxide dismutase (SOD) and catalase (CAT) were measured as antioaxidant enzymes in the liver tissues, and malondialdehyde (MDA) as oxidative stress marker, xanthine oxidase (XO) as an oxidant enzyme and glutathione peroxidase (GSH-Px) as antioaxidant enzyme were measured in the liver tissues and the plasma samples. Hepatic function tests were lower in treatment groups than control group (p<0.001 for ALT and AST). Plasma XO and MDA levels were lower in treatment groups than control group, but plasma GSH-Px levels were higher (p<0.05 for all). Tissue MDA levels were lower in treatment groups than control group, but tissue GSH-Px, SOD, CAT and XO levels were higher (p<0.05 for MDA and p<0.001 for others). Samples in control group histopathologically showed morphologic abnormalities specific to ischemia reperfusion. It has been found that both preoperative and intraoperative diosmin treatment decreases cellular damage and protects cells from toxic effects in liver IRI. As a conclusion, diosmin may be used as a protective agent against IRI in elective and emergent liver surgical operations. PMID:24289756

Intracellular drug delivery nanocarriers of glutathione-responsive degradable block copolymers having pendant disulfide linkages.

PubMed

Khorsand, Behnoush; Lapointe, Gabriel; Brett, Christopher; Oh, Jung Kwon

2013-06-10

Self-assembled micelles of amphiphilic block copolymers (ABPs) with stimuli-responsive degradation (SRD) properties have a great promise as nanotherapeutics exhibiting enhanced release of encapsulated therapeutics into targeted cells. Here, thiol-responsive degradable micelles based on a new ABP consisting of a pendant disulfide-labeled methacrylate polymer block (PHMssEt) and a hydrophilic poly(ethylene oxide) (PEO) block were investigated as effective intracellular nanocarriers of anticancer drugs. In response to glutathione (GSH) as a cellular trigger, the cleavage of pendant disulfide linkages in hydrophobic PHMssEt blocks of micellar cores caused the destabilization of self-assembled micelles due to change in hydrophobic/hydrophilic balance. Such GSH-triggered micellar destabilization changed their size distribution with an appearance of large aggregates and led to enhanced release of encapsulated anticancer drugs. Cell culture results from flow cytometry and confocal laser scanning microscopy for cellular uptake as well as cell viability measurements for high anticancer efficacy suggest that new GSH-responsive degradable PEO-b-PHMssEt micelles offer versatility in multifunctional drug delivery applications.

Assessment of glutathione levels in model solution and grape ferments supplemented with glutathione-enriched inactive dry yeast preparations using a novel UPLC-MS/MS method.

PubMed

Kritzinger, E C; Stander, M A; Du Toit, W J

2013-01-01

A novel, robust and fast ultra-high performance liquid chromatography-MS method has been developed for the simultaneous quantification of reduced glutathione (GSH) and oxidised glutathione (GSSG) in grape juice, wine and model wine solution. Sample preparation is minimal and does not require derivatisation. The method has very good performance in terms of sensitivity and selectivity. The limit of detection was 0.002 and 0.001 mg L(-1) for GSH and GSSG, respectively. The amount of GSH and GSSG released by commercial glutathione-enriched inactivated dry yeast preparations (GSH-IDYs) into a model solution was assessed. Significant differences in the amount of GSH and/or GSSG released into a model wine by different GSH-IDYs were observed, with ethanol influencing this release under certain conditions. The GSH and GSSG levels in grape juice fermentations supplemented with GSH-IDY were also assessed in relation to different addition times during fermentation. GSH-IDY addition can lead to elevated wine GSH levels, provided the supplementation is done early during alcoholic fermentation.

Polyphenolic composition of grape stem extracts affects antioxidant activity in endothelial and muscle cells.

PubMed

Goutzourelas, Nikolaos; Stagos, Dimitrios; Spanidis, Ypatios; Liosi, Maria; Apostolou, Anna; Priftis, Alexandros; Haroutounian, Serko; Spandidos, Demetrios A; Tsatsakis, Aristidis M; Kouretas, Demetrios

2015-10-01

The aim of the present study was the assessment of the antioxidant effects of polyphenolic extracts derived from the stems of three Greek grape varieties (Moshomayro, Mavrotragano and Mandilaria) in endothelial (EA.hy926) and muscle (C2C12) cells. We also investigated the effects of the polyphenolic composition on the antioxidant effects of the grape stem extracts. For this purpose, the endothelial and muscle cells were treated with low non-cytotoxic concentrations of the extracts for 24 h in order to assess the effects of the extracts on cellular redox status using oxidative stress biomarkers. The oxidative stress markers were thiobarbituric acid reactive substances (TBARS), protein carbonyl (CARB) levels, reactive oxygen species (ROS) levels and glutathione (GSH) levels. The results revealed that treatment of the EA.hy926 cells with Mandilaria extract significantly decreased the TBARS levels by 14.8% and the CARB levels by 25.9 %, while it increased the GSH levels by 15.8% compared to the controls. Moreover, treatment of the EA.hy926 cells with Mavrotragano extract significantly increased the GSH levels by 20.2%, while it significantly decreased the TBARS and CARB levels by 12.5% and 16.6%, respectively. Treatment of the C2C12 cells with Mandilaria extract significantly decreased the TBARS levels by 47.3 %, the CARB levels by 39.0 % and the ROS levels by 21.8%, while it increased the GSH levels by 22.6% compared to the controls. Moreover, treatment of the C2C12 cells with Mavrotragano significantly decreased the TBARS, CARB and ROS levels by 36.2%, 35.9% and 16.5%, respectively. In conclusion, to the best of our knowledgel, our results demonstrate for the first time that treatment with grape stem extracts at low concentrations improves the redox status of endothelial and muscle cells. Thus, grape stem extracts may be used for developing antioxidant food supplements or biofunctional foods. However, it was also found that the polyphenolic composition of grape stem extracts affects their antioxidant capacity. For example, the results suggested that trans-resveratrol, gallic acid, (+)-catechin, ferulic acid, caffeic acid, quercetin, coumaric acid and kaempferol may be essential for the antioxidant activity of grape stem extracts.

Electrochemical detection of glutathione based on Hg(2+)-mediated strand displacement reaction strategy.

PubMed

Lv, Yun; Yang, Lili; Mao, Xiaoxia; Lu, Mengjia; Zhao, Jing; Yin, Yongmei

2016-11-15

Glutathione (GSH) plays an important role in numerous cellular functions, and the abnormal GSH expression is closely related with many dangerous human diseases. In this work, we have proposed a simple but sensitive electrochemical method for quantitative detection of GSH based on an Hg(2+)-mediated strand displacement reaction. Owing to the specific binding of Hg(2+) with T-T mismatches, helper DNA can bind to 3' terminal of probe DNA 1 and initiate the displacement of probe DNA 2 immobilized on an electrode surface. However, Hg(2+)-mediated strand displacement reaction can be inhibited by the chelation of GSH with Hg(2+), thereby leading to an obvious electrochemical response obtained from methylene blue that is modified onto the probe DNA. Our method can sensitively detect GSH in a wide linear range from 0.5nM to 5μM with a low detection limit of 0.14nM, which can also easily distinguish target molecules in complex serum samples and even cell extractions. Therefore, this method may have great potential to monitor GSH in the physiological and pathological condition in the future. Copyright © 2016 Elsevier B.V. All rights reserved.

Repeated short-term stress synergizes the ROS signalling through up regulation of NFkB and iNOS expression induced due to combined exposure of trichloroethylene and UVB rays.

PubMed

Ali, Farrah; Sultana, Sarwat

2012-01-01

Restraint stress is known to catalyse the pathogenesis of the variety of chronic inflammatory disorders. The present study was designed to evaluate the effect of repeated short-term stress (RRS) on cellular transduction apart from oxidative burden and early tumour promotional biomarkers induced due to combined exposure of trichloroethylene (TCE) and Ultra-violet radiation (UVB). RRS leads to the increase in the expression of the stress responsive cellular transduction elements NFkB-p65 and activity of iNOS in the epidermal tissues of mice after toxicant exposure. RRS augments the steep depletion of the cellular antioxidant machinery which was evidenced by the marked depletion in GSH (Glutathione and GSH dependant enzymes), superoxide dismutase and catalase activity that were observed at significance level of P < 0.001 with increase in lipid peroxidation, H(2)O(2) and xanthine oxidase activity (P < 0.001) in the stressed animals and down regulation of DT-diaphorase activity (P < 0.001). Since, the induction of NFkB-p65 and inducible nitric oxide synthase expression mediated can lead to the hyperproliferation, we estimated a significant increment (P < 0.001) in the synthesis of polyamines in mice skin evidenced here by the ornithine decarboxylase which is the early marker of tumour promotion and further evaluated PCNA expression. All these findings cues towards the synergising ability of repeated short-term stress in the toxic response of TCE and UVB radiation.

L-cysteine supplementation upregulates glutathione (GSH) and vitamin D binding protein (VDBP) in hepatocytes cultured in high glucose and in vivo in liver, and increases blood levels of GSH, VDBP, and 25-hydroxy-vitamin D in Zucker diabetic fatty rats.

PubMed

Jain, Sushil K; Kanikarla-Marie, Preeti; Warden, Cassandra; Micinski, David

2016-05-01

Vitamin D binding protein (VDBP) status has an effect on and can potentially improve the status of 25(OH) vitamin D and increase the metabolic actions of 25(OH) vitamin D under physiological and pathological conditions. Diabetes is associated with lower levels of glutathione (GSH) and 25(OH) vitamin D. This study examined the hypothesis that upregulation of GSH will also upregulate blood levels of VDBP and 25(OH) vitamin D in type 2 diabetic rats. L-cysteine (LC) supplementation was used to upregulate GSH status in a FL83B hepatocyte cell culture model and in vivo using Zucker diabetic fatty (ZDF) rats. Results show that LC supplementation upregulates both protein and mRNA expression of VDBP and vitamin D receptor (VDR) and GSH status in hepatocytes exposed to high glucose, and that GSH deficiency, induced by glutamate cysteine ligase knockdown, resulted in the downregulation of GSH, VDBP, and VDR and an increase in oxidative stress levels in hepatocytes. In vivo, LC supplementation increased GSH and protein and mRNA expression of VDBP and vitamin D 25-hydroxylase (CYP2R1) in the liver, and simultaneously resulted in elevated blood levels of LC and GSH, as well as increases in VDBP and 25(OH) vitamin D levels, and decreased inflammatory biomarkers in ZDF rats compared with those in placebo-supplemented ZDF rats consuming a similar diet. LC supplementation may provide a novel approach by which to raise blood levels of VDBP and 25(OH) vitamin D in type 2 diabetes. © 2016 The Authors. Molecular Nutrition & Food Research Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The sphingosine 1-phosphate breakdown product, (2E)-hexadecenal, forms protein adducts and glutathione conjugates in vitro.

PubMed

Schumacher, Fabian; Neuber, Corinna; Finke, Hannah; Nieschalke, Kai; Baesler, Jessica; Gulbins, Erich; Kleuser, Burkhard

2017-08-01

Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes such as cell proliferation and apoptosis, can be irreversibly cleaved by S1P lyase, yielding phosphoethanolamine and (2 E )-hexadecenal (2 E HD). The latter metabolite, an α,β-unsaturated fatty aldehyde, may be susceptible to nucleophilic attack by cellular biomolecules. Hence, we studied whether 2 E HD forms reaction products with GSH and proteins in vitro. Using LC-MS/MS and stable isotopically labeled reference material, we identified a total of nine novel reaction products of 2 E HD in a cell-free approach: two GSH conjugates and seven l-amino acid adducts. Both GSH conjugates were also found in HepG2 cell lysates incubated with 2 E HD. Likewise, we detected four out of seven amino acid adducts released from the model protein, BSA, and proteins extracted from HepG2 cells. On this occasion, the 2 E HD Michael adduct with l-histidine proved to be the most prominent adduct. Most interestingly, inhibition of the enzymatically driven oxidative degradation of 2 E HD resulted in increased levels of both GSH conjugates and protein adducts in HepG2 cell lysates. Hence, our data provide new insights into sphingolipid metabolism and will be useful to investigate certain disorders linked to an impaired fatty aldehyde metabolism in more detail. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Cellular glutathione level does not predict ovarian cancer cells' resistance after initial or repeated exposure to cisplatin.

PubMed

Nikounezhad, Nastaran; Nakhjavani, Maryam; Shirazi, Farshad H

2017-05-01

Cisplatin resistance development is a major obstacle in ovarian cancer treatment. One of the most important mechanisms underlying cisplatin resistance is drug detoxification by glutathione. In the present study, the importance of initial or repeated exposure to cisplatin in glutathione dependent resistance was investigated. To this purpose, some cisplatin sensitive and resistant variants of human ovarian cancer cell lines providing an appropriate range of cisplatin sensitivity were selected. Clonogenic survival assay was performed to evaluate cisplatin resistance and intracellular contents of reduced (GSH) and oxidized (GSSG) glutathione were analyzed using an HPLC method. Our results indicated that the intracellular GSH and GSSG concentrations were nearly equal in A2780 and A2780CP cells, while the A2780CP cells showed 14 times more resistance than the A2780 cells after initial exposure to cisplatin. A2780-R1 and A2780-R3 cells which have been repeatedly exposed to cisplatin also showed no significant difference in glutathione content, even though A2780-R3 was about two times more resistant than A2780-R1. Moreover, intracellular GSH/GSSG ratio decreased in the resistant cells, reflecting a shift towards a more oxidizing intracellular environment indicative of oxidative stress. As a conclusion, it seems that although the intracellular glutathione concentration increases after repeated exposure to cisplatin, there is no clear correlation between the intracellular GSH content in ovarian cancer cells and their resistance to cisplatin neither after initial nor after repeated exposure to this drug.

3-Bromopyruvate induces rapid human prostate cancer cell death by affecting cell energy metabolism, GSH pool and the glyoxalase system.

PubMed

Valenti, Daniela; Vacca, Rosa A; de Bari, Lidia

2015-12-01

3-bromopyruvate (3-BP) is an anti-tumour drug effective on hepatocellular carcinoma and other tumour cell types, which affects both glycolytic and mitochondrial targets, depleting cellular ATP pool. Here we tested 3-BP on human prostate cancer cells showing, differently from other tumour types, efficient ATP production and functional mitochondrial metabolism. We found that 3-BP rapidly induced cultured androgen-insensitive (PC-3) and androgen-responsive (LNCaP) prostate cancer cell death at low concentrations (IC(50) values of 50 and 70 μM, respectively) with a multimodal mechanism of action. In particular, 3-BP-treated PC-3 cells showed a selective, strong reduction of glyceraldeide 3-phosphate dehydrogenase activity, due to the direct interaction of the drug with the enzyme. Moreover, 3-BP strongly impaired both glutamate/malate- and succinate-dependent mitochondrial respiration, membrane potential generation and ATP synthesis, concomitant with the inhibition of respiratory chain complex I, II and ATP synthase activities. The drastic reduction of cellular ATP levels and depletion of GSH pool, associated with significant increase in cell oxidative stress, were found after 3-BP treatment of PC-3 cells. Interestingly, the activity of both glyoxalase I and II, devoted to the elimination of the cytotoxic methylglyoxal, was strongly inhibited by 3-BP. Both N-acetylcysteine and aminoguanidine, GSH precursor and methylglyoxal scavenger, respectively, prevented 3-BP-induced PC-3 cell death, showing that impaired cell antioxidant and detoxifying capacities are crucial events leading to cell death. The provided information on the multi-target cytotoxic action of 3-BP, finally leading to PC-3 cell necrosis, might be useful for future development of 3-BP as a therapeutic option for prostate cancer treatment.

Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, X.; Li, L.; Zhang, L.

Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidativemore » stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H{sub 2}O{sub 2} generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H{sub 2}O{sub 2} generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H{sub 2}O{sub 2} accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.« less

Identification of glutathione adducts of α-chlorofatty aldehydes produced in activated neutrophils

PubMed Central

Duerr, Mark A.; Aurora, Rajeev; Ford, David A.

2015-01-01

α-Chlorofatty aldehydes (α-ClFALDs) are produced by hypochlorous acid targeting plasmalogens during neutrophil activation. This study investigated the reaction of the α-chlorinated carbon of α-ClFALD with the nucleophile, GSH. Utilizing ESI/MS/MS, the reaction product of GSH and the 16-carbon α-ClFALD, 2-chlorohexadecanal (2-ClHDA), was characterized. The resulting conjugate of 2-ClHDA and GSH (HDA-GSH) has an intact free aldehyde, and the chlorine at the α-carbon is ejected. Stable isotope-labeled [d4]HDA-GSH was synthesized, which further confirmed the structure, and was used to quantify natural α-ClFALD conjugates of GSH (FALD-GSH) using reverse-phase LC with detection by ESI/MS/MS using selected reaction monitoring. HDA-GSH is elevated in RAW 264.7 cells treated with physiologically relevant concentrations of exogenous 2-ClHDA. Furthermore, PMA-treated primary human neutrophils have elevated levels of HDA-GSH and the conjugate of 2-chlorooctadecanal (2-ClODA) and GSH (ODA-GSH), as well as elevated levels of 2-ClHDA and 2-ClODA. Production of both conjugates in PMA-stimulated neutrophils was reduced by 3-aminotriazole pretreatment, which also blocks endogenous α-ClFALD production. Additionally, plasma FALD-GSH levels were elevated in the K/BxN mouse arthritis model. Taken together, these studies demonstrate novel peptidoaldehydes derived from GSH and α-ClFALD in activated human neutrophils and in vivo in K/BxN mice. PMID:25814023

Oxidative stress, glutathione level and antioxidant response to heavy metals in multi-resistant pathogen, Candida tropicalis.

PubMed

Ilyas, Sidra; Rehman, Abdul

2015-01-01

In this study, we explored the multiple heavy metal-resistant yeast isolated from heavy metal-polluted environment. The isolated yeast showed maximum growth at 30 °C, pH 7.0, and the strain was identified as Candida tropicalis through 18S ribosomal RNA (rRNA) gene sequence analysis. Yeast cells grew well in medium containing different concentrations of heavy metal ions [CdCl₂, Pb(NO₃)₂, NaAsO₂, CuSO₄ and K₂Cr₂O₇]. Minimum inhibitory concentration (MIC) against different metal ions was ranged from 5 to 19 mM, and the metal resistance value against each metal observed by yeast cells was 5 mM (Cr), 10 mM (Cd), 15 mM (As), 14 mM (Cu) and 19 mM (Pb) and increased in the following order: Pb > Cu > As ≥ Cd > Cr. The total cellular glutathione, GSH/GSSG redox couple and metallothioneins like protein (MT) were assayed by growing cultures for 24 h and exposed to 100 mg/L of each heavy metal ion. Remarkable increase in γ-glutamylcysteinylglycine (GSH) level was determined in arsenic and cadmium treatment followed by chromium, lead and copper. Stressed cells had much more oxidized GSH than unstressed cells. GSH/GSSG ratio was significantly increased in cadmium and copper treatment in contrast to chromium, arsenic and lead. Statistical analysis revealed significantly higher cysteine level in all metal-treated samples as compared to control. Antioxidant glutathione transferase activity was not detected in metal-treated and untreated yeast samples. One-dimensional electrophoresis of proteins revealed marked differences in banding pattern of heavy metal-exposed yeast samples. A prominent 20 kDa band was observed in all treated samples suggesting that some differential proteins could be over-expressed during heavy metal treatment and might be involved in cell resistance mechanisms.

Phytochemical Ginkgolide B Attenuates Amyloid-β1-42 Induced Oxidative Damage and Altered Cellular Responses in Human Neuroblastoma SH-SY5Y Cells.

PubMed

Gill, Iqbal; Kaur, Sukhchain; Kaur, Navrattan; Dhiman, Monisha; Mantha, Anil K

2017-01-01

Oxidative stress is an upsurge in reactive oxygen/nitrogen species (ROS/RNS), which aggravates damage to cellular components viz. lipids, proteins, and nucleic acids resulting in impaired cellular functions and neurological pathologies including Alzheimer's disease (AD). In the present study, we have examined amyloid-β (Aβ)-induced oxidative stress responses, a major cause for AD, in the undifferentiated and differentiated human neuroblastoma SH-SY5Y cells. Aβ1-42-induced oxidative damage was evaluated on lipids by lipid peroxidation; proteins by protein carbonyls; antioxidant status by SOD and GSH enzyme activities; and DNA and RNA damage levels by evaluating the number of AP sites and 8-OHG base damages produced. In addition, the neuro-protective role of the phytochemical ginkgolide B (GB) in countering Aβ1-42-induced oxidative stress was assessed. We report that the differentiated cells are highly vulnerable to Aβ1-42-induced oxidative stress events as exerted by the deposition of Aβ in AD. Results of the current study suggest that the pre-treatment of GB, followed by Aβ1-42 treatment for 24 h, displayed neuro-protective potential, which countered Aβ1-42-induced oxidative stress responses in both undifferentiated and differentiated SH-SY5Y neuronal cells by: 1) hampering production of ROS and RNS; 2) reducing lipid peroxidation; 3) decreasing protein carbonyl content; 4) restoring antioxidant activities of SOD and GSH enzymes; and 5) maintaining genome integrity by reducing the oxidative DNA and RNA base damages. In conclusion, Aβ1-42 induces oxidative damage to the cellular biomolecules, which are associated with AD pathology, and are protected by the pre-treatment of GB against Aβ-toxicity. Taken together, this study advocates for phytochemical-based therapeutic interventions against AD.

E-cigarette aerosols induce lower oxidative stress in vitro when compared to tobacco smoke.

PubMed

Taylor, Mark; Carr, Tony; Oke, Oluwatobiloba; Jaunky, Tomasz; Breheny, Damien; Lowe, Frazer; Gaça, Marianna

2016-07-01

Tobacco smoking is a risk factor for various diseases. The underlying cellular mechanisms are not fully characterized, but include oxidative stress, apoptosis, and necrosis. Electronic-cigarettes (e-cigarettes) have emerged as an alternative to and a possible means to reduce harm from tobacco smoking. E-cigarette vapor contains significantly lower levels of toxicants than cigarette smoke, but standardized methods to assess cellular responses to exposure are not well established. We investigated whether an in vitro model of the airway epithelium (human bronchial epithelial cells) and commercially available assays could differentiate cellular stress responses to aqueous aerosol extracts (AqE) generated from cigarette smoke and e-cigarette aerosols. After exposure to AqE concentrations of 0.063-0.500 puffs/mL, we measured the intracellular glutathione ratio (GSH:GSSG), intracellular generation of oxidant species, and activation of the nuclear factor erythroid-related factor 2 (Nrf2)-controlled antioxidant response elements (ARE) to characterize oxidative stress. Apoptotic and necrotic responses were characterized by increases in caspase 3/7 activity and reductions in viable cell protease activities. Concentration-dependent responses indicative of oxidative stress were obtained for all endpoints following exposure to cigarette smoke AqE: intracellular generation of oxidant species increased by up to 83%, GSH:GSSG reduced by 98.6% and transcriptional activation of ARE increased by up to 335%. Caspase 3/7 activity was increased by up to 37% and the viable cell population declined by up to 76%. No cellular stress responses were detected following exposure to e-cigarette AqE. The methods used were suitably sensitive to be employed for comparative studies of tobacco and nicotine products.

Selenium supplementation to chronic kidney disease patients on hemodialysis does not induce the synthesis of plasma glutathione peroxidase.

PubMed

Zachara, Bronislaw A; Gromadzinska, Jolanta; Zbrog, Zbigniew; Swiech, Rafal; Wasowicz, Wojciech; Twardowska, Ewa; Jablonska, Ewa; Sobala, Wojciech

2009-01-01

Numerous authors have shown that selenium (Se) concentration and glutathione peroxidase (GSH-Px) activity in plasma of chronic kidney disease (CKD) patients are lower than in healthy subjects, but there are only few publications on the level of GSH-Px protein in those patients and no reports on the effect of Se supplementation to HD patients on the level of this enzyme. Se concentration and GSH-Px protein level in plasma were measured in a group of 30 CKD patients on hemodialysis (HD) supplemented with 200 microg Se/day for 3 months, and 28 patients on HD administered with placebo. Se concentration was measured by graphite furnace atomic absorption spectrometry and plasma GSH-Px protein level by the sandwich ELISA method using polyclonal antibody specific for human plasma GSH-Px. Se concentration in patients on placebo did not change throughout the 3-month study period, but increased significantly in Se supplemented group. Se supplementation to CKD patients on HD had no effect on the level of GSH-Px protein. The lack of GSH-Px protein in CKD patients on HD is not linked to Se deficiency since the level of this element increased after Se supplementation while enzyme protein level did not change. The damaged kidney of HD patients is unable to synthesize GSH-Px, even after induction with selenium.

Protective Effects of Black Rice Extracts on Oxidative Stress Induced by tert-Butyl Hydroperoxide in HepG2 Cells

PubMed Central

Lee, Seon-Mi; Choi, Youngmin; Sung, Jeehye; Kim, Younghwa; Jeong, Heon-Sang; Lee, Junsoo

2014-01-01

Black rice contains many biologically active compounds. The aim of this study was to investigate the protective effects of black rice extracts (whole grain extract, WGE and rice bran extract, RBE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. Cellular reactive oxygen species (ROS), antioxidant enzyme activities, malondialdehyde (MDA) and glutathione (GSH) concentrations were evaluated as biomarkers of cellular oxidative status. Cells pretreated with 50 and 100 μg/mL of WGE or RBE were more resistant to oxidative stress in a dose-dependent manner. The highest WGE and BRE concentrations enhanced GSH concentrations and modulated antioxidant enzyme activities (glutathione reductase, glutathione-S-transferase, catalase, and superoxide dismutase) compared to TBHP-treated cells. Cells treated with RBE showed higher protective effect compared to cells treated with WGE against oxidative insult. Black rice extracts attenuated oxidative insult by inhibiting cellular ROS and MDA increase and by modulating antioxidant enzyme activities in HepG2 cells. PMID:25580401

Sulforaphane Restores Cellular Glutathione Levels and Reduces Chronic Periodontitis Neutrophil Hyperactivity In Vitro

PubMed Central

Dias, Irundika H. K.; Chapple, Ian L. C.; Milward, Mike; Grant, Melissa M.; Hill, Eric; Brown, James; Griffiths, Helen R.

2013-01-01

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 . - by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients’ neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 . - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis. PMID:23826097

Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro.

PubMed

Dias, Irundika H K; Chapple, Ian L C; Milward, Mike; Grant, Melissa M; Hill, Eric; Brown, James; Griffiths, Helen R

2013-01-01

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 (. -) by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 (. -) production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis.

Validation of a Reversed-Phase High Performance Liquid Chromatography Method for the Simultaneous Analysis of Cysteine and Reduced Glutathione in Mouse Organs

PubMed Central

Brundu, Serena; Nencioni, Lucia; Celestino, Ignacio; Coluccio, Paolo; Palamara, Anna Teresa; Fraternale, Alessandra

2016-01-01

A depletion of reduced glutathione (GSH) has been observed in pathological conditions and in aging. Measuring GSH in tissues using mouse models is an excellent way to assess GSH depletion and the potential therapeutic efficacy of drugs used to maintain and/or restore cellular redox potential. A high performance liquid chromatography (HPLC) method for the simultaneous determination of GSH and cysteine (Cys) in mouse organs was validated according to USA and European standards. The method was based on separation coupled with ultraviolet detection and precolumn derivatization with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The required validation parameters, that are, selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, and stability, were studied for spleen, lymph nodes, pancreas, and brain. The results showed that the lower limits of quantification were 0.313 μM and 1.25 μM for Cys and GSH, respectively. Intraday and interday precisions were less than 11% and 14%, respectively, for both compounds. The mean extraction recoveries of Cys and GSH from all organs were more than 93% and 86%, respectively. Moreover, the stability of both analytes during sample preparation and storage was demonstrated. The method was accurate, reliable, consistent, and reproducible and it was useful to determine Cys and GSH in the organs of different mouse strains. PMID:26885246

Comparison of inhibition of murine leukaemia cell growth by 9-isothiocyanatoacridine and its cytosine adduct: involvement of thiols.

PubMed

Bajdichova, M; Paulikova, H; Jakubikova, J; Sabolova, D

2007-01-01

Cytotoxicity of two fluorescent acridine derivatives - 9-isothiocyanatoacridine (AcITC) and N-(9-acridinylthiocarbamoyl) cytosine (AcTCC) - a novel acridine compound, were investigated. Both substances have cytotoxic activity against the L1210 cellular line, IC50 values were in the micromolar range. Despite the high reactivity of AcITC towards thiols, its effects on leukemia cells were similar to naturally occurring isothiocyanates. AcITC changed the intracellular level of glutathione (GSH), and induced apoptosis. Arrest of cell cycle (G2/M-phase) was also observed. AcITC primarily reacted with -SH groups on cellular surface, and the study of the interaction of the isotiocyanate with human erythrocyte ghosts confirmed that the plasma membrane was the first place where AcITC bound. AcTCC does not react with cellular thiols; images obtained with fluorescent microscopy confirmed interaction of AcTCC with chromatine. Although AcTCC induced cellular arrest in the G2/M phase, apoptosis was not confirmed.

Effect of karanjin on 2,4,6-trinitrobenzenesulfonic acid-induced colitis in Balb/c mice.

PubMed

Patel, Praful Prakash; Trivedi, Naitikumar Devshankar

2017-01-01

The objective of this study is to evaluate the beneficial effect of karanjin for the treatment of experimental colitis. Colitis was induced in the Balb/c mice by rectal administration of 2% solution of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in 50% methanol. Karanjin (>98% pure) was administered in two different concentrations 100 and 200 mg/kg and sulfasalazine (100 mg/kg) as reference for 7 consecutive days to colitic mice. On the 8 day, mice were euthanized and degree of inflammation was assessed by macroscopic, microscopic, histology and biochemical estimation of myeloperoxidase (MPO), nitric oxide (NO), malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH) level were measured. Karanjin significantly and dose dependently ameliorate the macroscopic damage, histological changes such as cellular infiltration, tissue necrosis, mucosal and submucosal damage as compared to the TNBS control group. Karanjin reduces the activity of MPO, depressed MDA, and NO level and helps in restoring the level of CAT, SOD, and GSH to normal when compared to the TNBS colitis group. Result of the present study indicates that karanjin has the potential to cure colitis induced by intracolonic administration of TNBS.

Effect of karanjin on 2,4,6-trinitrobenzenesulfonic acid-induced colitis in Balb/c mice

PubMed Central

Patel, Praful Prakash; Trivedi, Naitikumar Devshankar

2017-01-01

OBJECTIVES: The objective of this study is to evaluate the beneficial effect of karanjin for the treatment of experimental colitis. METHODS: Colitis was induced in the Balb/c mice by rectal administration of 2% solution of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in 50% methanol. Karanjin (>98% pure) was administered in two different concentrations 100 and 200 mg/kg and sulfasalazine (100 mg/kg) as reference for 7 consecutive days to colitic mice. On the 8 day, mice were euthanized and degree of inflammation was assessed by macroscopic, microscopic, histology and biochemical estimation of myeloperoxidase (MPO), nitric oxide (NO), malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH) level were measured. RESULTS: Karanjin significantly and dose dependently ameliorate the macroscopic damage, histological changes such as cellular infiltration, tissue necrosis, mucosal and submucosal damage as compared to the TNBS control group. Karanjin reduces the activity of MPO, depressed MDA, and NO level and helps in restoring the level of CAT, SOD, and GSH to normal when compared to the TNBS colitis group. CONCLUSION: Result of the present study indicates that karanjin has the potential to cure colitis induced by intracolonic administration of TNBS. PMID:28706329

Antioxidant Machinery Differs between Melanic and Light Nestlings of Two Polymorphic Raptors

PubMed Central

Galván, Ismael; Gangoso, Laura; Grande, Juan M.; Negro, Juan J.; Rodríguez, Airam; Figuerola, Jordi; Alonso-Alvarez, Carlos

2010-01-01

Colour polymorphism results from the expression of multiallelic genes generating phenotypes with very distinctive colourations. Most colour polymorphisms are due to differences in the type or amount of melanins present in each morph, which also differ in several behavioural, morphometric and physiological attributes. Melanin-based colour morphs could also differ in the levels of glutathione (GSH), a key intracellular antioxidant, because of the role of this molecule in melanogenesis. As GSH inhibits the synthesis of eumelanin (i.e. the darkest melanin form), individuals of darker morphs are expected to have lower GSH levels than those of lighter morphs. We tested this prediction in nestlings of two polymorphic raptors, the booted eagle Hieraaetus pennatus and the Eleonora's falcon Falco eleonorae, both of which occur in two morphs differing in the extent of eumelanic plumage. As expected, melanic booted eagle nestlings had lower blood GSH levels than light morph eagle nestlings. In the Eleonora's falcon, however, melanic nestlings only had lower GSH levels after controlling for the levels of other antioxidants. We also found that melanic female eagle nestlings had higher levels of antioxidants other than GSH and were in better body condition than light female eagle nestlings. These findings suggest an adaptive response of melanic nestlings to compensate for reduced GSH levels. Nevertheless, these associations were not found in falcons, indicating species-specific particularities in antioxidant machinery. Our results are consistent with previous work revealing the importance of GSH on the expression of melanic characters that show continuous variation, and suggest that this pathway also applies to discrete colour morphs. We suggest that the need to maintain low GSH levels for eumelanogenesis in dark morph individuals may represent a physiological constraint that helps regulate the evolution and maintenance of polymorphisms. PMID:20976228

Antioxidant machinery differs between melanic and light nestlings of two polymorphic raptors.

PubMed

Galván, Ismael; Gangoso, Laura; Grande, Juan M; Negro, Juan J; Rodríguez, Airam; Figuerola, Jordi; Alonso-Alvarez, Carlos

2010-10-14

Colour polymorphism results from the expression of multiallelic genes generating phenotypes with very distinctive colourations. Most colour polymorphisms are due to differences in the type or amount of melanins present in each morph, which also differ in several behavioural, morphometric and physiological attributes. Melanin-based colour morphs could also differ in the levels of glutathione (GSH), a key intracellular antioxidant, because of the role of this molecule in melanogenesis. As GSH inhibits the synthesis of eumelanin (i.e. the darkest melanin form), individuals of darker morphs are expected to have lower GSH levels than those of lighter morphs. We tested this prediction in nestlings of two polymorphic raptors, the booted eagle Hieraaetus pennatus and the Eleonora's falcon Falco eleonorae, both of which occur in two morphs differing in the extent of eumelanic plumage. As expected, melanic booted eagle nestlings had lower blood GSH levels than light morph eagle nestlings. In the Eleonora's falcon, however, melanic nestlings only had lower GSH levels after controlling for the levels of other antioxidants. We also found that melanic female eagle nestlings had higher levels of antioxidants other than GSH and were in better body condition than light female eagle nestlings. These findings suggest an adaptive response of melanic nestlings to compensate for reduced GSH levels. Nevertheless, these associations were not found in falcons, indicating species-specific particularities in antioxidant machinery. Our results are consistent with previous work revealing the importance of GSH on the expression of melanic characters that show continuous variation, and suggest that this pathway also applies to discrete colour morphs. We suggest that the need to maintain low GSH levels for eumelanogenesis in dark morph individuals may represent a physiological constraint that helps regulate the evolution and maintenance of polymorphisms.

Effect of dietary glutamine on tumor glutathione levels and apoptosis-related proteins in DMBA-induced breast cancer of rats.

PubMed

Todorova, Valentina K; Harms, Stacy A; Kaufmann, Yihong; Luo, Shaoke; Luo, Kevin Q; Babb, Kirk; Klimberg, V Suzanne

2004-12-01

Glutamine (GLN) is a non-essential amino acid that is present in nearly every biochemical pathway and is the major intraorgan nitrogen carrier. GLN via glutamate, is one of the precursors for the synthesis of glutathione (GSH), the major endogenous antioxidant in mammalian cells, which protects them from oxidative injury and cell death. Cancer cells have higher GSH levels than the surrounding normal cells, which attributes to a higher rate of cell proliferation and resistance to chemotherapy. Therefore, selective tumor depletion of GSH presents a promising strategy in cancer treatment. Experimental studies have associated decreased GSH levels with inhibition of proliferation and stimulation of apoptosis. Previous results of our laboratory have provided evidence that dietary GLN diminished tumor development in implantable as well as 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer and elevated GSH in the host tissues. In this study we examined the effects of GLN on GSH levels in DMBA-induced mammary tumors and correlated the results with protein and mRNA expression of apoptosis-related proteins Bcl-2, Bax and caspase-3 in tumor cells. The results have shown that GLN supplementation caused a significant decrease in the tumor GSH levels and the ratio GSH/oxidized GSH (GSSG), accompanied by up-regulation of Bax and caspase-3, and down-regulation of Bcl-2. These findings suggest that dietary GLN supplementation suppresses mammary carcinogenesis by activation of apoptosis in tumor cells and this probably is a result of GSH down-regulation.

Identification of glutathione adducts of α-chlorofatty aldehydes produced in activated neutrophils.

PubMed

Duerr, Mark A; Aurora, Rajeev; Ford, David A

2015-05-01

α-Chlorofatty aldehydes (α-ClFALDs) are produced by hypochlorous acid targeting plasmalogens during neutrophil activation. This study investigated the reaction of the α-chlorinated carbon of α-ClFALD with the nucleophile, GSH. Utilizing ESI/MS/MS, the reaction product of GSH and the 16-carbon α-ClFALD, 2-chlorohexadecanal (2-ClHDA), was characterized. The resulting conjugate of 2-ClHDA and GSH (HDA-GSH) has an intact free aldehyde, and the chlorine at the α-carbon is ejected. Stable isotope-labeled [d4]HDA-GSH was synthesized, which further confirmed the structure, and was used to quantify natural α-ClFALD conjugates of GSH (FALD-GSH) using reverse-phase LC with detection by ESI/MS/MS using selected reaction monitoring. HDA-GSH is elevated in RAW 264.7 cells treated with physiologically relevant concentrations of exogenous 2-ClHDA. Furthermore, PMA-treated primary human neutrophils have elevated levels of HDA-GSH and the conjugate of 2-chlorooctadecanal (2-ClODA) and GSH (ODA-GSH), as well as elevated levels of 2-ClHDA and 2-ClODA. Production of both conjugates in PMA-stimulated neutrophils was reduced by 3-aminotriazole pretreatment, which also blocks endogenous α-ClFALD production. Additionally, plasma FALD-GSH levels were elevated in the K/BxN mouse arthritis model. Taken together, these studies demonstrate novel peptidoaldehydes derived from GSH and α-ClFALD in activated human neutrophils and in vivo in K/BxN mice. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

Evaluation of radio-protective effect of melatonin on whole body irradiation induced liver tissue damage.

PubMed

Shirazi, Alireza; Mihandoost, Ehsan; Ghobadi, Ghazale; Mohseni, Mehran; Ghazi-Khansari, Mahmoud

2013-01-01

Ionizing radiation interacts with biological systems to induce excessive fluxes of free radicals that attack various cellular components. Melatonin has been shown to be a direct free radical scavenger and indirect antioxidant via its stimulatory actions on the antioxidant system.The aim of this study was to evaluate the antioxidant role of melatonin against radiation-induced oxidative injury to the rat liver after whole body irradiation. In this experimental study,thirty-two rats were divided into four groups. Group 1 was the control group, group 2 only received melatonin (30 mg/kg on the first day and 30 mg/kg on the following days), group 3 only received whole body gamma irradiation of 10 Gy, and group 4 received 30 mg/kg melatonin 30 minutes prior to radiation plus whole body irradiation of 10 Gy plus 30 mg/kg melatonin daily through intraperitoneal (IP) injection for three days after irradiation. Three days after irradiation, all rats were sacrificed and their livers were excised to measure the biochemical parameters malondialdehyde (MDA) and glutathione (GSH). Each data point represents mean ± standard error on the mean (SEM) of at least eight animals per group. A one-way analysis of variance (ANOVA) was performed to compare different groups, followed by Tukey's multiple comparison tests (p<0.05). The results demonstrated that whole body irradiation induced liver tissue damage by increasing MDA levels and decreasing GSH levels. Hepatic MDA levels in irradiated rats that were treated with melatonin (30 mg/kg) were significantly decreased, while GSH levels were significantly increased, when compared to either of the control groups or the melatonin only group. The data suggest that administration of melatonin before and after irradiation may reduce liver damage caused by gamma irradiation.

Oxidative stress in the hydrocoral Millepora alcicornis exposed to CO2-driven seawater acidification

NASA Astrophysics Data System (ADS)

Luz, Débora Camacho; Zebral, Yuri Dornelles; Klein, Roberta Daniele; Marques, Joseane Aparecida; Marangoni, Laura Fernandes de Barros; Pereira, Cristiano Macedo; Duarte, Gustavo Adolpho Santos; Pires, Débora de Oliveira; Castro, Clovis Barreira e.; Calderon, Emiliano Nicolas; Bianchini, Adalto

2018-06-01

Global impacts are affecting negatively coral reefs' health worldwide. Ocean acidification associated with the increasing CO2 partial pressure in the atmosphere can potentially induce oxidative stress with consequent cellular damage in corals and hydrocorals. In the present study, parameters related to oxidative status were evaluated in the hydrocoral Millepora alcicornis exposed to three different levels of seawater acidification using a mesocosm system. CO2-driven acidification of seawater was performed until reaching 0.3, 0.6 and 0.9 pH units below the current pH of seawater pumped from the coral reef adjacent to the mesocosm. Therefore, treatments corresponded to control (pH 8.1), mild (pH 7.8), intermediate (pH 7.5) and severe (pH 7.2) seawater acidification. After 0, 16 and 30 d of exposure, hydrocorals were collected and the following parameters were analyzed in the holobiont: antioxidant capacity against peroxyl radicals (ACAP), total glutathione (GSHt) concentration, reduced (GSH) and oxidized (GSSG) glutathione ratio (GSH/GSSG), lipid peroxidation (LPO) and protein carbonyl group (PC) levels. ACAP was increased in hydrocorals after 16 d of exposure to intermediate levels of seawater acidification. GSHt and GSH/GSSG did not change over the experimental period. LPO was increased at any level of seawater acidification, while PC content was increased in hydrocorals exposed to intermediate and severe seawater acidification for 30 d. These findings indicate that the antioxidant defense system of M. alcicornis is capable of coping with acidic conditions for a short period of time (16 d). Additionally, they clearly show that a long-term (30 d) exposure to seawater acidification induces oxidative stress with consequent oxidative damage to lipids and proteins, which could compromise hydrocoral health.

The promising anticancer drug 3-bromopyruvate is metabolized through glutathione conjugation which affects chemoresistance and clinical practice: An evidence-based view.

PubMed

El Sayed, Salah Mohamed; Baghdadi, Hussam; Zolaly, Mohammed; Almaramhy, Hamdi H; Ayat, Mongi; Donki, Jagadish G

2017-03-01

3-Bromopyruvate (3BP) is a promising effective anticancer drug against many different tumors in children and adults. 3BP exhibited strong anticancer effects in both preclinical and human studies e.g. energy depletion, oxidative stress, anti-angiogenesis, anti-metastatic effects, targeting cancer stem cells and antagonizing the Warburg effect. There is no report about 3BP metabolism to guide researchers and oncologists to improve clinical practice and prevent drug resistance. In this article, we provide evidences that 3BP is metabolized through glutathione (GSH) conjugation as a novel report where 3BP was confirmed to be attached to GSH followed by permanent loss of pharmacological effects in a picture similar to cisplatin. Both cisplatin and 3BP are alkylating agents. Reported decrease in endogenous cellular GSH content upon 3BP treatment was confirmed to be due to the formation of 3BP-GSH complex i.e. GSH consumption for conjugation with 3BP. Cancer cells having high endogenous GSH exhibit resistance to 3BP while 3BP sensitive cells acquire resistance upon adding exogenous GSH. Being a thiol blocker, 3BP may attack thiol groups in tissues and serum proteins e.g. albumin and GSH. That may decrease 3BP-induced anticancer effects and the functions of those proteins. We proved here that 3BP metabolism is different from metabolism of hydroxypyruvate that results from metabolism of D-serine using D-amino acid oxidase. Clinically, 3BP administration should be monitored during albumin infusion and protein therapy where GSH should be added to emergency medications. GSH exerts many physiological effects and is safe for human administration both orally and intravenously. Based on that, reported GSH-induced inhibition of 3BP effects makes 3BP effects reversible, easily monitored and easily controlled. This confers a superiority of 3BP over many anticancer agents. Copyright © 2017 Elsevier Ltd. All rights reserved.

An in vitro toxicity evaluation of gold-, PLLA- and PCL-coated silica nanoparticles in neuronal cells for nanoparticle-assisted laser-tissue soldering.

PubMed

Koch, Franziska; Möller, Anja-M; Frenz, Martin; Pieles, Uwe; Kuehni-Boghenbor, Kathrin; Mevissen, Meike

2014-08-01

The uptake of silica (Si) and gold (Au) nanoparticles (NPs) engineered for laser-tissue soldering in the brain was investigated using microglial cells and undifferentiated and differentiated SH-SY5Y cells. It is not known what effects NPs elicit once entering the brain. Cellular uptake, cytotoxicity, apoptosis, and the potential induction of oxidative stress by means of depletion of glutathione levels were determined after NP exposure at concentrations of 10(3) and 10(9)NPs/ml. Au-, silica poly (ε-caprolactone) (Si-PCL-) and silica poly-L-lactide (Si-PLLA)-NPs were taken up by all cells investigated. Aggregates and single NPs were found in membrane-surrounded vacuoles and the cytoplasm, but not in the nucleus. Both NP concentrations investigated did not result in cytotoxicity or apoptosis, but reduced glutathione (GSH) levels predominantly at 6 and 24h, but not after 12 h of NP exposure in the microglial cells. NP exposure-induced GSH depletion was concentration-dependent in both cell lines. Si-PCL-NPs induced the strongest effect of GSH depletion followed by Si-PLLA-NPs and Au-NPs. NP size seems to be an important characteristic for this effect. Overall, Au-NPs are most promising for laser-assisted vascular soldering in the brain. Further studies are necessary to further evaluate possible effects of these NPs in neuronal cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

Glutathione-Disrupted Biofilms of Clinical Pseudomonas aeruginosa Strains Exhibit an Enhanced Antibiotic Effect and a Novel Biofilm Transcriptome.

PubMed

Klare, William; Das, Theerthankar; Ibugo, Amaye; Buckle, Edwina; Manefield, Mike; Manos, Jim

2016-08-01

Pseudomonas aeruginosa infections result in high morbidity and mortality rates for individuals with cystic fibrosis (CF), with premature death often occurring. These infections are complicated by the formation of biofilms in the sputum. Antibiotic therapy is stymied by antibiotic resistance of the biofilm matrix, making novel antibiofilm strategies highly desirable. Within P. aeruginosa biofilms, the redox factor pyocyanin enhances biofilm integrity by intercalating with extracellular DNA. The antioxidant glutathione (GSH) reacts with pyocyanin, disrupting intercalation. This study investigated GSH disruption by assaying the physiological effects of GSH and DNase I on biofilms of clinical CF isolates grown in CF artificial sputum medium (ASMDM+). Confocal scanning laser microscopy showed that 2 mM GSH, alone or combined with DNase I, significantly disrupted immature (24-h) biofilms of Australian epidemic strain (AES) isogens AES-1R and AES-1M. GSH alone greatly disrupted mature (72-h) AES-1R biofilms, resulting in significant differential expression of 587 genes, as indicated by RNA-sequencing (RNA-seq) analysis. Upregulated systems included cyclic diguanylate and pyoverdine biosynthesis, the type VI secretion system, nitrate metabolism, and translational machinery. Biofilm disruption with GSH revealed a cellular physiology distinct from those of mature and dispersed biofilms. RNA-seq results were validated by biochemical and quantitative PCR assays. Biofilms of a range of CF isolates disrupted with GSH and DNase I were significantly more susceptible to ciprofloxacin, and increased antibiotic effectiveness was achieved by increasing the GSH concentration. This study demonstrated that GSH, alone or with DNase I, represents an effective antibiofilm treatment when combined with appropriate antibiotics, pending in vivo studies. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Glutathione-Disrupted Biofilms of Clinical Pseudomonas aeruginosa Strains Exhibit an Enhanced Antibiotic Effect and a Novel Biofilm Transcriptome

PubMed Central

Das, Theerthankar; Ibugo, Amaye; Buckle, Edwina; Manefield, Mike; Manos, Jim

2016-01-01

Pseudomonas aeruginosa infections result in high morbidity and mortality rates for individuals with cystic fibrosis (CF), with premature death often occurring. These infections are complicated by the formation of biofilms in the sputum. Antibiotic therapy is stymied by antibiotic resistance of the biofilm matrix, making novel antibiofilm strategies highly desirable. Within P. aeruginosa biofilms, the redox factor pyocyanin enhances biofilm integrity by intercalating with extracellular DNA. The antioxidant glutathione (GSH) reacts with pyocyanin, disrupting intercalation. This study investigated GSH disruption by assaying the physiological effects of GSH and DNase I on biofilms of clinical CF isolates grown in CF artificial sputum medium (ASMDM+). Confocal scanning laser microscopy showed that 2 mM GSH, alone or combined with DNase I, significantly disrupted immature (24-h) biofilms of Australian epidemic strain (AES) isogens AES-1R and AES-1M. GSH alone greatly disrupted mature (72-h) AES-1R biofilms, resulting in significant differential expression of 587 genes, as indicated by RNA-sequencing (RNA-seq) analysis. Upregulated systems included cyclic diguanylate and pyoverdine biosynthesis, the type VI secretion system, nitrate metabolism, and translational machinery. Biofilm disruption with GSH revealed a cellular physiology distinct from those of mature and dispersed biofilms. RNA-seq results were validated by biochemical and quantitative PCR assays. Biofilms of a range of CF isolates disrupted with GSH and DNase I were significantly more susceptible to ciprofloxacin, and increased antibiotic effectiveness was achieved by increasing the GSH concentration. This study demonstrated that GSH, alone or with DNase I, represents an effective antibiofilm treatment when combined with appropriate antibiotics, pending in vivo studies. PMID:27161630

Cynaropicrin targets the trypanothione redox system in Trypanosoma brucei.

PubMed

Zimmermann, Stefanie; Oufir, Mouhssin; Leroux, Alejandro; Krauth-Siegel, R Luise; Becker, Katja; Kaiser, Marcel; Brun, Reto; Hamburger, Matthias; Adams, Michael

2013-11-15

In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei-the causative agent of Human African Trypanosomiasis-by a so far unknown mechanism. We hypothesized that CYNs α,β-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)2), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)2 redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC-MS/MS analysis was established to quantify intra-cellular CYN, T(SH)2, GSH, as well as GS-CYN and T(S-CYN)2 adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)2 pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)2 redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)2 redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase.

PubMed

Kim, Sun Yee; Park, Jeen-Woo

2003-03-01

Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP+-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP+-dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.

Arsenic induces apoptosis in mouse liver is mitochondria dependent and is abrogated by N-acetylcysteine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santra, Amal; Chowdhury, Abhijit; Ghatak, Subhadip

2007-04-15

Arsenicosis, caused by arsenic contamination of drinking water supplies, is a major public health problem in India and Bangladesh. Chronic liver disease, often with portal hypertension occurs in chronic arsenicosis, contributes to the morbidity and mortality. The early cellular events that initiate liver cell injury due to arsenicosis have not been studied. Our aim was to identify the possible mechanisms related to arsenic-induced liver injury in mice. Liver injury was induced in mice by arsenic treatment. The liver was used for mitochondrial oxidative stress, mitochondrial permeability transition (MPT). Evidence of apoptosis was sought by TUNEL test, caspase assay and histology.more » Pretreatment with N-acetyl-L-cysteine (NAC) was done to modulate hepatic GSH level. Arsenic treatment in mice caused liver injury associated with increased oxidative stress in liver mitochondria and alteration of MPT. Altered MPT facilitated cytochrome c release in the cytosol, activation of caspase 9 and caspase 3 activities and apoptotic cell death. Pretreatment of NAC to arsenic-treated mice abrogated all these alteration suggesting a glutathione (GSH)-dependent mechanism. Oxidative stress in mitochondria and inappropriate MPT are important in the pathogenesis of arsenic induced apoptotic liver cell injury. The phenomenon is GSH dependent and supplementation of NAC might have beneficial effects.« less

Non-invasive imaging of the levels and effects of glutathione on the redox status of mouse brain using electron paramagnetic resonance imaging.

PubMed

Emoto, Miho C; Matsuoka, Yuta; Yamada, Ken-Ichi; Sato-Akaba, Hideo; Fujii, Hirotada G

2017-04-15

Glutathione (GSH) is the most abundant non-protein thiol that buffers reactive oxygen species in the brain. GSH does not reduce nitroxides directly, but in the presence of ascorbates, addition of GSH increases ascorbate-induced reduction of nitroxides. In this study, we used electron paramagnetic resonance (EPR) imaging and the nitroxide imaging probe, 3-methoxycarbonyl-2,2,5,5-tetramethyl-piperidine-1-oxyl (MCP), to non-invasively obtain spatially resolved redox data from mouse brains depleted of GSH with diethyl maleate compared to control. Based on the pharmacokinetics of the reduction reaction of MCP in the mouse heads, the pixel-based rate constant of its reduction reaction was calculated as an index of the redox status in vivo and mapped as a "redox map". The obtained redox maps from control and GSH-depleted mouse brains showed a clear change in the brain redox status, which was due to the decreased levels of GSH in brains as measured by a biochemical assay. We observed a linear relationship between the reduction rate constant of MCP and the level of GSH for both control and GSH-depleted mouse brains. Using this relationship, the GSH level in the brain can be estimated from the redox map obtained with EPR imaging. Copyright © 2017 Elsevier Inc. All rights reserved.

Apocynin protects against neurological damage induced by quinolinic acid by an increase in glutathione synthesis and Nrf2 levels.

PubMed

Cruz-Álvarez, Silvia; Santana-Martínez, Ricardo; Avila-Chávez, Euclides; Barrera-Oviedo, Diana; Hernández-Pando, Rogelio; Pedraza-Chaverri, José; Maldonado, Perla D

2017-05-14

Apocynin (APO) is a well-known NADPH oxidase (NOX) inhibitor. However, several studies have reported its ability to increase glutathione (GSH) levels. Due to GSH is a major non-enzymatic antioxidant in brain, the aim of this study was to evaluate, in the striatum of control and quinolinic acid (QUIN) injected rats, the effect of APO administration on: (1) GSH levels, (2) activity of some enzymes involved in the GSH metabolism, and (3) nuclear factor erythroid-2-related factor 2 (Nrf2) mRNA levels. Animals received QUIN 240nmol in right striatum and APO (5mg/kg, i.p.), 30min before and 60min after intrastriatal injection. APO treatment prevented the QUIN-induced histological damage to the striatum. In control rats, APO treatment increased GSH and Nrf2 mRNA levels and the activities of gamma-glutamylcysteine ligase (γ-GCL), glutathione-S-transferase (GST) and glutathione peroxidase (GPx). On the other hand, APO treatment prevented the QUIN-induced decrease in GSH and Nrf2 levels, and in γ-GCL and GPx activities. These data indicate that APO is able to increase GSH levels and the activity of proteins involved in its metabolism, which could be associated with its ability to increase the Nrf2 mRNA levels. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Curcumin attenuates insulin resistance in hepatocytes by inducing Nrf2 nuclear translocation.

PubMed

Zhao, Shu-Guang; Li, Qiang; Liu, Zhen-Xiong; Wang, Jing-Jie; Wang, Xv-Xia; Qin, Ming; Wen, Qin-Sheng

2011-01-01

NF-E2-Related Factor-2 (Nrf2) is a transcription factor that plays a crucial role in the cellular protection against oxidative stress. Curcumin has been reported to induce Nrf2 nuclear translocation and upregulate the expression of numerous reactive oxygen species (ROS) detoxifying and antioxidant genes in hepatocytes. This study was designed to investigate whether curcumin-induced Nrf2 nuclear translocation could reduce ROS-mediated insulin resistance in cultured LO2 hepatocytes. Human LO2 hepatocytes were incubated with curcumine and glucose oxidase (GO) in the presence/absence of wortmannin (a phosphatidyinositol 3-kinase (PI3K) inhibitor), oxidative stress, cellular damage, Nrf2 nuclear translocation and insulin resistance were measured. GO exposure significantly increased intracellular ROS, glutathione (GSH) depletion, malondialdehyde (MDA) formation, and increased activities of cellular lactate dehydrogenase (LDH) and aspartate amino transferase (AST), as well as causing insulin resistance. Curcumin pretreatment significantly attenuated these disturbances in intracellular ROS, liver enzyme activity and significantly antagonized the lipid peroxidation, GSH depletion and insulin resistance induced by GO in LO2 hepatocytes. These effects paralleled Nrf2 nuclear translocation induced by curcumin. Wortmannin partially blocked curcumin-induced Nrf2 nuclear translocation. In addition, wortmannin prevented curcumin-induced improvements in intracellular ROS, MDA formation, GSH depletion, liver enzyme activity and insulin resistance in cultured LO2 hepatocytes. These findings suggest that curcumin could reduce ROS-mediated insulin resistance in hepatocytes, at least in part through nuclear translocation of Nrf2.

[Selenium requirements of dairy goats].

PubMed

Dercksen, D P; Counotte, G H M; Hazebroek, M Klein; Arts, W; van Rijn, T

2007-06-15

Selenium is an essential part of the enzyme glutathione-peroxidase (GSH-Px) and plays an important role in the intracellular aspecific immune defence. Reference values for blood levels of GSH-Px are not available for dairy goats. The EU has authorized the addition of selenium (as E), in the form of sodium selenite or sodium selenate, to animal feeds, to a maximum of 0.5 mg selenium/kg complete feed. Dairy goats given feed containing the maximum level of selenium (0.5 mg/kg) had GSH-Px levels of more than 1000 U/g Hb. The reference values for GSH-Px in cattle, horses, and pigs are between 120 and 600 U/g Hb. Newborn kids had GSH-Px levels between 350 and 400 U/g Hb, comparable with those ofnewborn calves. In conclusion, the addition of selenium to feeds for dairy goats in amounts authorized by the EU leads to blood GSH-Px levels that are substantially higher than those in other species, such as horses, cattle, and pigs. Thus the maximum level of supplemental selenium in feeds for dairy goats should be less than 0.5 mg/kg.

Long-wavelength TCF-based fluorescence probes for the detection and intracellular imaging of biological thiols.

PubMed

Sedgwick, Adam C; Gardiner, Jordan E; Kim, Gyoungmi; Yevglevskis, Maksims; Lloyd, Matthew D; Jenkins, A Toby A; Bull, Steven D; Yoon, Juyoung; James, Tony D

2018-05-08

Two 'turn on' TCF-based fluorescence probes were developed for the detection of biological thiols (TCF-GSH and TCFCl-GSH). TCF-GSH was shown to have a high sensitivity towards glutathione (GSH) with a 0.28 μM limit of detection. Unfortunately, at higher GSH concentrations the fluorescence intensity of TCF-GSH decreased and toxicity was observed for TCF-GSH in live cells. However, TCFCl-GSH was shown to be able to detect GSH at biologically relevant concentrations with a 0.45 μM limit of detection. No toxicity was found for TCFCl-GSH and a clear 'turn on' with good photostability was observed for the exogenous addition of GSH, Cys and HCys. Furthermore, TCFCl-GSH was used to evaluate the effects of drug treatment on the levels of GSH in live cells.

L-Cysteine metabolism and its nutritional implications.

PubMed

Yin, Jie; Ren, Wenkai; Yang, Guan; Duan, Jielin; Huang, Xingguo; Fang, Rejun; Li, Chongyong; Li, Tiejun; Yin, Yulong; Hou, Yongqing; Kim, Sung Woo; Wu, Guoyao

2016-01-01

L-Cysteine is a nutritionally semiessential amino acid and is present mainly in the form of L-cystine in the extracellular space. With the help of a transport system, extracellular L-cystine crosses the plasma membrane and is reduced to L-cysteine within cells by thioredoxin and reduced glutathione (GSH). Intracellular L-cysteine plays an important role in cellular homeostasis as a precursor for protein synthesis, and for production of GSH, hydrogen sulfide (H(2)S), and taurine. L-Cysteine-dependent synthesis of GSH has been investigated in many pathological conditions, while the pathway for L-cysteine metabolism to form H(2)S has received little attention with regard to prevention and treatment of disease in humans. The main objective of this review is to highlight the metabolic pathways of L-cysteine catabolism to GSH, H(2)S, and taurine, with special emphasis on therapeutic and nutritional use of L-cysteine to improve the health and well-being of animals and humans. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In vitro toxicity of nanoparticles in BRL 3A rat liver cells.

PubMed

Hussain, S M; Hess, K L; Gearhart, J M; Geiss, K T; Schlager, J J

2005-10-01

This study was undertaken to address the current deficient knowledge of cellular response to nanosized particle exposure. The study evaluated the acute toxic effects of metal/metal oxide nanoparticles proposed for future use in industrial production methods using the in vitro rat liver derived cell line (BRL 3A). Different sizes of nanoparticles such as silver (Ag; 15, 100 nm), molybdenum (MoO(3); 30, 150 nm), aluminum (Al; 30, 103 nm), iron oxide (Fe(3)O(4); 30, 47 nm), and titanium dioxide (TiO(2); 40 nm) were evaluated for their potential toxicity. We also assessed the toxicity of relatively larger particles of cadmium oxide (CdO; 1 microm), manganese oxide (MnO(2); 1-2 microm), and tungsten (W; 27 microm), to compare the cellular toxic responses with respect to the different sizes of nanoparticles with different core chemical compositions. For toxicity evaluations, cellular morphology, mitochondrial function (MTT assay), membrane leakage of lactate dehydrogenase (LDH assay), reduced glutathione (GSH) levels, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were assessed under control and exposed conditions (24h of exposure). Results showed that mitochondrial function decreased significantly in cells exposed to Ag nanoparticles at 5-50 microg/ml. However, Fe(3)O(4), Al, MoO(3) and TiO(2) had no measurable effect at lower doses (10-50 microg/ml), while there was a significant effect at higher levels (100-250 microg/ml). LDH leakage significantly increased in cells exposed to Ag nanoparticles (10-50 microg/ml), while the other nanoparticles tested displayed LDH leakage only at higher doses (100-250 microg/ml). In summary the Ag was highly toxic whereas, MoO(3) moderately toxic and Fe(3)O(4), Al, MnO(2) and W displayed less or no toxicity at the doses tested. The microscopic studies demonstrated that nanoparticle-exposed cells at higher doses became abnormal in size, displaying cellular shrinkage, and an acquisition of an irregular shape. Due to toxicity of silver, further study conducted with reference to its oxidative stress. The results exhibited significant depletion of GSH level, reduced mitochondrial membrane potential and increase in ROS levels, which suggested that cytotoxicity of Ag (15, 100 nm) in liver cells is likely to be mediated through oxidative stress.

High level of reduced glutathione contributes to detoxification of lipid peroxide-derived reactive carbonyl species in transgenic Arabidopsis overexpressing glutathione reductase under aluminum stress.

PubMed

Yin, Lina; Mano, Jun'ichi; Tanaka, Kiyoshi; Wang, Shiwen; Zhang, Meijuan; Deng, Xiping; Zhang, Suiqi

2017-10-01

Lipid peroxide-derived reactive carbonyl species (RCS), generated downstream of reactive oxygen species (ROS), are critical damage-inducing species in plant aluminum (Al) toxicity. In mammals, RCS are scavenged primarily by glutathione (reduced form of glutathione, GSH), but in plant Al stress, contribution of GSH to RCS detoxification has not been evaluated. In this study, Arabidopsis plants overexpressing the gene AtGR1 (accession code At3g24170), encoding glutathione reductase (GR), were generated, and their performance under Al stress was examined. These transgenic plants (GR-OE plants) showed higher GSH levels and GSH/GSSG (oxidized form of GSH) ratio, and an improved Al tolerance as they suffered less inhibition of root growth than wild-type under Al stress. Exogenous application of 4-hydroxy-2-nonenal, an RCS responsible for Al toxicity in roots, markedly inhibited root growth in wild-type plants. GR-OE plants suffered significantly smaller inhibition, indicating that the enhanced GSH level increased the capacity of RCS detoxification. The generation of H 2 O 2 due to Al stress in GR-OE plants was lower by 26% than in wild-type. Levels of various RCS, such as malondialdehyde, butyraldehyde, phenylacetaldehyde, (E)-2-heptenal and n-octanal, were suppressed by more than 50%. These results indicate that high levels of GSH and GSH/GSSG ratio by GR overexpression contributed to the suppression of not only ROS, but also RCS. Thus, the maintenance of GSH level by overexpressing GR reinforces dual detoxification functions in plants and is an efficient approach to enhance Al tolerance. © 2017 Scandinavian Plant Physiology Society.

Elevation of Glucose 6-Phosphate Dehydrogenase Activity Induced by Amplified Insulin Response in Low Glutathione Levels in Rat Liver

PubMed Central

Taniguchi, Misako; Mori, Nobuko; Iramina, Chizuru

2016-01-01

Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level, γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule. PMID:27597985

Glutathione: new roles in redox signaling for an old antioxidant

PubMed Central

Aquilano, Katia; Baldelli, Sara; Ciriolo, Maria R.

2014-01-01

The physiological roles played by the tripeptide glutathione have greatly advanced over the past decades superimposing the research on free radicals, oxidative stress and, more recently, redox signaling. In particular, GSH is involved in nutrient metabolism, antioxidant defense, and regulation of cellular metabolic functions ranging from gene expression, DNA and protein synthesis to signal transduction, cell proliferation and apoptosis. This review will be focused on the role of GSH in cell signaling by analysing the more recent advancements about its capability to modulate nitroxidative stress, autophagy, and viral infection. PMID:25206336

Glutathione: new roles in redox signaling for an old antioxidant.

PubMed

Aquilano, Katia; Baldelli, Sara; Ciriolo, Maria R

2014-01-01

The physiological roles played by the tripeptide glutathione have greatly advanced over the past decades superimposing the research on free radicals, oxidative stress and, more recently, redox signaling. In particular, GSH is involved in nutrient metabolism, antioxidant defense, and regulation of cellular metabolic functions ranging from gene expression, DNA and protein synthesis to signal transduction, cell proliferation and apoptosis. This review will be focused on the role of GSH in cell signaling by analysing the more recent advancements about its capability to modulate nitroxidative stress, autophagy, and viral infection.

Early cellular responses against tributyltin chloride exposure in primary cultures derived from various brain regions.

PubMed

Mitra, Sumonto; Siddiqui, Waseem A; Khandelwal, Shashi

2014-05-01

Tributyltin (TBT) is a potent biocide and commonly used in various industrial sectors. Humans are mainly exposed through the food chain. We have previously demonstrated tin accumulation in brain following TBT-chloride (TBTC) exposure. In this study, effect of TBTC on dissociated cells from different brain regions was evaluated. Cytotoxicity assay (MTT), mode of cell death (Annexin V/PI assay), oxidative stress parameters (ROS and lipid peroxidation), reducing power of the cell (GSH), mitochondrial membrane potential (MMP) and intracellular Ca(2+) were evaluated to ascertain the effect of TBTC. Expression of glial fibrillary acidic protein (GFAP) was measured to understand the effect on astroglial cells. TBTC as low as 30 nM was found to reduce GSH levels, whereas higher doses of 300 and 3000 nM induced ROS generation and marked loss in cell viability mainly through apoptosis. Striatum showed higher susceptibility than other regions, which may have further implications on various neurological aspects. Copyright © 2014 Elsevier B.V. All rights reserved.

Curcumin Pretreatment Induces Nrf2 and an Antioxidant Response and Prevents Hemin-Induced Toxicity in Primary Cultures of Cerebellar Granule Neurons of Rats

PubMed Central

González-Reyes, Susana; Guzmán-Beltrán, Silvia; Medina-Campos, Omar Noel; Pedraza-Chaverri, José

2013-01-01

Curcumin is a bifunctional antioxidant derived from Curcuma longa. This study identifies curcumin as a neuroprotectant against hemin-induced damage in primary cultures of cerebellar granule neurons (CGNs) of rats. Hemin, the oxidized form of heme, is a highly reactive compound that induces cellular injury. Pretreatment of CGNs with 5–30 μM curcumin effectively increased by 2.3–4.9 fold heme oxygenase-1 (HO-1) expression and by 5.6–14.3-fold glutathione (GSH) levels. Moreover, 15 μM curcumin attenuated by 55% the increase in reactive oxygen species (ROS) production, by 94% the reduction of GSH/glutathione disulfide (GSSG) ratio, and by 49% the cell death induced by hemin. The inhibition of heme oxygenase system or GSH synthesis with tin mesoporphyrin and buthionine sulfoximine, respectively, suppressed the protective effect of curcumin against hemin-induced toxicity. These data strongly suggest that HO-1 and GSH play a major role in the protective effect of curcumin. Furthermore, it was found that 24 h of incubation with curcumin increases by 1.4-, 2.3-, and 5.2-fold the activity of glutathione reductase, glutathione S-transferase and superoxide dismutase, respectively. Additionally, it was found that curcumin was capable of inducing nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation into the nucleus. These data suggest that the pretreatment with curcumin induces Nrf2 and an antioxidant response that may play an important role in the protective effect of this antioxidant against hemin-induced neuronal death. PMID:24454990

Factors affecting the presence of dissolved glutathione in estuarine waters.

PubMed

Tang, Degui; Shafer, Martin M; Karner, Dawn A; Overdier, Joel; Armstrong, David E

2004-08-15

We investigated factors influencing the presence of the thiol glutathione (GSH) in estuarine waters. Our study addressed thiol phase-association, the biological release from algal cultures, and the role of copper in both thiol release and preservation. Our measurements in three diverse estuaries in the continental United States (San Diego Bay, Cape Fear Estuary, and Norfolk Estuary) show that dissolved GSH, present at sub-nanomolar levels, is preferentially partitioned into the ultra-filtrate fraction (<1 kDa) in comparison with dissolved organic carbon (DOC). Concentrations of GSH generally increased with increases in total copper (Cu)levels, although large variability was observed among estuaries. In 30-h exposure experiments, release of dissolved GSH from the diatom Thalassiosira weissflogii into organic ligand-free experimental media was a strong function of added Cu concentration. The released GSH increased from about 0.02 to 0.27 fmol/cell as Cu was increased from the background level (0.5 nM) to 310 nM in the modified Aquil media. However, excretion of GSH was lower (up to 0.13 fmol/cell) when cells were grown in surface waters of San Diego Bay, despite much higher total Cu concentrations. Experiments conducted in-situ in San Diego Bay water indicated that high concentrations of added Cu destabilized GSH, while both Mn(II) and natural colloids promoted GSH stability. In contrast, laboratory experiments in synthetic media indicated that moderate levels of added Cu enhanced GSH stability.

A high-fat diet differentially regulates glutathione phenotypes in the obesity-prone mouse strains DBA/2J, C57BL/6J, and AKR/J.

PubMed

Norris, Katie M; Okie, Whitney; Kim, Woo Kyun; Adhikari, Roshan; Yoo, Sarah; King, Stephanie; Pazdro, Robert

2016-12-01

The ubiquitous tripeptide glutathione (GSH) is a critical component of the endogenous antioxidant defense system. Tissue GSH concentrations and redox status (GSH/GSSG) are genetically controlled, but it is unclear whether interactions between genetic background and diet affect GSH homeostasis. The current study tested the hypothesis that a high-fat diet regulates GSH homeostasis in a manner dependent on genetic background. At 4 months of age, female mice representing 3 obesity-prone inbred strains-C57BL/6J (B6), DBA/2J (D2), and AKR/J (AKR)-were randomly assigned to consume a control (10% energy from fat) or high-fat (62% energy from fat) diet for 10 weeks (n=5/diet per strain). Tissue GSH levels, GSSG levels, and GSH/GSSG were quantified, and hepatic expression of GSH-related enzymes was evaluated by quantitative reverse transcription polymerase chain reaction. The high-fat diet caused a decrease in hepatic GSH/GSSG in D2 mice. In contrast, B6 mice exhibited a decrease in GSSG levels in the liver and kidney, as well as a resultant increase in renal GSH/GSSG. AKR mice also exhibited increased renal GSH/GSSG on a high-fat diet. Finally, the high-fat diet induced a unique gene expression response in D2 mice compared with B6 and AKR. The D2 response was characterized by up-regulation of glutamate-cysteine ligase modifier subunit and down-regulation of glutathione reductase, whereas the B6 and AKR responses were characterized by up-regulation of glutathione peroxidase 1. Two-way analysis of variance analyses confirmed several diet-strain interactions within the GSH system, and linear regression models highlighted relationships between body mass and GSH outcomes as well. Overall, our data indicate that dietary fat regulates the GSH system in a strain-dependent manner. Copyright © 2016 Elsevier Inc. All rights reserved.

H2O2 mediates ALA-induced glutathione and ascorbate accumulation in the perception and resistance to oxidative stress in Solanum lycopersicum at low temperatures.

PubMed

Liu, Tao; Hu, Xiaohui; Zhang, Jiao; Zhang, Junheng; Du, Qingjie; Li, Jianming

2018-02-15

Low temperature is a crucial factor influencing plant growth and development. The chlorophyll precursor, 5-aminolevulinic acid (ALA) is widely used to improve plant cold tolerance. However, the interaction between H 2 O 2 and cellular redox signaling involved in ALA-induced resistance to low temperature stress in plants remains largely unknown. Here, the roles of ALA in perceiving and regulating low temperature-induced oxidative stress in tomato plants, together with the roles of H 2 O 2 and cellular redox states, were characterized. Low concentrations (10-25 mg·L - 1 ) of ALA enhanced low temperature-induced oxidative stress tolerance of tomato seedlings. The most effective concentration was 25 mg·L - 1 , which markedly increased the ratio of reduced glutathione and ascorbate (GSH and AsA), and enhanced the activities of superoxide dismutase, catalase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. Furthermore, gene expression of respiratory burst oxidase homolog1 and H 2 O 2 content were upregulated with ALA treatment under normal conditions. Treatment with exogenous H 2 O 2 , GSH, and AsA also induced plant tolerance to oxidative stress at low temperatures, while inhibition of GSH and AsA syntheses significantly decreased H 2 O 2 -induced oxidative stress tolerance. Meanwhile, scavenging or inhibition of H 2 O 2 production weakened, but did not eliminate, GSH- or AsA- induced tomato plant tolerance to oxidative stress at low temperatures. Appropriate concentrations of ALA alleviated the low temperature-induced oxidative stress in tomato plants via an antioxidant system. The most effective concentration was 25 mg·L - 1 . The results showed that H 2 O 2 induced by exogenous ALA under normal conditions is crucial and may be the initial step for perception and signaling transmission, which then improves the ratio of GSH and AsA. GSH and AsA may then interact with H 2 O 2 signaling, resulting in enhanced antioxidant capacity in tomato plants at low temperatures.

Local and systemic oxidative stress and glucocorticoid receptor levels in chronic obstructive pulmonary disease patients

PubMed Central

Zeng, Mian; Li, Yue; Jiang, Yujie; Lu, Guifang; Huang, Xiaomei; Guan, Kaipan

2013-01-01

BACKGROUND: Previous studies have indicated that oxidative stress plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). OBJECTIVES: To study local and systemic oxidative stress status in COPD patients, and to clarify the relationship between local and systemic oxidative stress. METHODS: Lipid peroxide malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and GSH peroxidase (GSH-PX) levels in induced sputum and plasma, as well as glucocorticoid receptor (GR) levels in peripheral blood leukocytes were examined in 43 acute exacerbation of COPD patients (group A), 35 patients with stable COPD (group B) and 28 healthy controls (14 smokers [group C]; 14 nonsmokers [group D]). RESULTS: MDA levels in induced sputum and plasma decreased progressively in groups A to D, with significant differences between any two groups (P<0.001). GSH, SOD and GSH-PX levels in both induced sputum and plasma increased progressively in groups A to D, with significant differences between any two groups (P<0.001). GR levels in peripheral blood leukocytes decreased progressively in groups D to A (all comparisons P<0.001). Pearson analysis revealed strong correlations between MDA, GSH, SOD and GSH-PX levels in plasma and induced sputum. The activity of SOD in plasma and sputum were both positively correlated with GR levels (partial correlation coefficients 0.522 and 0.574, respectively [P<0.001]). CONCLUSIONS: Oxidative stress levels were elevated in COPD patients. There was a correlation between local and systemic oxidative status in COPD, and between decreased SOD activity and decreased GR levels in COPD patients. PMID:23457673

Glutathione and glutathione S-transferase levels in patients with liver metastases of colorectal cancer and other hepatic disorders.

PubMed

Baltruskeviciene, Edita; Kazbariene, Birutė; Badaras, Robertas; Bagdonaitė, Loreta; Krikštaponienė, Aurelija; Zdanavičius, Linas; Aleknavicius, Eduardas; Didziapetrienė, Janina

2016-07-01

Glutathione and glutathione S-transferases (GST) are involved in cell defence against reactive oxygen species, which induces oxidative stress and are associated with different chronic diseases. The aim of the present study was to determine the differences in reduced glutathione (GSH) and GST levels in patients with different liver diseases. Overall, 114 patients were enrolled in this study: 58 patients with colorectal cancer (18 without and 40 with liver metastases), 27 with liver steatosis, 29 with alcoholic cirrhosis and a group of 40 healthy volunteers. The levels of GSH and GST in blood serum were evaluated by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's guidelines. Significant differences in GSH and GST levels were observed in most of the groups compared to the healthy volunteers (GSH: 52.72 µg/mL, GST: 0.53 ng/mL): with hepatic steatosis (GSH: 17.04 µg/mL, p < 0.001; GST: 5.89 ng/mL, p < 0.001), alcoholic cirrhosis (GSH: 62.04 µg/mL, p < 0.003; GST: 0.94 ng/mL, p < 0.001) and liver metastases (GSH: 37.84 µg/mL, p < 0.001, GST: 1.25 ng/mL, p=0.747). The different GSH and GST levels in patients with colorectal cancer liver metastases, liver steatosis and alcoholic cirrhosis indicate the differences in antioxidative system damage and its compensatory possibilities and could serve as potential biomarkers for its correction.

Oxidative stress and dysfunctional NRF2 underlie pachyonychia congenita phenotypes

PubMed Central

Kerns, Michelle L.; Hakim, Jill M.C.; Lu, Rosemary G.; Guo, Yajuan; Berroth, Andreas; Kaspar, Roger L.

2016-01-01

Palmoplantar keratoderma (PPK) are debilitating lesions that arise in individuals with pachyonychia congenita (PC) and feature upregulation of danger-associated molecular patterns and skin barrier regulators. The defining features of PC-associated PPK are reproduced in mice null for keratin 16 (Krt16), which is commonly mutated in PC patients. Here, we have shown that PPK onset is preceded by oxidative stress in footpad skin of Krt16–/– mice and correlates with an inability of keratinocytes to sustain nuclear factor erythroid–derived 2 related factor 2–dependent (NRF2-dependent) synthesis of the cellular antioxidant glutathione (GSH). Additionally, examination of plantar skin biopsies from individuals with PC confirmed the presence of high levels of hypophosphorylated NRF2 in lesional tissue. In Krt16–/– mice, genetic ablation of Nrf2 worsened spontaneous skin lesions and accelerated PPK development in footpad skin. Hypoactivity of NRF2 in Krt16–/– footpad skin correlated with decreased levels or activity of upstream NRF2 activators, including PKCδ, receptor for activated C kinase 1 (RACK1), and p21. Topical application of the NRF2 activator sulforaphane to the footpad of Krt16–/– mice prevented the development of PPK and normalized redox balance via regeneration of GSH from existing cellular pools. Together, these findings point to oxidative stress and dysfunctional NRF2 as contributors to PPK pathogenesis, identify K16 as a regulator of NRF2 activation, and suggest that pharmacological activation of NRF2 should be further explored for PC treatment. PMID:27183391

Therapeutic potential of alpha-ketoglutarate against acetaminophen-induced hepatotoxicity in rats

PubMed Central

Mehra, Lalita; Hasija, Yasha; Mittal, Gaurav

2016-01-01

Objective: Alpha-ketoglutarate (α-KG) is a cellular intermediary metabolite of Krebs cycle, involved in energy metabolism, amino acid synthesis, and nitrogen transport. It is available over-the-counter and marketed as a nutritional supplement. There is a growing body of evidence to suggest that dietary α-KG has the potential to maintain cellular redox status and thus can protect various oxidative stress induced disease states. The aim of the present study was to investigate the hepatoprotective role of α-KG in acetaminophen (APAP) induced toxicity in rats. Materials and Methods: Animals were divided into three groups of six animals each. Group I (Vehicle control): Normal Saline, Group II (APAP): A single intraperitoneal injection of 0.6 g/kg, Group III (APAP + α-KG): APAP as in Group II with α-KG treatment at a dose of 2 g/kg, orally for 5 days. Then the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) with oxidative stress markers including malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and histopathology were analyzed. Results: The results indicate that APAP caused significant elevations in ALT, AST, ALP, and MDA levels, while GSH, SOD, and CAT were significantly depleted while co-administration of α-KG showed a significant (P < 0.05) reduction in the severity of these damages. Histologically, the liver showed inflammation and necrosis after APAP treatment, which were significantly restored with co-administration of α-KG. Conclusion: These results indicate the possible therapeutic potential of α-KG in protecting liver damage by APAP in rats. PMID:28216953

Comparison of gene expression signatures of diamide, H2O2 and menadione exposed Aspergillus nidulans cultures – linking genome-wide transcriptional changes to cellular physiology

PubMed Central

Pócsi, István; Miskei, Márton; Karányi, Zsolt; Emri, Tamás; Ayoubi, Patricia; Pusztahelyi, Tünde; Balla, György; Prade, Rolf A

2005-01-01

Background In addition to their cytotoxic nature, reactive oxygen species (ROS) are also signal molecules in diverse cellular processes in eukaryotic organisms. Linking genome-wide transcriptional changes to cellular physiology in oxidative stress-exposed Aspergillus nidulans cultures provides the opportunity to estimate the sizes of peroxide (O22-), superoxide (O2•-) and glutathione/glutathione disulphide (GSH/GSSG) redox imbalance responses. Results Genome-wide transcriptional changes triggered by diamide, H2O2 and menadione in A. nidulans vegetative tissues were recorded using DNA microarrays containing 3533 unique PCR-amplified probes. Evaluation of LOESS-normalized data indicated that 2499 gene probes were affected by at least one stress-inducing agent. The stress induced by diamide and H2O2 were pulse-like, with recovery after 1 h exposure time while no recovery was observed with menadione. The distribution of stress-responsive gene probes among major physiological functional categories was approximately the same for each agent. The gene group sizes solely responsive to changes in intracellular O22-, O2•- concentrations or to GSH/GSSG redox imbalance were estimated at 7.7, 32.6 and 13.0 %, respectively. Gene groups responsive to diamide, H2O2 and menadione treatments and gene groups influenced by GSH/GSSG, O22- and O2•- were only partly overlapping with distinct enrichment profiles within functional categories. Changes in the GSH/GSSG redox state influenced expression of genes coding for PBS2 like MAPK kinase homologue, PSK2 kinase homologue, AtfA transcription factor, and many elements of ubiquitin tagging, cell division cycle regulators, translation machinery proteins, defense and stress proteins, transport proteins as well as many enzymes of the primary and secondary metabolisms. Meanwhile, a separate set of genes encoding transport proteins, CpcA and JlbA amino acid starvation-responsive transcription factors, and some elements of sexual development and sporulation was ROS responsive. Conclusion The existence of separate O22-, O2•- and GSH/GSSG responsive gene groups in a eukaryotic genome has been demonstrated. Oxidant-triggered, genome-wide transcriptional changes should be analyzed considering changes in oxidative stress-responsive physiological conditions and not correlating them directly to the chemistry and concentrations of the oxidative stress-inducing agent. PMID:16368011

Thioredoxin Uses a GSH-independent Route to Deglutathionylate Endothelial Nitric-oxide Synthase and Protect against Myocardial Infarction*

PubMed Central

Subramani, Jaganathan; Kundumani-Sridharan, Venkatesh; Hilgers, Rob H. P.; Owens, Cade; Das, Kumuda C.

2016-01-01

Reversible glutathionylation plays a critical role in protecting protein function under conditions of oxidative stress generally and for endothelial nitric-oxide synthase (eNOS) specifically. Glutathione-dependent glutaredoxin-mediated deglutathionylation of eNOS has been shown to confer protection in a model of heart damage termed ischemia-reperfusion injury, motivating further study of eNOS deglutathionylation in general. In this report, we present evidence for an alternative mechanism of deglutathionylation. In this pathway thioredoxin (Trx), a small cellular redox protein, is shown to rescue eNOS from glutathionylation during ischemia-reperfusion in a GSH-independent manner. By comparing mice with global overexpression of Trx and mice with cardiomyocyte-specific overexpression of Trx, we demonstrate that vascular Trx-mediated deglutathionylation of eNOS protects against ischemia-reperfusion-mediated myocardial infarction. Trx deficiency in endothelial cells promoted eNOS glutathionylation and reduced its enzymatic activity, whereas increased levels of Trx led to deglutathionylated eNOS. Thioredoxin-mediated deglutathionylation of eNOS in the coronary artery in vivo protected against reperfusion injury, even in the presence of normal levels of GSH. We further show that Trx directly interacts with eNOS, and we confirmed that Cys-691 and Cys-910 are the glutathionylated sites, as mutation of these cysteines partially rescued the decrease in eNOS activity, whereas mutation of a distal site, Cys-384, did not. Collectively, this study shows for the first time that Trx is a potent deglutathionylating protein in vivo and in vitro that can deglutathionylate proteins in the presence of high levels of GSSG in conditions of oxidative stress. PMID:27587398

Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase.

PubMed

Mohr, S; Hallak, H; de Boitte, A; Lapetina, E G; Brüne, B

1999-04-02

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.

Glutathione levels in plasma, saliva and gingival crevicular fluid after periodontal therapy in obese and normal weight individuals.

PubMed

Öngöz Dede, F; Bozkurt Doğan, Ş; Balli, U; Avci, B; Durmuşlar, M C; Baratzade, T

2016-12-01

The purpose of this study was to investigate the effects of obesity on reduced and oxidized glutathione (GSH and GSSG) levels in the gingival crevicular fluid, plasma and saliva of patients with chronic periodontitis and to evaluate the changes after nonsurgical periodontal therapy. The study included 60 patients: 30 patients with chronic periodontitis (15 obese patients and 15 normal weight patients) and 30 healthy control subjects (15 obese patients and 15 normal weight patients). Gingival crevicular fluid, plasma and saliva samples were collected, and clinical periodontal measurements were recorded at baseline and at the first month after periodontal therapy from patients with chronic periodontitis. GSH and GSSG levels were analyzed with spectrophotometry. The GSH levels in the plasma, saliva and gingival crevicular fluid in obese individuals with chronic periodontitis were lower than in normal weight individuals at baseline (p < 0.01). There was a significant difference in the GSH/GSSG ratio in plasma and gingival crevicular fluid between the obese and normal weight groups at baseline (p < 0.01). The GSH levels in plasma, gingival crevicular fluid and saliva were significantly increased in both chronic periodontitis groups after nonsurgical periodontal therapy (p < 0.01). A significant positive correlation was found between GSH levels in saliva, plasma and gingival crevicular fluid in all groups (p < 0.001). The study revealed that obesity in patients with chronic periodontitis is associated with decreased GSH levels and the GSH/GSSG ratio. Moreover, nonsurgical periodontal therapy may be helpful for improvement in glutathione values in obese and normal weight individuals with chronic periodontitis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Oxidative stress markers at birth: Analyses of a neonatal population.

PubMed

Giuffrè, Mario; Rizzo, Manfredi; Scaturro, Giusy; Pitruzzella, Alessandro; Marino Gammazza, Antonella; Cappello, Francesco; Corsello, Giovanni; Li Volti, Giovanni

2015-01-01

In order to further understand neonatal stress and, thus, control it efficaciously, there is a need for more information on the manifestations of stress at the molecular level in the newborn, with particular regard to oxidants, and anti-oxidant and anti-stress mechanisms, including mitochondrial heat shock protein-chaperones such as Hsp60. We investigated patterns of anti-oxidants, biomarkers of oxidative stress, and Hsp60 levels in sera from newborns and found significant associations between glutathione (GSH) levels and gestational age, delivery modality, and lipid hydroperoxydes (LOOH) level. LOOH levels and spontaneous (vaginal) delivery were independently associated with increased GSH levels when these were above the median. Hsp60 and LOOH levels were positively correlated whereas Hsp60 and GSH levels were inversely correlated in spontaneously delivered newborns; in contrast, Hsp60 and GSH levels were positively correlated in newborns delivered by cesarea. Our results point to new directions in the search for definite patterns of GSH, LOOH, and Hsp60 in the newborn's serum that might have functional and diagnostic significance and that could help in the monitoring of newborn health during and after delivery. In addition, the data provide a starting basis for investigating the precise roles and interplay of GSH and Hsp60 in the maintenance of an optimal redox balance at birth to cope with the stress inherent to delivery, and also for investigating the predictive value of any given pattern of GSH, LOOH, and Hsp60 at birth with regard to health status and risk of disease in adult life. Copyright © 2015 Elsevier GmbH. All rights reserved.

Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2*

PubMed Central

Brechbuhl, Heather M.; Gould, Neal; Kachadourian, Remy; Riekhof, Wayne R.; Voelker, Dennis R.; Day, Brian J.

2010-01-01

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2′,5′-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2′,5′-dihydroxychalcone (2′,5′-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 μm), indicating competitive inhibition. In contrast, 2′,5′-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter. PMID:20332504

Glutathione transport is a unique function of the ATP-binding cassette protein ABCG2.

PubMed

Brechbuhl, Heather M; Gould, Neal; Kachadourian, Remy; Riekhof, Wayne R; Voelker, Dennis R; Day, Brian J

2010-05-28

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2',5'-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2',5'-dihydroxychalcone (2',5'-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 microM), indicating competitive inhibition. In contrast, 2',5'-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.

Neuroprotective Activities of Saffron and Crocin.

PubMed

Soeda, Shinji; Aritake, Kosuke; Urade, Yoshihiro; Sato, Hiroshi; Shoyama, Yukihiro

2016-01-01

We first considered that saffron is really safety food because it has a long-use history. The neuroprotective activities of saffron and its major constituent, crocin, are separately discussed in vitro and in vivo. We reviewed the inhibitory activities of crocin against PC-12 cell apoptosis. The oxidative stress decreased the cellular levels of glutathione (GSH) which is an inhibitor of neutral sphingomyelinase (N-SMase). Therefore, the level of GSH was assayed by the addition of crocin resulted in the activation of glutathione reductase (GR). It became evident that crocin treatment prevents the N-SMase activation resulting in the decrease of ceramide release. From these evidences we summarized the role of crocin for neuronal cell death. We used the ethanol-blocking assay system for learning and memory activities. The effect of saffron and crocin on improving ethanol-induced impairment of learning behaviors of mice in passive avoidance tasks has been clear. Further, we did make clear that saffron and crocin prevent the inhibitory effect of ethanol on long-term potentiation (LTP) in the dentate gyrus. Finally we found that 100 mg/kg of crocin gave non-rapid eye movement sleep (non-REM sleep) although mice were started to be active during night time.

Mitochondrial GSH replenishment as a potential therapeutic approach for Niemann Pick type C disease.

PubMed

Torres, Sandra; Matías, Nuria; Baulies, Anna; Nuñez, Susana; Alarcon-Vila, Cristina; Martinez, Laura; Nuño, Natalia; Fernandez, Anna; Caballeria, Joan; Levade, Thierry; Gonzalez-Franquesa, Alba; Garcia-Rovés, Pablo; Balboa, Elisa; Zanlungo, Silvana; Fabrías, Gemma; Casas, Josefina; Enrich, Carlos; Garcia-Ruiz, Carmen; Fernández-Checa, José C

2017-04-01

Niemann Pick type C (NPC) disease is a progressive lysosomal storage disorder caused by mutations in genes encoding NPC1/NPC2 proteins, characterized by neurological defects, hepatosplenomegaly and premature death. While the primary biochemical feature of NPC disease is the intracellular accumulation of cholesterol and gangliosides, predominantly in endolysosomes, mitochondrial cholesterol accumulation has also been reported. As accumulation of cholesterol in mitochondria is known to impair the transport of GSH into mitochondria, resulting in mitochondrial GSH (mGSH) depletion, we investigated the impact of mGSH recovery in NPC disease. We show that GSH ethyl ester (GSH-EE), but not N-acetylcysteine (NAC), restored the mGSH pool in liver and brain of Npc1 -/- mice and in fibroblasts from NPC patients, while both GSH-EE and NAC increased total GSH levels. GSH-EE but not NAC increased the median survival and maximal life span of Npc1 -/- mice. Moreover, intraperitoneal therapy with GSH-EE protected against oxidative stress and oxidant-induced cell death, restored calbindin levels in cerebellar Purkinje cells and reversed locomotor impairment in Npc1 -/- mice. High-resolution respirometry analyses revealed that GSH-EE improved oxidative phosphorylation, coupled respiration and maximal electron transfer in cerebellum of Npc1 -/- mice. Lipidomic analyses showed that GSH-EE treatment had not effect in the profile of most sphingolipids in liver and brain, except for some particular species in brain of Npc1 -/- mice. These findings indicate that the specific replenishment of mGSH may be a potential promising therapy for NPC disease, worth exploring alone or in combination with other options. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

PubMed

Kim, Min-Hyun; Kim, Aryung; Yu, Ji Hoon; Lim, Joo Weon; Kim, Hyeyoung

2014-05-01

To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T). We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA. The cells were cultured with or without glutamine or GSH. ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting. While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and cultured without glutamine showed higher levels of ROS and IL-8 than those transfected with negative control siRNA; increased levels of ROS and IL-8 were suppressed by the treatment of glutamine. Glutamine deprivation induces ROS production, NF-κB activation, and IL-8 expression as well as a reduction in GSH in A-T fibroblasts, all of which are attenuated by glutamine supplementation.

Changes in the antioxidant metabolism in the embryonic development of the common South American toad Bufo arenarum: differential responses to pesticide in early embryos and autonomous-feeding larvae.

PubMed

Ferrari, Ana; Anguiano, Liliana; Lascano, Cecilia; Sotomayor, Verónica; Rosenbaum, Enrique; Venturino, Andrés

2008-01-01

Amphibians may be critically challenged by aquatic contaminants during their embryonic development. Many classes of compounds, including organophosphorus pesticides, are able to cause oxidative stress that affects the delicate cellular redox balance regulating tissue modeling. We determined the progression of antioxidant defenses during the embryonic development of the South American common toad, Bufo arenarum. Superoxide dismutase (SOD) and catalase (CAT) activities were high in the unfertilized eggs, and remained constant during the first stages of development. SOD showed a significant increase when the gills were completely active and opercular folds began to form. Reductase (GR) activity was low in the oocytes and increased significantly when gills and mouth were entirely developed and the embryos presented a higher exposure to pro-oxidant conditions suggesting an environmental control. Reduced glutathione (GSH) content was also initially low, and rose continuously pointing out an increasing participation of GSH-related enzymes in the control of oxidative stress. GSH peroxidases and GSH-S-transferases showed relatively high and constant activities, probably related to lipid peroxide control. B. arenarum embryos have plenty of yolk platelets containing lipids, which provide the energy and are actively transferred to the newly synthesized membranes during the early embryonic development. Exposure to the pro-oxidant pesticide malathion during 48 h did not significantly affect the activity of antioxidant enzymes in early embryos, but decreased the activities of CAT, GR, and the pool of GSH in larvae. Previous work indicated that lipid peroxide levels were kept low in malathion-exposed larvae, thus we conclude that oxidative stress is overcome by the antioxidant defenses. The increase in the antioxidant metabolism observed in the posthatching phase of development of B. arenarum embryo, thus constitutes a defense against natural and human-generated pro-oxidants present in the aquatic environment.

Derivatives of xanthic acid are novel antioxidants: application to synaptosomes.

PubMed

Lauderback, Christopher M; Drake, Jennifer; Zhou, Daohong; Hackett, Janna M; Castegna, Alessandra; Kanski, Jaroslaw; Tsoras, Maria; Varadarajan, Sridhar; Butterfield, D Allan

2003-04-01

Xanthic acids have long been known to act as reducing agents. Recently, D609, a tricyclodecanol derivative of xanthic acid, has been reported to have anti-apoptotic and anti-inflammatory properties that are attributed to specific inhibition of phosphatidyl choline phospholipase C (PC-PLC). However, because oxidative stress is involved in both of these cellular responses, the possibility that xanthates may act as antioxidants was investigated in the current study. Finding that xanthates efficiently scavenge hydroxyl radicals, the mechanism by which D609 and other xanthate derivatives may protect against oxidative damage was further examined. The xanthates studied, especially D609, mimic glutathione (GSH). Xanthates scavenge hydroxyl radicals and hydrogen peroxide, form disulfide bonds (dixanthogens), and react with electrophilic products of lipid oxidation (acrolein) in a manner similar to GSH. Further, upon disulfide formation, dixanthogens are reduced by glutathione reductase to a redox active xanthate. Supporting its role as an antioxidant, D609 significantly (p < 0.01) reduces free radical-induced changes in synaptosomal lipid peroxidation (TBARs), protein oxidation (protein carbonyls), and protein conformation. Thus, in addition to inhibitory effects on PC-PLC, D609 may prevent cellular apoptotic and inflammatory cascades by acting as antioxidants and novel GSH mimics. These results are discussed with reference to potential therapeutic application of D609 in oxidative stress conditions.

MRP proteins as potential mediators of heavy metal resistance in zebrafish cells.

PubMed

Long, Yong; Li, Qing; Wang, Youhui; Cui, Zongbin

2011-04-01

Acquired resistance of mammalian cells to heavy metals is closely relevant to enhanced expression of several multidrug resistance-associated proteins (MRP), but it remains unclear whether MRP proteins confer resistance to heavy metals in zebrafish. In this study, we obtained zebrafish (Danio rerio) fibroblast-like ZF4 cells with resistance to toxic heavy metals after chronic cadmium exposure and selection for 6months. These cadmium-resistant cells (ZF4-Cd) were maintained in 5μM cadmium and displayed cross-resistance to cadmium, mercury, arsenite and arsenate. ZF4-Cd cells remained the resistance to heavy metals after protracted culture in cadmium-free medium. In comparison with ZF4-WT cells, ZF4-Cd cells exhibited accelerated rate of cadmium excretion, enhanced activity of MRP-like transport, elevated expression of abcc2, abcc4 and mt2 genes, and increased content of cellular GSH. Inhibition of MRP-like transport activity, GSH biosynthesis and GST activity significantly attenuated the resistance of ZF4-Cd cells to heavy metals. The results indicate that some of MRP transporters are involved in the efflux of heavy metals conjugated with cellular GSH and thus play crucial roles in heavy metal detoxification of zebrafish cells. Copyright © 2010 Elsevier Inc. All rights reserved.

Protective effects of anethole dithiolethione against oxidative stress-induced cytotoxicity in human Jurkat T cells.

PubMed

Khanna, S; Sen, C K; Roy, S; Christen, M O; Packer, L

1998-07-01

The protective effects of anethole dithiolethione (ADT) against H2O2- or 4-hydroxynonenal (HNE)-induced cytotoxicity in human Jurkat T cells were investigated. Jurkat T cells were pretreated with ADT (10-50 microM) for 18 hr and then challenged with H202 or HNE for up to 4 hr. Cytotoxicity was assessed by measuring: 1) leakage of lactate dehydrogenase from cells to medium; and 2) exclusion of the DNA intercalating fluorescent probe propidium iodide by viable cells. Pretreatment of cells with ADT (10 or 25 microM) for 18 hr significantly protected cells against H202- or HNE-induced cytotoxicity. Treatment of cells with ADT (10-50 microM) for 72 hr significantly increased the activities of catalase and glutathione reductase. The maximum effect of ADT treatment on the activity of these enzymes was observed when cells were treated with 25 microM of ADT for 72 hr. A significant increase in cellular GSH was observed in cells that were treated with ADT for 72 hr. Using monobromobimane as a thiol probe, we consistently observed that cells pretreated for 18 hr with ADT (25 or 50 microM) had also increased total thiol content. Exposure of Jurkat T cells to H202 or HNE resulted in a time-dependent decrease in cellular GSH. ADT (10-50 microM, 18 hr) pretreatment circumvented H202-dependent lowering of cellular GSH. In conclusion, ADT proved to be a potent cytoprotective thiol antioxidant with multifaceted mechanisms of action, suggesting that the drug has a remarkable therapeutic potential.

Oxidative Stress Markers Correlate with Renal Dysfunction and Thrombocytopenia in Severe Leptospirosis

PubMed Central

Araújo, Alan M.; Reis, Eliana A. G.; Athanazio, Daniel A.; Ribeiro, Guilherme S.; Hagan, José E.; Araujo, Guilherme C.; Damião, Alcineia O.; Couto, Nicolli S.; Ko, Albert I.; Noronha-Dutra, Alberto; Reis, Mitermayer G.

2014-01-01

Leptospirosis is a zoonotic disease that causes severe manifestations such as Weil's disease and pulmonary hemorrhage syndrome. The aim of this study was to evaluate whether reactive oxygen species (ROS) production and antioxidant reduced glutathione (GSH) levels are related to complications in patients hospitalized with leptospirosis. The ROS production and GSH levels were measured in blood samples of 12 patients and nine healthy controls using chemiluminescence and absorbance assays. We found that ROS production was higher and GSH levels were lower in leptospirosis patients compared with healthy individuals. Among patients, GSH depletion was correlated with thrombocytopenia and elevated serum creatinine, whereas a strong positive correlation was observed between ROS production and elevated serum potassium. Additional investigation of the biological significance of ROS production and GSH levels is warranted as they may guide the development of novel adjuvant therapies for leptospirosis targeting oxidative stress. PMID:24493675

Formation of hydrogen peroxide in cell culture media by apple polyphenols and its effect on antioxidant biomarkers in the colon cell line HT-29.

PubMed

Bellion, Phillip; Olk, Melanie; Will, Frank; Dietrich, Helmut; Baum, Matthias; Eisenbrand, Gerhard; Janzowski, Christine

2009-10-01

Beneficial health effects of diets containing fruits have partly been attributed to polyphenols which display a spectrum of bioactive effects, including antioxidant activity. However, polyphenols can also exert prooxidative effects in vitro. In this study, polyphenol-mediated hydrogen peroxide (H(2)O(2)) formation was determined after incubation of apple juice extracts (AEs) and polyphenols in cell culture media. Effects of extracellular H(2)O(2 )on total glutathione (tGSH; =GSH + GSSG) and cellular reactive oxygen species (ROS) level of HT-29 cells were studied by coincubation +/- catalase (CAT). AEs ( > or =30 microg/mL) significantly generated H(2)O(2) in DMEM, depending on their composition. Similarly, H(2)O(2) was measured for individual apple polyphenols/degradation products (phenolic acids > epicatechin, flavonols > dihydrochalcones). Highest concentrations were generated by compounds bearing the o-catechol moiety. H(2)O(2) formation was found to be pH dependent; addition of CAT caused a complete decomposition of H(2)O(2) whereas superoxide dismutase was less/not effective. At incubation of HT-29 cells with quercetin (1-100 microM), generated H(2)O(2) slightly contributed to antioxidant cell protection by modulation of tGSH- and ROS-level. In conclusion, H(2)O(2) generation in vitro by polyphenols has to be taken into consideration when interpreting results of such cell culture experiments. Unphysiologically high polyphenol concentrations, favoring substantial H(2)O(2 )formation, are not expected to be met in vivo, even under conditions of high end nutritional uptake.

Demethylation of neferine in human liver microsomes and formation of quinone methide metabolites mediated by CYP3A4 accentuates its cytotoxicity.

PubMed

Shen, Qi; Zuo, Minjuan; Ma, Li; Tian, Ye; Wang, Lu; Jiang, Huidi; Zhou, Quan; Zhou, Hui; Yu, Lushan; Zeng, Su

2014-12-05

Neferine is a bisbenzylisoquinoline alkaloid isolated from the seed embryos of Nelumbonucifera Gaertn (Lotus) with various potent pharmacological effects. Recently, neferine has attracted attention for its anti-tumor activities. Our study explored its metabolism and cytotoxicity mechanism. Approaches using chemical inhibitors and recombinant human enzymes to characterize the involved enzymes and kinetic studies indicated that the demethylation of neferine by cytochrome P450 (CYP) 2D6 and CYP3A4 fitted a biphasic kinetic profile. Glutathione (GSH) was used as a trapping agent to identify reactive metabolites of neferine, and four novel GSH conjugates were detected with [M+H](+) ions at m/z 902.4, 916.2, 916.1, and 930.4. Based on its structure containing para-methylene phenol and results from a product ion scan, GSH tends to conjugate with C9' after undergoing oxidative metabolism to form the binding site predominated by CYP3A4. Furthermore, the addition of recombinant human GSTA1, GSTT1, and GSTP1 had little effect on the production of the GSH conjugates. In a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay, combined with the GSH modulators l-buthionine sulfoximine or N-acetyl-l-cysteine, neferine treatment of MDCK-hCYP3A4 and HepG2 cells revealed that CYP3A4 expression and cellular GSH content could cause an EC50 shift. Metabolic activation mediated by CYP3A4 and GSH depletion significantly enhanced neferine-induced cytotoxicity. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Effects of polysaccharide peptides from COV-1 strain of Coriolus versicolor on glutathione and glutathione-related enzymes in the mouse.

PubMed

Yeung, John H K; Or, Penelope M Y

2007-06-01

The effects of polysaccharide peptide (PSP), an immunomodulator isolated from Coriolus versicolor COV-1, on glutathione (GSH) and GSH-related enzymes was investigated in C57 mouse. Administration of PSP (1-4 micromole/kg, i.p.) produced a transient, dose-dependent depletion (10-37%) of hepatic GSH, with no effect on serum glutamic-pyruvic transaminase (SGPT) activity. Blood GSH was depleted (6-25%) at 3 h, followed by a rebound increase above the control GSH level (20%) at 18 h. The GSSG/GSH ratio, a measure of oxidative stress, was increased 3 h after PSP treatment but returned to normal levels at 24 h. Sub-chronic treatment of PSP (1-4 micromole/kg/day, i.p.) for seven days did not produce any significant changes in hepatic GSH levels and the GSSG/GSH ratio when measured 24 h after the final dose of PSP. PSP had little effect on glutathione transferase (GST), glutathione reductase (GSSG reductase) and glutathione peroxidase (GPX) activities in the liver. However, a dose-dependent increase in blood GPX activity (30-48%) was observed at 3h, which coincided with the increase in the GSSG/GSH ratio. The increase in blood GPX activity may be a responsive measure to deal with the transient oxidative stress induced by PSP treatment. The results showed that PSP only caused a transient perturbation on hepatic glutathione without affecting the GSH-related enzymes such as GST, GSSG reductase and GPX. The observed changes in blood GSH simply reflected the intra-organ translocation of glutathione, as the glutathione-related enzymes were not significantly affected by PSP treatment.

Enhanced reactive oxygen species overexpression by CuO nanoparticles in poorly differentiated hepatocellular carcinoma cells

NASA Astrophysics Data System (ADS)

Kung, Mei-Lang; Hsieh, Shu-Ling; Wu, Chih-Chung; Chu, Tian-Huei; Lin, Yu-Chun; Yeh, Bi-Wen; Hsieh, Shuchen

2015-01-01

Copper oxide nanoparticles (CuO NPs) are known to exhibit toxic effects on a variety of cell types and organs. To determine the oxidative impact of CuO NPs on hepatocellular carcinoma (HCC) cells, well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) cells were exposed to CuO NPs. Cell viability assay showed that the median inhibition concentration (IC50) for SK-Hep-1 and HepG2 cells was 25 μg ml-1 and 85 μg ml-1, respectively. Cellular fluorescence intensity using DCFH-DA staining analysis revealed significant intracellular reactive oxygen species (ROS) generation of up to 242% in SK-Hep-1 cells, compared with 86% in HepG2 cells. HPLC analysis demonstrated that a CuO NP treatment caused cellular GSH depletion of 58% and a GSH/GSSG ratio decrease to ~0.1 in SK-Hep-1 cells. The oxidative stress caused by enhanced superoxide anion production was observed in both HepG2 (146%) and SK-Hep-1 (192%) cells. The Griess assay verified that CuO NPs induced NO production (170%) in SK-Hep-1 cells. Comet assay and western blot further demonstrated that CuO NPs induced severe DNA strand breakage (70%) in SK-Hep-1 cells and caused DNA damage via increased γ-H2AX levels. These results suggest that well-differentiated HepG2 cells possess a robust antioxidant defense system against CuO NP-induced ROS stress and exhibit more tolerance to oxidative stress. Conversely, poorly differentiated SK-Hep-1 cells exhibited a deregulated antioxidant defense system that allowed accumulation of CuO NP-induced ROS and resulted in severe cytotoxicity.Copper oxide nanoparticles (CuO NPs) are known to exhibit toxic effects on a variety of cell types and organs. To determine the oxidative impact of CuO NPs on hepatocellular carcinoma (HCC) cells, well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) cells were exposed to CuO NPs. Cell viability assay showed that the median inhibition concentration (IC50) for SK-Hep-1 and HepG2 cells was 25 μg ml-1 and 85 μg ml-1, respectively. Cellular fluorescence intensity using DCFH-DA staining analysis revealed significant intracellular reactive oxygen species (ROS) generation of up to 242% in SK-Hep-1 cells, compared with 86% in HepG2 cells. HPLC analysis demonstrated that a CuO NP treatment caused cellular GSH depletion of 58% and a GSH/GSSG ratio decrease to ~0.1 in SK-Hep-1 cells. The oxidative stress caused by enhanced superoxide anion production was observed in both HepG2 (146%) and SK-Hep-1 (192%) cells. The Griess assay verified that CuO NPs induced NO production (170%) in SK-Hep-1 cells. Comet assay and western blot further demonstrated that CuO NPs induced severe DNA strand breakage (70%) in SK-Hep-1 cells and caused DNA damage via increased γ-H2AX levels. These results suggest that well-differentiated HepG2 cells possess a robust antioxidant defense system against CuO NP-induced ROS stress and exhibit more tolerance to oxidative stress. Conversely, poorly differentiated SK-Hep-1 cells exhibited a deregulated antioxidant defense system that allowed accumulation of CuO NP-induced ROS and resulted in severe cytotoxicity. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05843g

Decreased glutathione biosynthesis contributes to EGFR T790M-driven erlotinib resistance in non-small cell lung cancer.

PubMed

Li, Hongde; Stokes, William; Chater, Emily; Roy, Rajat; de Bruin, Elza; Hu, Yili; Liu, Zhigang; Smit, Egbert F; Heynen, Guus Jje; Downward, Julian; Seckl, Michael J; Wang, Yulan; Tang, Huiru; Pardo, Olivier E

2016-01-01

Epidermal growth factor receptor (EGFR) inhibitors such as erlotinib are novel effective agents in the treatment of EGFR-driven lung cancer, but their clinical impact is often impaired by acquired drug resistance through the secondary T790M EGFR mutation. To overcome this problem, we analysed the metabonomic differences between two independent pairs of erlotinib-sensitive/resistant cells and discovered that glutathione (GSH) levels were significantly reduced in T790M EGFR cells. We also found that increasing GSH levels in erlotinib-resistant cells re-sensitised them, whereas reducing GSH levels in erlotinib-sensitive cells made them resistant. Decreased transcription of the GSH-synthesising enzymes (GCLC and GSS) due to the inhibition of NRF2 was responsible for low GSH levels in resistant cells that was directly linked to the T790M mutation. T790M EGFR clinical samples also showed decreased expression of these key enzymes; increasing intra-tumoural GSH levels with a small-molecule GST inhibitor re-sensitised resistant tumours to erlotinib in mice. Thus, we identified a new resistance pathway controlled by EGFR T790M and a therapeutic strategy to tackle this problem in the clinic.

Cellular defense against UVB-induced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase.

PubMed

Jo, Seung-Hee; Lee, So-Hyun; Chun, Hang Suk; Lee, Su Min; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

2002-03-29

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury. (c)2002 Elsevier Science (USA).

Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo

PubMed Central

Timme-Laragy, Alicia R.; Goldstone, Jared V.; Imhoff, Barry R.; Stegeman, John J.; Hahn, Mark E.; Hansen, Jason M.

2013-01-01

Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous non-protein antioxidant defense molecule is the tri-peptide glutathione (γ-glutamyl-cysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0–5 days post-fertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione (GSH, GSSG) using HPLC, and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0–120 hours of zebrafish development (including mature oocytes, fertilization, mid-blastula transition, gastrulation, somitogenesis, pharyngula, pre-hatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12 hours post fertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12 h, and then oscillated around −190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (−220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study provides a foundation for understanding the redox regulation of developmental signaling and investigating the effects of oxidative stress during embryogenesis. PMID:23770340

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conotte, R.; Colet, J.-M., E-mail: jean-marie.colet@umons.ac.be

The main curative treatment of colorectal cancer remains the surgery. However, when metastases are suspected, surgery is followed by a preventive chemotherapy using oxaliplatin which, unfortunately, may cause liver sinusoidal obstruction syndrome (SOS). Such hepatic damage is barely detected during or after chemotherapy due to a lack of effective diagnostic procedures, but liver biopsy. The primary objective of the present study was to identify potential early diagnosis biomarkers of SOS using a metabonomic approach. SOS was induced in rats by monocrotaline, a prototypical toxic substance. {sup 1}H NMR spectroscopy analysis of urine samples collected from rats treated with monocrotaline showedmore » significant metabolic changes as compared to controls. During a first phase, cellular protective mechanisms such as an increased synthesis of GSH (reduced taurine) and the recruitment of cell osmolytes in the liver (betaine) were seen. In the second phase, the disturbance of the urea cycle (increased ornithine and urea reduction) leading to the depletion of NO, the alteration in the GSH synthesis (increased creatine and GSH precursors (glutamate, dimethylglycine and sarcosine)), and the liver necrosis (decrease taurine and increase creatine) all indicate the development of SOS. - Highlights: • Urine metabonomic profiles of SOS have been identified. • Urine osmoprotectants and anti-oxidants indicated an initial liver protection. • Liver necrosis was demonstrated by increased urine levels of taurine and creatine. • NO depletion was suggested by changes in ornithine and urea.« less

Auranofin induces apoptosis and necrosis in HeLa cells via oxidative stress and glutathione depletion.

PubMed

You, Bo Ra; Shin, Hye Rim; Han, Bo Ram; Kim, Suhn Hee; Park, Woo Hyun

2015-02-01

Auranofin (Au), an inhibitor of thioredoxin reductase, is a known anti‑cancer drug. In the present study, the anti‑growth effect of Au on HeLa cervical cancer cells was examined in association with levels of reactive oxygen species (ROS) and glutathione (GSH). Au inhibited the growth of HeLa cells with an IC50 of ~2 µM at 24 h. This agent induced apoptosis and necrosis, accompanied by the cleavage of poly (ADP‑ribose) polymerase and loss of mitochondrial membrane potential. The pan‑caspase inhibitor, benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone, prevented apoptotic cell death and each of the assessed caspase inhibitors inhibited necrotic cell death induced by Au. With respect to the levels of ROS and GSH, Au increased intracellular O2•- in the HeLa cells and induced GSH depletion. The pan‑caspase inhibitor reduced the levels of O2•- and GSH depletion in Au‑treated HeLa cells. The antioxidant, N‑acetyl cysteine, not only attenuated apoptosis and necrosis in the Au‑treated HeLa cells, but also decreased the levels of O2•- and GSH depletion in the cells. By contrast, L‑buthionine sulfoximine, a GSH synthesis inhibitor, intensified cell death O2•- and GSH depletion in the Au‑treated HeLa cells. In conclusion, Au induced apoptosis and necrosis in HeLa cells via the induction of oxidative stress and the depletion of GSH.

DNA damage and glutathione level in children with asthma bronchiale: effect of antiasthmatic therapy.

PubMed

Hasbal, Canan; Aksu, Bagdagul Y; Himmetoglu, Solen; Dincer, Yildiz; Koc, Eylem E; Hatipoglu, Sami; Akcay, Tulay

2010-06-01

When the production of reactive oxygen species (ROS) exceeds the capacity of antioxidant defences, a condition known as oxidative stress occurs and it has been implicated in many pathological conditions including asthma. Interaction of ROS with DNA may result in mutagenic oxidative base modifications such as 8-hydroxydeoxyguanosine (8-oxo-dGuo) and DNA strand breaks. Reduced glutathione (GSH) serves as a powerful antioxidant against harmful effects of ROS. The aim of this study was to describe DNA damage as level of DNA strand breaks and formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites, which reflects oxidative DNA damage and GSH level in children with mild-to-moderate persistent asthma; and to examine the effect of antiasthmatic therapy on these DNA damage parameters and GSH level. Before and after 8 wk of antiasthmatic therapy blood samples were taken, DNA strand breaks and Fpg-sensitive sites in peripheral leukocytes were determined by comet assay, GSH level of whole blood was measured by spectrophotometric method. DNA strand breaks and Fpg-sensitive sites in the asthma group were found to be increased as compared with control group. GSH level in the asthma group was not significantly different from those in the control group. Levels of strand breaks, Fpg-sensitive sites and GSH were found to be decreased in the asthma group after the treatment. In conclusion, oxidative DNA damage (strand breaks and Fpg-sensitive sites) is at a high level in children with asthma. DNA damage parameters and GSH level were found to be decreased after therapy. Our findings imply that antiasthmatic therapy including glucocorticosteroids not only controls asthma but also decreases mutation risk in children with asthma bronchiale.

Phytoextraction of toxic metals: a central role for glutathione.

PubMed

Seth, C S; Remans, T; Keunen, E; Jozefczak, M; Gielen, H; Opdenakker, K; Weyens, N; Vangronsveld, J; Cuypers, A

2012-02-01

Phytoextraction has a promising potential as an environmentally friendly clean-up method for soils contaminated with toxic metals. To improve the development of efficient phytoextraction strategies, better knowledge regarding metal uptake, translocation and detoxification in planta is a prerequisite. This review highlights our current understanding on these mechanisms, and their impact on plant growth and health. Special attention is paid to the central role of glutathione (GSH) in this process. Because of the high affinity of metals to thiols and as a precursor for phytochelatins (PCs), GSH is an essential metal chelator. Being an important antioxidant, a direct link between metal detoxification and the oxidative challenge in plants growing on contaminated soils is observed, where GSH could be a key player. In addition, as redox couple, oxidized and reduced GSH transmits specific information, in this way tuning cellular signalling pathways under environmental stress conditions. Possible improvements of phytoextraction could be achieved by using transgenic plants or plant-associated microorganisms. Joined efforts should be made to cope with the challenges faced with phytoextraction in order to successfully implement this technique in the field. © 2011 Blackwell Publishing Ltd.

Chemoresponsive Colloidosomes via Ag⁺ Soldering of Surface-Assembled Nanoparticle Monolayers.

PubMed

Liu, Miao; Tian, Qian; Li, Yulin; You, Bo; Xu, An; Deng, Zhaoxiang

2015-04-28

Colloidosomes with a hollow interior and a porous plasmonic shell are highly desired for many applications including nanoreactors, surface-enhanced Raman scattering (SERS), photothermal therapy, and controlled drug release. We herein report a silica nanosphere-templated electrostatic self-assembly in conjunction with a newly developed Ag(+) soldering to fabricate gold colloidosomes toward multifunctionality and stimuli-responsibility. The gold colloidosomes are capable of capturing a nanosized object and releasing it via structural dissociation upon responding to a biochemical input (GSH, glutathione) at a concentration close to its cellular level. In addition, the colloidosomes have a tunable nanoporous shell composed of strongly coupled gold nanoparticles, which exhibit broadened near-infrared plasmon resonance. These features along with the simplicity and high tunability of the fabrication process make the gold colloidosomes quite promising for applications in a chemical or cellular environment.

Evaluation of glutathione level in gingival crevicular fluid in periodontal health, in chronic periodontitis and after nonsurgical periodontal therapy: A clinicobiochemical study

PubMed Central

Savita, A. M.; Sarun, E.; Arora, Shivli; Krishnan, Swathi

2015-01-01

Context: Periodontitis is predominantly due to exaggerated host response to pathogenic microorganisms and their products which causes an imbalance between the reactive oxygen species-antioxidant in gingival crevicular fluid (GCF). Glutathione is an important redox regulator in GCF and maintenance of stable reduced glutathione (GSH):oxidized glutathione (GSSG) ratio is essential for periodontal health. Aims: The present study was undertaken to evaluate and compare the level of glutathione and redox balance (GSH: GSSG ratio) in GCF of chronic periodontitis patients, periodontally healthy controls and also to evaluate the effect of nonsurgical periodontal therapy on the level of glutathione and redox balance during 3 months postoperative visit. Study Design: Baseline GCF samples were collected from 20 chronic periodontitis patients and 20 periodontally healthy subjects for GSH and GSSG levels estimation. Periodontitis patients were recalled 3 months postnonsurgical periodontal therapy to re-sample GCF. Materials and Methods: GSH and GSSG levels were measured by high-performance liquid chromatography. The values were statistically analyzed by Paired t-test. Results: The mean GSH and GSSG values in GCF were found to be significantly lower in periodontitis patients pre- and 3 months post-nonsurgical periodontal therapy, compared with those in the control group subjects. In addition, the successful nonsurgical therapy even though leading to a significant improvement in the GSH and GSSG levels, does not restore glutathione concentration to the levels seen in healthy subjects. Conclusion: Successful nonsurgical periodontal therapy leads to significant improvement in the redox balance (GSH: GSSG ratio) in chronic periodontitis patients. PMID:26097356

Effects of salinity on the toxicity and biotransformation of L-selenomethionine in Japanese medaka (Oryzias latipes) embryos: mechanisms of oxidative stress.

PubMed

Lavado, Ramon; Shi, Dalin; Schlenk, Daniel

2012-02-01

Previous studies in mammals have shown that organoselenium depletes the cellular antioxidant, glutathione (GSH) due to activation of organoselenides to organoselenoxides by flavin-containing monooxygenases (FMO). Since FMO tends to be induced in euryhaline fish exposed to hypersaline conditions, the developmental toxicity of salinity and organoselenium was examined in the euryhaline fish Japanese medaka (Oryzias latipes). FMO activity, GSH, and selenium concentrations in Japanese medaka embryos were measured following a 24-h exposure to 0.05 mM L-selenomethionine (SeMet) under different saline conditions: freshwater (<0.5 dS/m), 4.2, 6.7, and 16.8 dS/m. Concentrations of GSH and the hatch-out ratio of the SeMet-treated embryos decreased in a salinity dependent manner. While SeMet treatment led to accumulation within embryos, selenium concentrations were unaltered by salinity treatment. Compared to freshwater-exposed embryos, microsomes from embryos at 6.7 and 16.8 dS/m had enhanced oxidation of SeMet to the selenoxide (10- and 14.3-fold, respectively), which correlated with GSH depletion. The results show that increased SeMet oxidation by hypersaline conditions with subsequent GSH depletion may play an important role in the developmental toxicity of selenomethionine. Copyright © 2011 Elsevier B.V. All rights reserved.

Redox-Sensitive Cerium Oxide Nanoparticles Protect Human Keratinocytes from Oxidative Stress Induced by Glutathione Depletion.

PubMed

Singh, Ragini; Karakoti, Ajay S; Self, William; Seal, Sudipta; Singh, Sanjay

2016-11-22

Cerium oxide nanoparticles (CeNPs) have gathered much attention in the biomedical field due to its unique antioxidant property. It can protect cells and tissues from oxidative stress induced damage due to its autoregenerative redox cycle. Our study explores the antioxidant and antigenotoxic behavior of PEGylated CeNPs toward oxidative insult produced by buthionine sulfoximine (BSO) in human keratinocytes (HaCaT cells). BSO inhibits the γ-glutamylcysteinesynthetase (γ-GCS) enzyme and thus acts as a glutathione (GSH) depleting agent to modulate the cellular redox potential. GSH is a natural ROS scavenger present in the mammalian cells, and its depletion causes generation of reactive oxygen species (ROS). In this study, we challenged HaCaT cells (keratinocytes) with BSO to alter the redox potential within the cell and monitored toxicity, ROS generation, and nuclear fragmentation. We also followed changes in expressions of related proteins and genes. We found that PEGylated CeNPs can protect HaCaT cells from BSO-induced oxidative damage. BSO-exposed cells, preincubated with PEGylated CeNPs, showed better cell survival and significant decrease in the intracellular levels of ROS. We also observed decrease in lactate dehydrogenase (LDH) release and nuclear fragmentation in CeNP-treated cells that were challenged with BSO as compared to treatment with BSO alone. Exposure of HaCaT cells with BSO leads to altered expression of antioxidant genes and proteins, i.e., thioredoxin reductase (TrxR) and peroxiredoxin 6 (Prx6) whereas, in our study, pretreatment of PEGylated CeNPs reduces the need for induction of genes that produce enzymes involved in the defense against oxidative stress. Since, growing evidence argued the involvement of ROS in mediating death of mammalian cells in several ailments, our finding reinforces the use of PEGylated CeNPs as a potent pharmacological agent under the lower cellular GSH/GSSG ratios for the treatment of diseases mediated by free radicals.

Reactive oxygen species are crucial for hydroxychavicol toxicity toward KB epithelial cells.

PubMed

Jeng, J H; Wang, Y J; Chang, W H; Wu, H L; Li, C H; Uang, B J; Kang, J J; Lee, J J; Hahn, L J; Lin, B R; Chang, M C

2004-01-01

Betel quid (BQ) chewing shows a strong correlation to the incidence of oral submucous fibrosis (OSF), leukoplakia and oral cancer. BQ contains mainly areca nut, lime, Piper betle leaf (PBL) and the inflorescence of P. betle (IPB). Hydroxychavicol (4-allyl-catechol, HC), as a major phenolic compound in PBL and IPB, is shown to induce oxidative stress, glutathione (GSH) depletion and cell cycle deregulation. Using bivariate BrdU/PI flow cytometry, KB cells in DNA synthesis (S phase) are shown to be sensitive to the toxic effect of HC and show cell cycle arrest and apoptosis following exposure to 0.1 and 0.3 mM HC. HC-induced apoptosis and cell cycle arrest are associated with mitochondrial membrane potential (delta Psim) depolarization as revealed by a decrease in rhodamine fluorescence. N-acetyl-L-cysteine (1 mM), superoxide dismutase (100 U/ml) and catalase (1000 U/ml) were effective in prevention of HC-induced GSH depletion (as indicated by chloromethylfluorescein fluorescence), reactive oxygen species (ROS) production (by dichlorofluorescein fluorescence), cell cycle arrest and apoptosis. However, dimethylthiourea (2 mM), neocuproine (1 mM), 1,10-phenanthroline (200 microM) and desferrioxamine (0.5 mM) showed little effect on HC-induced cell changes. HC elevated the cellular and mitochondrial GSH levels at moderate concentrations (0.05-0.1 mM), whereas at a concentration of 0.3 mM, inhibitory effects were noted. These results indicate that HC consumption may be associated with BQ-chewing-related oral mucosal diseases via GSH depletion, ROS production, mitochondrial dysfunction, cell cycle disturbance and the induction of apoptosis. These events are related to the production of superoxide radicals and hydrogen peroxide.

Methylphenidate clinically oral doses improved brain and heart glutathione redox status and evoked renal and cardiac tissue injury in rats.

PubMed

Loureiro-Vieira, Sara; Costa, Vera Marisa; Duarte, José Alberto; Duarte-Araújo, Margarida; Gonçalves-Monteiro, Salomé; Maria de Lourdes, Bastos; Carvalho, Félix; Capela, João Paulo

2018-04-01

Methylphenidate (MPH) is a first-line stimulant drug to treat attention deficit hyperactivity disorder (ADHD). Overdiagnosis of ADHD and MPH abuse lead to serious concerns about the possible long-term adverse consequences of MPH in healthy children and adolescents. We aimed to evaluate MPH effects in adolescent male Wistar rats (postnatal day 40) using an oral dose scheme (2 daily MPH doses 5 mg/kg in a 5% sucrose solution, 5 h apart, for 7 days) that mimics the therapeutic doses given to human adolescents. Twenty-four hours after the last MPH administration, rats were sacrificed and brain areas [cerebellum, prefrontal cortex (PFC), hippocampus, and striatum], peripheral organs (liver, heart, and kidneys), and blood were collected for biochemical and histological analysis. MPH treatment did not alter rats' body temperature or weight, neither food or water intake throughout the experiment. The ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) significantly increased in the PFC and hippocampus of MPH-treated rats, meanwhile protein carbonylation remained unchanged in the brain. In the heart, the GSH/GSSG ratio and GSH levels were significantly increased, with decreased GSSG, while histology revealed significant damage, namely interstitial edema, vascular congestion, and presence of a fibrin-like material in the interstitial space. In the kidneys, MPH treatment resulted in extensive necrotic areas with cellular disorganization and cell infiltration, and immunohistochemistry analysis revealed a marked activation of nuclear factor-ĸB. This study showed that clinically relevant oral MPH doses improve the GSH redox status in the brain and heart, but evoke heart and kidney tissue damage to adolescent rats. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Plasma glutathione of HIV⁺ patients responded positively and differently to dietary supplementation with cysteine or glutamine.

PubMed

Borges-Santos, Maria Dorotéia; Moreto, Fernando; Pereira, Paulo Câmara Marques; Ming-Yu, Yong; Burini, Roberto Carlos

2012-07-01

Patients with positivity for the human immunodeficiency virus (HIV⁺) present low concentrations of antioxidant nutrients, including total glutathione (GSH) and its precursors. We investigated the responses of the sulfur-containing amino acid pathway to cysteine and glutamine (Gln) dietary supplements in patients with HIV⁺ compared with healthy controls. Twelve treated patients (six men and six women, 22-45 y old) and 20 healthy controls (10 men and 10 women, 20-59 y old) were randomly assigned to 7-d dietary supplements containing N-acetylcysteine (NAC; 1 g/d) or Gln (20 g/d), with a 7-d washout period ingesting their usual diet. Blood samples were drawn after an overnight fast. High-performance liquid chromatographic plasma analysis of sulfur-containing amino acids (methionine, homocysteine, cysteine, and taurine), GSH, oxidized GSH, and serine, glycine, glutamic acid, and Gln was carried out moments before and after 7-d supplementations. Statistical comparisons were undertaken between groups and between dietary supplements (P < 0.05). Patients with HIV⁺ showed higher oxidized GSH and lower concentrations of GSH and all amino acids except homocysteine. The HIV⁺ group responded to the NAC by increased levels of sulfur-containing amino acids and GSH and equalized taurine and GSH levels in the control group. The Gln supplements also equalized the levels of GSH, Gln, and glycine in the control group. An increase in GSH may be attained by NAC or Gln supplementation, with NAC acting by increasing cysteine levels and Gln likely acting by replenishing the glycine pool (trial registered at http://www.clinicaltrials.gov, identifier NCT00910442). Copyright © 2012 Elsevier Inc. All rights reserved.

Environmentally persistent free radicals amplify ultrafine particle mediated cellular oxidative stress and cytotoxicity

PubMed Central

Balakrishna, Shrilatha; Lomnicki, Slawo; McAvey, Kevin M; Cole, Richard B; Dellinger, Barry; Cormier, Stephania A

2009-01-01

Background Combustion generated particulate matter is deposited in the respiratory tract and pose a hazard to the lungs through their potential to cause oxidative stress and inflammation. We have previously shown that combustion of fuels and chlorinated hydrocarbons produce semiquinone-type radicals that are stabilized on particle surfaces (i.e. environmentally persistent free radicals; EPFRs). Because the composition and properties of actual combustion-generated particles are complex, heterogeneous in origin, and vary from day-to-day, we have chosen to use surrogate particle systems. In particular, we have chosen to use the radical of 2-monochlorophenol (MCP230) as the EPFR because we have previously shown that it forms a EPFR on Cu(II)O surfaces and catalyzes formation of PCDD/F. To understand the physicochemical properties responsible for the adverse pulmonary effects of combustion by-products, we have exposed human bronchial epithelial cells (BEAS-2B) to MCP230 or the CuO/silica substrate. Our general hypothesis was that the EPFR-containing particle would have greater toxicity than the substrate species. Results Exposure of BEAS-2B cells to our combustion generated particle systems significantly increased reactive oxygen species (ROS) generation and decreased cellular antioxidants resulting in cell death. Resveratrol treatment reversed the decline in cellular glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels for both types of combustion-generated particle systems. Conclusion The enhanced cytotoxicity upon exposure to MCP230 correlated with its ability to generate more cellular oxidative stress and concurrently reduce the antioxidant defenses of the epithelial cells (i.e. reduced GSH, SOD activity, and GPx). The EPFRs in MCP230 also seem to be of greater biological concern due to their ability to induce lipid peroxidation. These results are consistent with the oxidizing nature of the CuO/silica ultrafine particles and the reducing nature and prolonged environmental and biological lifetimes of the EPFRs in MCP230. PMID:19374750

Glutathione and abscisic acid supplementation influences somatic embryo maturation and hormone endogenous levels during somatic embryogenesis in Podocarpus lambertii Klotzsch ex Endl.

PubMed

Fraga, Hugo Pacheco de Freitas; Vieira, Leila do Nascimento; Puttkammer, Catarina Corrêa; Dos Santos, Henrique Pessoa; Garighan, Julio de Andrade; Guerra, Miguel Pedro

2016-12-01

Here we propose a protocol for embryogenic cultures induction, proliferation and maturation for the Brazilian conifer Podocarpus lambertii, and investigated the effect of abscisic acid (ABA) and glutathione (GSH) supplementation on the maturation phase. ABA, zeatin (Z) and salicylic acid (SA) endogenous levels were quantified. Number of somatic embryos obtained in ABA-supplemented treatment was significant higher than in ABA-free treatment, showing the relevance of ABA supplementation during somatic embryos maturation. Histological analysis showed the stereotyped sequence of developmental stages in conifer somatic embryos, reaching the late torpedo-staged embryo. GSH supplementation in maturation culture medium improved the somatic embryos number and morphological features. GSH 0mM and GSH 0.1mM treatments correlated with a decreased ABA endogenous level during maturation, while GSH 0.5mM treatment showed constant levels. All treatments resulted in decreased Z endogenous levels, supporting the concept that cytokinins are important during the initial cell division but not for the later stages of embryo development. The lowest SA levels found in GSH 0.5mM treatment were coincident with early embryonic development, and this treatment resulted in the highest development of somatic embryos. Thus, a correlation between lower SA levels and improved somatic embryo formation can be hypothesized. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Bcl-2 is a novel interacting partner for the 2-oxoglutarate carrier and a key regulator of mitochondrial glutathione

PubMed Central

Wilkins, Heather M.; Marquardt, Kristin; Lash, Lawrence H.; Linseman, Daniel A.

2011-01-01

Despite making up only a minor fraction of the total cellular glutathione, recent studies indicate that the mitochondrial glutathione pool is essential for cell survival. Selective depletion of mitochondrial glutathione is sufficient to sensitize cells to mitochondrial oxidative stress (MOS)1 and intrinsic apoptosis. Glutathione is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. Therefore, regulation of mitochondrial glutathione transport is a key factor in maintaining the antioxidant status of mitochondria. Bcl-2 is resident in the outer mitochondrial membrane where it acts as a central regulator of the intrinsic apoptotic cascade. In addition, Bcl-2 displays an antioxidant-like function that has been linked experimentally to the regulation of cellular glutathione content. We have previously demonstrated a novel interaction between recombinant Bcl-2 and reduced glutathione (GSH) which was antagonized by either Bcl-2 homology-3 domain (BH3) mimetics or a BH3-only protein, recombinant Bim. These previous findings prompted us to investigate if this novel Bcl-2/GSH interaction might play a role in regulating mitochondrial glutathione transport. Incubation of primary cultures of cerebellar granule neurons (CGNs) with the BH3 mimetic, HA14-1, induced MOS and caused specific depletion of the mitochondrial glutathione pool. Bcl-2 was co-immunoprecipitated with GSH following chemical cross-linking in CGNs and this Bcl-2/GSH interaction was antagonized by pre-incubation with HA14-1. Moreover, both HA14-1 and recombinant Bim inhibited GSH transport into isolated rat brain mitochondria. To further investigate a possible link between Bcl-2 function and mitochondrial glutathione transport, we next examined if Bcl-2 associated with the 2-oxoglutarate carrier (OGC), an inner mitochondrial membrane protein known to transport glutathione in liver and kidney. Following co-transfection of CHO cells, Bcl-2 was co-immunoprecipitated with OGC and this novel interaction was significantly enhanced by glutathione monoethylester (GSH-MEE). Similarly, recombinant Bcl-2 interacted with recombinant OGC in the presence of GSH. Bcl-2 and OGC co-transfection in CHO cells significantly increased the mitochondrial glutathione pool. Finally, the ability of Bcl-2 to protect CHO cells from apoptosis induced by hydrogen peroxide was significantly attenuated by the OGC inhibitor phenylsuccinate. These data suggest that GSH binding by Bcl-2 enhances its affinity for the OGC. Bcl-2 and OGC appear to act in a coordinated manner to increase the mitochondrial glutathione pool and enhance resistance of cells to oxidative stress. We conclude that regulation of mitochondrial glutathione transport is a principal mechanism by which Bcl-2 suppresses MOS. PMID:22115789

Antioxidant status in delayed healing type of wounds

PubMed Central

Rasik, Anamika M; Shukla, Arti

2000-01-01

This investigation studied the contribution of antioxidants in delaying healing in excision cutaneous wounds (8 mm) in diabetic, aged and immunocompromised animals. Skin levels of catalase, glutathione (GSH), ascorbic acid (AA) and vitamin E in streptozotocin-induced diabetic rat were lower as compared to nondiabetics. The 7-d wound tissue of diabetic rats showed an increased vitamin E level along with depleted GSH content. In aged rats (18 months old), higher levels of skin superoxide dismutase (SOD), glutathione peroxidase (Gpx) and thiobarbituric acid reactive substances (TBARS) and lower levels of catalase and GSH were found as compared to their values in young rats (3–4 months old). The levels of SOD, GPx, catalase, AA, GSH and vitamin E in 7-d wound tissue of aged rats were significantly lower in comparison to those in young rats. However, TBARS were elevated in these wound tissues. The non-wounded skin of immunocompromised (athymic) mice showed lower levels of SOD, catalase, and TBARS and higher GSH and GPx levels in comparison to those present in normal mouse skin. Surprisingly, the analysis of 7-d wound tissue showed higher levels of SOD, catalase, GPx, and GSH and lower TBARS level in athymic mice compared to the wound tissue of normal mice. Thus low levels of antioxidants accompanied by raised levels of markers of free radical damage play a significant role in delaying wound healing in aged rats. In diabetic rats reduced glutathione levels may have a contributory role in delaying the healing process. However, in immunocompromised mice the antioxidant status following injury showed an adapted response. PMID:10971747

Enhanced GSH synthesis by Bisphenol A exposure promoted DNA methylation process in the testes of adult rare minnow Gobiocypris rarus.

PubMed

Yuan, Cong; Zhang, Yingying; Liu, Yan; Zhang, Ting; Wang, Zaizhao

2016-09-01

DNA methylation is a commonly studied epigenetic modification. The mechanism of BPA on DNA methylation is poorly understood. The present study aims to explore whether GSH synthesis affects DNA methylation in the testes of adult male rare minnow Gobiocypris rarus in response to Bisphenol A (BPA). Male G. rarus was exposed to 1, 15 and 225μgL(-1) BPA for 7 days. The levels of global DNA methylation, hydrogen peroxide (H2O2) and glutathione (GSH) in the testes were analyzed. Meanwhile, the levels of enzymes involved in DNA methylation and de novo GSH synthesis, and the substrate contents for GSH production were measured. Furthermore, gene expression profiles of the corresponding genes of all studied enzymes were analyzed. Results indicated that BPA at 15 and 225μgL(-1) caused hypermethylation of global DNA in the testes. The 15μgL(-1) BPA resulted in significant decrease of ten-eleven translocation proteins (TETs) while 225μgL(-1) BPA caused significant increase of DNA methyltransferase proteins (DNMTs). Moreover, 225μgL(-1) BPA caused significant increase of H2O2 and GSH levels, and the de novo GSH synthesis was enhanced. These results indicated that the significant decrease of the level of TETs may be sufficient to cause the DNA hypermethylation by 15μgL(-1) BPA. However, the significantly increased of DNMTs contributed to the significant increase of DNA methylation levels by 225μgL(-1) BPA. Moreover, the elevated de novo GSH synthesis may promote the DNA methylation process. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of Electromagnetic Radiation Use on Oxidant/Antioxidant Status and DNA Turn-over Enzyme Activities in Erythrocytes and Heart, Kidney, Liver, and Ovary Tissues From Rats: Possible Protective Role of Vitamin C.

PubMed

Devrim, Erdinç; Ergüder, Imge B; Kılıçoğlu, Bülent; Yaykaşlı, Emine; Cetin, Recep; Durak, Ilker

2008-01-01

ABSTRACT In this study, the aim was to investigate possible effects of Electromagnetic Radiation (EMR) use on oxidant and antioxidant status in erythrocytes and kidney, heart, liver, and ovary tissues from rats, and possible protective role of vitamin C. For this aim, 40 Wistar albino female rats were used throughout the study. The treatment group was exposed to EMR in a frequency of 900 MHz, the EMR plus vitamin C group was exposed to the same EMR frequency and given vitamin C (250 mg/kg/day) orally for 4 weeks. There were 10 animals in each group including control and vitamin C groups. At the end of the study period, blood samples were obtained from the animals to get erythrocyte sediments. Then the animals were sacrificed and heart, kidney, liver, and ovary tissues were removed. Malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), xanthine oxidase (XO), and adenosine deaminase (ADA) enzyme activities were measured in the tissues and erythrocytes. It was observed that MDA level, XO, and GSH-Px activities significantly increased in the EMR group as compared with those of the control group in the erythrocytes. In the kidney tissues, it was found that MDA level and CAT activity significantly increased, whereas XO and ADA activities decreased in the cellular phone group as compared with those of the control group. However, in the heart tissues it was observed that MDA level, ADA, and XO activities significantly decreased in the cellular phone group as compared with those of the control group. The results suggest that EMR at the frequency generated by a cell phone causes oxidative stress and peroxidation in the erythrocytes and kidney tissues from rats. In the erythrocytes, vitamin C seems to make partial protection against the oxidant stress.

Cell phone use and parotid salivary gland alterations: no molecular evidence.

PubMed

de Souza, Fabrício T A; Correia-Silva, Jeane F; Ferreira, Efigênia F; Siqueira, Elisa C; Duarte, Alessandra P; Gomez, Marcus Vinícius; Gomez, Ricardo S; Gomes, Carolina C

2014-07-01

The association between cell phone use and the development of parotid tumors is controversial. Because there is unequivocal evidence that the microenvironment is important for tumor formation, we investigated in the parotid glands whether cell phone use alters the expression of gene products related to cellular stress. We used the saliva produced by the parotid glands of 62 individuals to assess molecular alterations compatible with cellular stress, comparing the saliva from the gland exposed to cell phone radiation (ipsilateral) to the saliva from the opposite, unexposed parotid gland (contralateral) of each individual. We compared salivary flow, total protein concentration, p53, p21, reactive oxygen species (ROS), and salivary levels of glutathione (GSH), heat shock proteins 27 and 70, and IgA between the ipsilateral and contralateral parotids. No difference was found for any of these parameters, even when grouping individuals by period of cell phone use in years or by monthly average calls in minutes. We provide molecular evidence that the exposure of parotid glands to cell phone use does not alter parotid salivary flow, protein concentration, or levels of proteins of genes that are directly or indirectly affected by heat-induced cellular stress. ©2014 American Association for Cancer Research.

Inhibitory Effect of Glutathione on Oxidative Liver Injury Induced by Dengue Virus Serotype 2 Infections in Mice

PubMed Central

Wang, Juan; Chen, Yanlei; Gao, Na; Wang, Yisong; Tian, Yanping; Wu, Jiangman; Zhang, Junlei; Zhu, Junping; Fan, Dongying; An, Jing

2013-01-01

The pathogenesis of dengue virus (DV) infection has not been completely defined and change of redox status mediated by depletion of glutathione (GSH) in host cell is a common result of viral infection. Our previous study has demonstrated that DV serotype 2 (DV2) infection alters host intracellular GSH levels, and exogenous GSH inhibits viral production by modulating the activity of NF-κB in HepG2 cells. GSH is the most powerful intracellular antioxidant and involved in viral infections. Thus, this study was to investigate whether DV2 infection can induce alteration in redox balance and effect of GSH on the disease in HepG2 xenografts SCID mice. Our results revealed that mice infected with DV2 showed alterations in oxidative stress by increasing the level of malondialdehyde (MDA), an end product of lipid peroxidation, and GSSG/GSH ratio. DV2-infected mice also showed a decrease in the activity of catalase (CAT) and total superoxide dismutase (T-SOD) in the serum and/or observed organs, especially the liver. Moreover, DV2 infection resulted in elevated serum levels of the cytokines tumor necrosis factor-α and interlukin-6 and obvious histopathological changes in the liver. The administration of exogenous GSH significantly reversed all of the aforementioned pathological changes and prevented significant liver damage. Furthermore, in vitro treatment of HepG2 cells with antioxidants such as GSH inhibited viral entry as well as the production of reactive oxygen species in HepG2 cells. These results suggest that GSH prevents DV2-induced oxidative stress and liver injury in mice by inhibiting proinflammatory cytokine production, and GSH and may be a promising therapeutic agent for prevention of oxidative liver damage during DV infection. PMID:23383181

Depletion by styrene of glutathione in plasma and bronchioalveolar lavage fluid of non-Swiss albino (NSA) mice.

PubMed

Carlson, Gary P

2010-01-01

Styrene is a widely used chemical, but it is known to produce lung and liver damage in mice. This may be related to oxidative stress associated with the decrease in the levels of reduced glutathione (GSH) in the target tissues. The purpose of this study was to investigate the effect of styrene and its primary metabolites R-styrene oxide (R-SO) and S-styrene oxide (S-SO) on GSH levels in the lung lumen, as determined by amounts of GSH in bronchioalveolar lavage fluid (BALF) and in plasma. When non-Swiss albino (NSA) mice were administered styrene (600 mg/kg, ip), there was a significant fall in GSH levels in both BALF and plasma within 3 h. These returned to control levels by 12 h. The active metabolite R-SO (300 mg/kg, ip) also produced significant decreases in GSH in both BALF and plasma, but S-SO was without marked effect. Since GSH is a principal antioxidant in the lung epithelial lining fluid, this fall due to styrene may exert a significant influence on the ability of the lung to buffer oxidative damage.

Effects of S-Adenosylmethionine and Its Combinations With Taurine and/or Betaine on Glutathione Homeostasis in Ethanol-induced Acute Hepatotoxicity

PubMed Central

Lee, Seo Yeon; Ko, Kwang Suk

2016-01-01

Background Exposure to ethanol abuse and severe oxidative stress are risk factors for hepatocarcinoma. The aim of this study was to evaluate the effects of S-adenosylmethionine (SAMe) and its combinations with taurine and/or betaine on the level of glutathione (GSH), a powerful antioxidant in the liver, in acute hepatotoxicity induced by ethanol. Methods To examine the effects of SAMe and its combinations with taurine and/or betaine on ethanol-induced hepatotoxicity, AML12 cells and C57BL/6 mice were pretreated with SAMe, taurine, and/or betaine, followed by ethanol challenge. Cell viability was detected with an MTT assay. GSH concentration and mRNA levels of GSH synthetic enzymes were measured using GSH reductase and quantitative real-time reverse transcriptase-PCR. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured with commercially available kits. Results Pretreatment of SAMe, with or without taurine and/or betaine, attenuated decreases in GSH levels and mRNA expression of the catalytic subunit of glutamate-cysteine ligase (GCL), the rate-limiting enzyme for GSH synthesis, in ethanol-treated cells and mice. mRNA levels of the modifier subunit of GCL and glutathione synthetase were increased in mice treated with SAMe combinations. SAMe, taurine, and/or betaine pretreatment restored serum ALT and AST levels to control levels in the ethanol-treated group. Conclusions Combinations of SAMe with taurine and/or betaine have a hepatoprotective effect against ethanol-induced liver injury by maintaining GSH homeostasis. PMID:27722142

Acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress

PubMed Central

2013-01-01

Background A variety of oncogenic and environmental factors alter tumor metabolism to serve the distinct cellular biosynthetic and bioenergetic needs present during oncogenesis. Extracellular acidosis is a common microenvironmental stress in solid tumors, but little is known about its metabolic influence, particularly when present in the absence of hypoxia. In order to characterize the extent of tumor cell metabolic adaptations to acidosis, we employed stable isotope tracers to examine how acidosis impacts glucose, glutamine, and palmitate metabolism in breast cancer cells exposed to extracellular acidosis. Results Acidosis increased both glutaminolysis and fatty acid β-oxidation, which contribute metabolic intermediates to drive the tricarboxylic acid cycle (TCA cycle) and ATP generation. Acidosis also led to a decoupling of glutaminolysis and novel glutathione (GSH) synthesis by repressing GCLC/GCLM expression. We further found that acidosis redirects glucose away from lactate production and towards the oxidative branch of the pentose phosphate pathway (PPP). These changes all serve to increase nicotinamide adenine dinucleotide phosphate (NADPH) production and counter the increase in reactive oxygen species (ROS) present under acidosis. The reduced novel GSH synthesis under acidosis may explain the increased demand for NADPH to recycle existing pools of GSH. Interestingly, acidosis also disconnected novel ribose synthesis from the oxidative PPP, seemingly to reroute PPP metabolites to the TCA cycle. Finally, we found that acidosis activates p53, which contributes to both the enhanced PPP and increased glutaminolysis, at least in part, through the induction of G6PD and GLS2 genes. Conclusions Acidosis alters the cellular metabolism of several major metabolites, which induces a significant degree of metabolic inflexibility. Cells exposed to acidosis largely rely upon mitochondrial metabolism for energy generation to the extent that metabolic intermediates are redirected away from several other critical metabolic processes, including ribose and glutathione synthesis. These alterations lead to both a decrease in cellular proliferation and increased sensitivity to ROS. Collectively, these data reveal a role for p53 in cellular metabolic reprogramming under acidosis, in order to permit increased bioenergetic capacity and ROS neutralization. Understanding the metabolic adaptations that cancer cells make under acidosis may present opportunities to generate anti-tumor therapeutic agents that are more tumor-specific. PMID:24359630

Liposomal Glutathione Supplementation Restores TH1 Cytokine Response to Mycobacterium tuberculosis Infection in HIV-Infected Individuals.

PubMed

Ly, Judy; Lagman, Minette; Saing, Tommy; Singh, Manpreet Kaur; Tudela, Enrique Vera; Morris, Devin; Anderson, Jessica; Daliva, John; Ochoa, Cesar; Patel, Nishita; Pearce, Daniel; Venketaraman, Vishwanath

2015-11-01

Cytokines are signaling biomolecules that serve as key regulators of our immune system. CD4(+) T-cells can be grouped into 2 major categories based on their cytokine profile: T-helper 1 (TH1) subset and T-helper 2 (TH2) subset. Protective immunity against HIV infection requires TH1-directed CD4 T-cell responses, mediated by cytokines, such as interleukin-1β (IL-1β), IL-12, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). Cytokines released by the TH1 subset of CD4 T-cells are considered important for mediating effective immune responses against intracellular pathogens such as Mycobacterium tuberculosis (M. tb). Oxidative stress and redox imbalance that occur during HIV infection often lead to inappropriate immune responses. Glutathione (GSH) is an antioxidant present in nearly all cells and is recognized for its function in maintaining redox homeostasis. Our laboratory previously reported that individuals with HIV infection have lower levels of GSH. In this study, we report a link between lower levels of GSH and dysregulation of TH1- and TH2-associated cytokines in the plasma samples of HIV-positive subjects. Furthermore, we demonstrate that supplementing individuals with HIV infection for 13 weeks with liposomal GSH (lGSH) resulted in a significant increase in the levels of TH1 cytokines, IL-1β, IL-12, IFN-γ, and TNF-α. lGSH supplementation in individuals with HIV infection also resulted in a substantial decrease in the levels of free radicals and immunosuppressive cytokines, IL-10 and TGF-β, relative to those in a placebo-controlled cohort. Finally, we determined the effects of lGSH supplementation in improving the functions of immune cells to control M. tb infection by conducting in vitro assays using peripheral blood mononuclear cells collected from HIV-positive individuals at post-GSH supplementation. Our studies establish a correlation between low levels of GSH and increased susceptibility to M. tb infection through TH2-directed response, which may be relieved with lGSH supplementation enhancing the TH1 response.

Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Yoon Sik, E-mail: yumshak@naver.com; Seo, Hyun Wook, E-mail: suruk@naver.com; Jung, Guhung, E-mail: drjung@snu.ac.kr

2015-02-13

Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study thatmore » Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H{sub 2}O{sub 2} and GSH modulate HBV capsid assembly. • H{sub 2}O{sub 2} facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H{sub 2}O{sub 2} and GSH induce conformation change of Hsp90.« less

The role of glutathione in DNA damage by potassium bromate in vitro.

PubMed

Parsons, J L; Chipman, J K

2000-07-01

We have investigated the role of reduced glutathione (GSH) in the genetic toxicity of the rodent renal carcinogen potassium bromate (KBrO(3)). A statistically significant increase in the concentration of 8-oxodeoxyguanosine (8-oxodG) relative to deoxyguanosine was measured following incubation of calf thymus DNA with KBrO(3) and GSH or N-acetylcysteine (NACys). This was dependent on these thiols and was associated with the loss of GSH and production of oxidized glutathione. A short-lived (<6 min) intermediate was apparent which did not react with the spin trap dimethylpyrroline N-oxide. DNA oxidation was not evident when potassium chlorate (KClO(3)) or potassium iodate (KIO(3)) were used instead of KBrO(3), though GSH depletion also occurred with KIO(3), but not with KClO(3). Other reductants and thiols in combination with KBrO(3) did not cause a significant increase in DNA oxidation. DNA strand breakage was also induced by KBrO(3) in human white blood cells (5 mM) and rat kidney epithelial cells (NRK-52E, 1.5 mM). This was associated with an apparent small depletion of thiols in NRK-52E cells at 15 min and with an elevation of 8-oxodG at a delayed time of 24 h. Depletion of intra-cellular GSH by diethylmaleate in human lymphocytes decreased the amount of strand breakage induced by KBrO(3). Extracellular GSH, however, protected against DNA strand breakage by KBrO(3), possibly due to the inability of the reactive product to enter the cell. In contrast, membrane-permeant NACys enhanced KBrO(3)-induced DNA strand breakage in these cells. DNA damage by KBrO(3) is therefore largely dependent on access to intracellular GSH.

Increased ophthalmic acid production is supported by amino acid catabolism under fasting conditions in mice.

PubMed

Kobayashi, Sho; Lee, Jaeyong; Takao, Toshifumi; Fujii, Junichi

2017-09-23

Glutathione (GSH) plays pivotal roles in antioxidation and detoxification. The transsulfuration pathway, in conjunction with methionine metabolism, produces equimolar amounts of cysteine (Cys) and 2-oxobutyric acid (2OB). The resulting 2OB is then converted into 2-aminobutyric acid (2AB) by a transaminase and is utilized as a substitute for Cys by the GSH-synthesizing machinery to produce ophthalmic acid (OPT). By establishing a method for simultaneously measuring Cys, GSH, and OPT by liquid chromatography-mass spectrometry, we found that fasting causes an elevation in OPT levels in the liver and blood plasma, even though the levels of Cys and GSH are decreased. Autophagy was activated, but the levels of GSH/OPT-synthesizing enzymes remained unchanged. After 6 h of fasting, the mice were given 1% 2AB and/or 5% glucose in the drinking water for an additional 24 h and the above metabolites analyzed. 2AB administration caused an increase in OPT levels, and, when glucose was co-administered with 2AB, the levels of OPT were elevated further but GSH levels were decreased somewhat. These results suggest that, while Cys is utilized for glyconeogenesis under fasting conditions, reaching levels that were insufficient for the synthesis of GSH, 2OB was preferentially converted to 2AB via amino acid catabolism and was utilized as a building block for OPT. Thus the consumption of Cys and the parallel elevation of 2AB under fasting conditions appeared to force γ-glutamylcysteine synthetase to form γ-glutamyl-2AB, despite the fact that the enzyme has a higher Km value for 2AB than Cys. Copyright © 2017 Elsevier Inc. All rights reserved.

Oxidative stress responses in gills of tilapia (Oreochromis niloticus) at different salinities

NASA Astrophysics Data System (ADS)

Handayani, Kiki Syaputri; Novianty, Zahra; Saputri, Miftahul Rohmah; Irawan, Bambang; Soegianto, Agoes

2017-08-01

The objective of present study is to evaluate the impact of different salinities on the levels of CAT, GSH and MDA of the gills of Nile tilapia (Oreochromis niloticus). Nile tilapia was treated by exposure to salinities concentration 0 ‰, 5 ‰ and 10 ‰. Research models were weakened and sacrificed, then took the left and right sides of the gills. The result of gills homogenity was centrifuged for supernatan, then supernatan was proceed with testing levels of CAT, GSH and MDA by ELISA assay methods. The levels of CAT in gills were significantly higher at 10 ‰ than at 5 ‰ and 0 ‰. The levels of GSH in gills were significantly higher at 0 ‰ than 5 ‰. The levels of GSH in gills at 5 ‰ and 10 ‰ salinities were not significantly different. The levels of MDA in gills at salinity 10 ‰ and 5 ‰ were higher than in control gills at 0 ‰ salinities. This occurs because the salinity of 10 ‰ salinity was optimal for live of fish tilapia. In conclusion, salinity impact the increasing of CAT, GSH, and MDA levels in gills of Nile tilapia.

Effects of Transportation on Antioxidant Status in Cynomolgus Macaques (Macaca fascicularis).

PubMed

Pan, Xueying; Lu, Liang; Zeng, Xiancheng; Chang, Yan; Hua, Xiuguo

2016-01-01

To evaluate the effects of transportation on oxidative stress in cynomolgus monkeys, we measured serum levels of reduced glutathione (GSH), malondialdehyde, and protein carbonyl (PC) and the activities of total antioxidant capacity (TAOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase in cynomolgus macaques before transportation (day 0), on the day of arrival (day 1), and on days 7, 14, and 21 after transportation. Compared with that on day 0, TAOC and catalase activities on days 1, 7, and 14 after transportation were significantly decreased, reached their nadirs on day 7, and increased thereafter to reach their pretransportation levels by day 21 after transportation. Compared with day 0 levels, mean SOD activity and GSH concentration were decreased significantly on day 1; they thereafter increased to reach their pretransportation measures by day 7 after transportation. In contrast, PC and malondialdehyde concentrations in serum and the activity of GSH-Px were increased on day 1 compared with day 0 and thereafter decreased to reach their pretransportation levels by day 14 after transportation. In summary, GSH, TAOC, catalase, and SOD levels decreased and malondialdehyde, PC, and GSH-Px concentrations increased in cynomolgus macaques after transportation. These results suggest that transportation might imbalance oxidant and antioxidant levels to create excess oxidative stress in cynomolgus macaques. Therefore, cynomolgus macaques should have at least 21 d to recover after transportation and regain their healthy status.

Effects of Transportation on Antioxidant Status in Cynomolgus Macaques (Macaca fascicularis)

PubMed Central

Pan, Xueying; Lu, Liang; Zeng, Xiancheng; Chang, Yan; Hua, Xiuguo

2016-01-01

To evaluate the effects of transportation on oxidative stress in cynomolgus monkeys, we measured serum levels of reduced glutathione (GSH), malondialdehyde, and protein carbonyl (PC) and the activities of total antioxidant capacity (TAOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase in cynomolgus macaques before transportation (day 0), on the day of arrival (day 1), and on days 7, 14, and 21 after transportation. Compared with that on day 0, TAOC and catalase activities on days 1, 7, and 14 after transportation were significantly decreased, reached their nadirs on day 7, and increased thereafter to reach their pretransportation levels by day 21 after transportation. Compared with day 0 levels, mean SOD activity and GSH concentration were decreased significantly on day 1; they thereafter increased to reach their pretransportation measures by day 7 after transportation. In contrast, PC and malondialdehyde concentrations in serum and the activity of GSH-Px were increased on day 1 compared with day 0 and thereafter decreased to reach their pretransportation levels by day 14 after transportation. In summary, GSH, TAOC, catalase, and SOD levels decreased and malondialdehyde, PC, and GSH-Px concentrations increased in cynomolgus macaques after transportation. These results suggest that transportation might imbalance oxidant and antioxidant levels to create excess oxidative stress in cynomolgus macaques. Therefore, cynomolgus macaques should have at least 21 d to recover after transportation and regain their healthy status. PMID:27657707

Determination of glutathione in human plasma using high-performance liquid chromatography with electrochemical detection with a carbon-epoxy resin composite electrode chemically modified with cobalt phthalocyanine.

PubMed

Wring, S A; Hart, J P; Birch, B J

1989-12-01

High-performance liquid chromatography with electrochemical detection (LCEC), incorporating a novel carbon-epoxy resin working electrode modified with cobalt phthalocyanine, has been employed for preliminary studies directed towards the determination of normal circulating levels of reduced glutathione (GSH) in human plasma. The mobile phase consisted of 0.05 M phosphate buffer (pH 3) containing 0.1% m/m ethylenediaminetetraacetic acid (EDTA); the calibration graph was linear in the range 0.24-30.7 ng of GSH injected. The mean recovery of GSH added to a control serum over the physiological concentration range (0.38-3.07 ng ml-1) was 99%; this was achieved following a simple sample pre-treatment method, prior to LCEC, involving chelation of divalent cations with EDTA and subsequent acidification with orthophosphoric acid. Using the LCEC method, the mean circulating level of GSH in plasma, found in three normal subjects, was 2.69 microM, GSH; this indicates that the method might be applicable to the determination of depressed circulating levels of GSH.

Effect of patchouli alcohol on the regulation of heat shock-induced oxidative stress in IEC-6 cells.

PubMed

Liu, Xiaoxi; Jiang, Linshu; Liu, Fenghua; Chen, Yuping; Xu, Lei; Li, Deyin; Ma, Yunfei; Li, Huanrong; Xu, Jianqin

2016-08-01

Purpose Patchouli alcohol (PA) is used to treat gastrointestinal dysfunction. The purpose of this study was to ascertain the function of PA in the regulated process of oxidative stress in rat intestinal epithelial cells (IEC-6). Materials and methods Oxidative stress was stimulated by exposing IEC-6 cells to heat shock (42 °C for 3 h). IEC-6 cells in treatment groups were pretreated with various concentrations of PA (10, 40, and 80 ng/mL) for 3 h before heat shock. Results Heat shock caused damage to the morphology of IEC-6 cells, and increased reactive oxygen species (ROS) level and malondialdehyde (MDA) content. Moreover, mRNA and protein expression by target genes related to oxidative stress in heat shock were also altered. Specifically, the mRNA expression by HSP70, HSP90, GSH-px, NRF2 nd HO-1were all increased, and Nrf2 and Keap1 protein expression were increased after heat shock. However, pretreatment with PA weakened the level of damage to the cellular morphology, and decreased the MDA content caused by heat shock, indicating PA had cytoprotective activities. Pretreatment with PA at high dose significantly increased generation of intracellular ROS. Compared with the heat shock group alone, PA pretreatment significantly decreased the mRNA expression by HSP70, HSP90, SOD, CAT, GSH-px, KEAP1 and HO-1. Furthermore, the high dose of PA significantly increased Nrf2 protein expression, while both the intermediate and high dose of PA significantly increased HO-1 protein expression. Conclusion Heat-shock-induced oxidative stress in IEC-6 cells, and PA could alleviate the Nrf2-Keap1 cellular oxidative stress responses.

Aldosterone Activates Transcription Factor Nrf2 in Kidney Cells Both In Vitro and In Vivo

PubMed Central

Oteiza, Patricia I.; Link, Samuel; Hey, Valentin; Stopper, Helga; Schupp, Nicole

2014-01-01

Abstract Aims: An increased kidney cancer risk was found in hypertensive patients, who frequently exhibit hyperaldosteronism, known to contribute to kidney injury, with oxidative stress playing an important role. The capacity of kidney cells to up-regulate transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2), a key regulator of the cellular antioxidative defense, as a prevention of aldosterone-induced oxidative damage was investigated both in vitro and in vivo. Results: Aldosterone activated Nrf2 and increased the expression of enzymes involved in glutathione (GSH) synthesis and detoxification. This activation depended on the mineralocorticoid receptor (MR) and oxidative stress. In vitro, Nrf2 activation, GSH amounts, and target gene levels decreased after 24 h, while oxidant levels remained high. Nrf2 activation could not protect cells against oxidative DNA damage, as aldosterone-induced double-strand breaks and 7,8-dihydro-8-oxo-guanine (8-oxodG) lesions steadily rose. The Nrf2 activator sulforaphane enhanced the Nrf2 response both in vitro and in vivo, thereby preventing aldosterone-induced DNA damage. In vivo, Nrf2 activation further had beneficial effects on the aldosterone-caused blood pressure increase and loss of kidney function. Innovation: This is the first study showing the activation of Nrf2 by aldosterone. Moreover, the results identify sulforaphane as a substance that is capable of preventing aldosterone-induced damage both in vivo and in vitro. Conclusion: Aldosterone-induced Nrf2 adaptive response cannot neutralize oxidative actions of chronically increased aldosterone, which, therefore could be causally involved in the increased cancer incidence of hypertensive individuals. Enhancing the cellular antioxidative defense with sulforaphane might exhibit beneficial effects. Antioxid. Redox Signal. 21, 2126–2142. PMID:24512358

A dual-mode nanosensor based on carbon quantum dots and gold nanoparticles for discriminative detection of glutathione in human plasma.

PubMed

Shi, Yupeng; Pan, Yi; Zhang, Heng; Zhang, Zhaomin; Li, Mei-Jin; Yi, Changqing; Yang, Mengsu

2014-06-15

Glutathione (GSH) plays key roles in biological systems and serves many cellular functions. Since biothiols all incorporate thiol, carboxylic and amino groups, discriminative detection of GSH over cysteine (Cys) and homocysteine (Hcy) is still challenging. We herein report a dual-mode nanosensor with both colorimetric and fluorometric readout based on carbon quantum dots and gold nanoparticles for discriminative detection of GSH over Cys/Hcy. The proposed sensing system consists of AuNPs and fluorescent carbon quantum dots (CQDs), where CQDs function as fluorometric reporter, and AuNPs serve a dual function as colorimetric reporter and fluorescence quencher. The mechanism of the nanosensor is based on two distance-dependent phenomenons, color change of AuNPs and FRET. Through controlling the surface properties of as-prepared nanoparticles, the addition of CQDs into AuNPs colloid solution might induce the aggregation of AuNPs and CQDs, leading to AuNPs color changing from red to blue and CQDs fluorescence quench. However, the presence of GSH can protect AuNPs from being aggregated and enlarge the inter-particle distance, which subsequently produces color change and fluorescent signal recovery. The nanosensor described in this report reflects on its simplicity and flexibility, where no further surface functionalization is required for the as-prepared nanoparticles, leading to less laborious and more cost-effective synthesis. The proposed dual-mode nanosensor demonstrated highly selectivity toward GSH, and allows the detection of GSH as low as 50 nM. More importantly, the nanosensor could not only function in aqueous solution for GSH detection with high sensitivity but also exhibit sensitive responses toward GSH in complicated biological environments, demonstrating its potential in bioanalysis and biodection, which might be significant in disease diagnosis in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

Targeting multidrug resistance protein 1 (MRP1, ABCC1): past, present, and future.

PubMed

Cole, Susan P C

2014-01-01

The human ATP-binding cassette transporter multidrug resistance protein 1 (MRP1), encoded by ABCC1, was initially identified because of its ability to confer multidrug resistance in lung cancer cells. It is now established that MRP1 plays a role in protecting certain tissues from xenobiotic insults and that it mediates the cellular efflux of the proinflammatory cysteinyl leukotriene C4 as well as a vast array of other endo- and xenobiotic organic anions. Many of these are glutathione (GSH) or glucuronide conjugates, the products of Phase II drug metabolism. MRP1 also plays a role in the cellular efflux of the reduced and oxidized forms of GSH and thus contributes to the many physiological and pathophysiological processes influenced by these small peptides, including oxidative stress. In this review, the pharmacological and physiological aspects of MRP1 are considered in the context of the current status and future prospects of pharmacological and genetic modulation of MRP1 activity.

Neuropilin-1-targeted gold nanoparticles enhance therapeutic efficacy of platinum(IV) drug for prostate cancer treatment.

PubMed

Kumar, Anil; Huo, Shuaidong; Zhang, Xu; Liu, Juan; Tan, Aaron; Li, Shengliang; Jin, Shubin; Xue, Xiangdong; Zhao, YuanYuan; Ji, Tianjiao; Han, Lu; Liu, Hong; Zhang, XiaoNing; Zhang, Jinchao; Zou, Guozhang; Wang, Tianyou; Tang, Suoqin; Liang, Xing-Jie

2014-05-27

Platinum-based anticancer drugs such as cisplatin, oxaliplatin, and carboplatin are some of the most potent chemotherapeutic agents but have limited applications due to severe dose-limiting side effects and a tendency for cancer cells to rapidly develop resistance. The therapeutic index can be improved through use of nanocarrier systems to target cancer cells efficiently. We developed a unique strategy to deliver a platinum(IV) drug to prostate cancer cells by constructing glutathione-stabilized (Au@GSH) gold nanoparticles. Glutathione (GSH) has well-known antioxidant properties, which lead to cancer regression. Here, we exploit the advantages of both the antioxidant properties and high surface-area-to-volume ratio of Au@GSH NPs to demonstrate their potential for delivery of a platinum(IV) drug by targeting the neuropilin-1 receptor (Nrp-1). A lethal dose of a platinum(IV) drug functionalized with the Nrp-1-targeting peptide (CRGDK) was delivered specifically to prostate cancer cells in vitro. Targeted peptide ensures specific binding to the Nrp-1 receptor, leading to enhanced cellular uptake level and cell toxicity. The nanocarriers were themselves nontoxic, but exhibited high cytotoxicity and increased efficacy when functionalized with the targeting peptide and drug. The uptake of drug-loaded nanocarriers is dependent on the interaction with Nrp-1 in cell lines expressing high (PC-3) and low (DU-145) levels of Nrp-1, as confirmed through inductively coupled plasma mass spectrometry and confocal microscopy. The nanocarriers have effective anticancer activity, through upregulation of nuclear factor kappa-B (NF-κB) protein (p50 and p65) expression and activation of NF-κB-DNA-binding activity. Our preliminary investigations with platinum(IV)-functionalized gold nanoparticles along with a targeting peptide hold significant promise for future cancer treatment.

Blood cell oxidative stress precedes hemolysis in whole blood-liver slice co-cultures of rat, dog, and human tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vickers, Alison E.M., E-mail: vickers_alison@allergan.co; Sinclair, John R.; Fisher, Robyn L.

A novel in vitro model to investigate time-dependent and concentration-dependent responses in blood cells and hemolytic events is studied for rat, dog, and human tissues. Whole blood is co-cultured with a precision-cut liver slice. Methimazole (MMI) was selected as a reference compound, since metabolism of its imidazole thione moiety is linked with hematologic disorders and hepatotoxicity. An oxidative stress response occurred in all three species, marked by a decline in blood GSH levels by 24 h that progressed, and preceded hemolysis, which occurred at high MMI concentrations in the presence of a liver slice with rat (>= 1000 muM atmore » 48 h) and human tissues (>= 1000 muM at 48 h, >= 750 muM at 72 h) but not dog. Human blood-only cultures exhibited a decline of GSH levels but minimal to no hemolysis. The up-regulation of liver genes for heme degradation (Hmox1 and Prdx1), iron cellular transport (Slc40a1), and GSH synthesis and utilization (mGST1 and Gclc) were early markers of the oxidative stress response. The up-regulation of the Kupffer cell lectin Lgals3 gene expression indicated a response to damaged red blood cells, and Hp (haptoglobin) up-regulation is indicative of increased hemoglobin uptake. Up-regulation of liver IL-6 and IL-8 gene expression suggested an activation of an inflammatory response by liver endothelial cells. In summary, MMI exposure led to an oxidative stress response in blood cells, and an up-regulation of liver genes involved with oxidative stress and heme homeostasis, which was clearly separate and preceded frank hemolysis.« less

Depletion of liver glutathione levels in rats: a potential confound of nose-only inhalation.

PubMed

Fechter, Laurence D; Nelson-Miller, Alisa; Gearhart, Caroline

2008-07-01

Nose-only inhalation exposure chambers offer key advantages to whole-body systems, particularly when aerosol or mixed aerosol-vapor exposures are used. Specifically, nose-only chambers provide enhanced control over the route of exposure and dose by minimizing the deposition of particles either on the subjects skin/fur or on surfaces of a whole-body exposure system. In the current series of experiments, liver, brain, and lung total glutathione (GSH) levels were assessed following either nose-only or whole-body exposures to either jet fuel or to clean, filtered air. The data were compared to untreated control subjects. Acute nose-only inhalation exposures of rats resulted in a significant depletion of liver GSH levels both in subjects that were exposed to clean, filtered air as well as those exposed to JP-8 jet fuel and to a synthetic jet fuel. Glutathione levels were not altered in lung or brain tissue. Whole-body inhalation exposure had no effect on GSH levels in any tissue for any of the treatment groups. A second experiment demonstrated that the loss of GSH did not occur if rats were anaesthetized prior to and during nose-only exposure to clean, filtered air or to mixed hydrocarbons. These data appear to be consistent with studies demonstrating depletion in liver GSH levels among rats subjected to restraint stress. Finally, the depletion of GSH that was observed in liver following a single acute exposure was reduced following five daily exposures to clean, filtered air, suggesting the possibility of habituation to restraint in the nose-only exposure chamber. The finding that placement in a nose-only exposure chamber per se yields liver GSH depletion raises the possibility of an interaction between this mode of toxicant exposure and the toxicological effects of certain inhaled test substances.

In vitro evaluation of cellular responses induced by ZnO nanoparticles, zinc ions and bulk ZnO in fish cells.

PubMed

Fernández, Dolores; García-Gómez, Concepción; Babín, Mar

2013-05-01

Zinc oxide nanoparticles (ZnO-NPs) are inevitably released into the environment and are potentially dangerous for aquatic life. However, the potential mechanisms of cytotoxicity of zinc nanoparticles remain unclear. Studying the toxicity of ZnO-NPs with In vitro systems will help to determine their interactions with cellular biomolecules. The aim of this study was to evaluate the cytotoxic potentials of ZnO-NPs in established fish cell lines (RTG-2, RTH-149 and RTL-W1) and compare them with those of bulk ZnO and Zn(2+) ions. Membrane function (CFDA-AM assay), mitochondrial function (MTT assay), cell growth (KBP assay), cellular stress (β-galactosidase assay), reductase enzyme activity (AB assay), reactive oxygen species (ROS), total glutathione cellular content (tGSH assay) and glutathione S-transferase (GST) activities were assessed for all cell lines. ZnO-NPs cytotoxicity was greater than those of bulk ZnO and Zn(2+). ZnO-NPs induced oxidative stress is dependent on their dose. Low cost tests, such as CFDA-AM, ROS, GST activity and tGSH cell content test that use fish cell lines, may be used to detect oxidative stress and redox status changes. Particle dissolution of the ZnO-NPs did not appear to play an important role in the observed toxicity in this study. Published by Elsevier B.V.

The role of Hibiscus sabdariffa L. (Roselle) in maintenance of ex vivo murine bone marrow-derived hematopoietic stem cells.

PubMed

Abdul Hamid, Zariyantey; Lin Lin, Winnie Hii; Abdalla, Basma Jibril; Bee Yuen, Ong; Latif, Elda Surhaida; Mohamed, Jamaludin; Rajab, Nor Fadilah; Paik Wah, Chow; Wak Harto, Muhd Khairul Akmal; Budin, Siti Balkis

2014-01-01

Hematopoietic stem cells- (HSCs-) based therapy requires ex vivo expansion of HSCs prior to therapeutic use. However, ex vivo culture was reported to promote excessive production of reactive oxygen species (ROS), exposing HSCs to oxidative damage. Efforts to overcome this limitation include the use of antioxidants. In this study, the role of Hibiscus sabdariffa L. (Roselle) in maintenance of cultured murine bone marrow-derived HSCs was investigated. Aqueous extract of Roselle was added at varying concentrations (0-1000 ng/mL) for 24 hours to the freshly isolated murine bone marrow cells (BMCs) cultures. Effects of Roselle on cell viability, reactive oxygen species (ROS) production, glutathione (GSH) level, superoxide dismutase (SOD) activity, and DNA damage were investigated. Roselle enhanced the survival (P < 0.05) of BMCs at 500 and 1000 ng/mL, increased survival of Sca-1(+) cells (HSCs) at 500 ng/mL, and maintained HSCs phenotype as shown from nonremarkable changes of surface marker antigen (Sca-1) expression in all experimental groups. Roselle increased (P < 0.05) the GSH level and SOD activity but the level of reactive oxygen species (ROS) was unaffected. Moreover, Roselle showed significant cellular genoprotective potency against H2O2-induced DNA damage. Conclusively, Roselle shows novel property as potential supplement and genoprotectant against oxidative damage to cultured HSCs.

Oxidative stress response in SH-SY5Y cells exposed to short-term 1800 MHz radiofrequency radiation.

PubMed

Marjanovic Cermak, Ana Marija; Pavicic, Ivan; Trosic, Ivancica

2018-01-28

The exact mechanism that could explain the effects of radiofrequency (RF) radiation exposure at non-thermal level is still unknown. Increasing evidence suggests a possible involvement of reactive oxygen species (ROS) and development of oxidative stress. To test the proposed hypothesis, human neuroblastoma cells (SH-SY5Y) were exposed to 1800 MHz short-term RF exposure for 10, 30 and 60 minutes. Electric field strength within Gigahertz Transverse Electromagnetic cell (GTEM) was 30 V m -1 and specific absorption rate (SAR) was calculated to be 1.6 W kg -1 . Cellular viability was measured by MTT assay and level of ROS was determined by fluorescent probe 2',7'-dichlorofluorescin diacetate. Concentrations of malondialdehyde and protein carbonyls were used to assess lipid and protein oxidative damage and antioxidant activity was evaluated by measuring concentrations of total glutathione (GSH). After radiation exposure, viability of irradiated cells remained within normal physiological values. Significantly higher ROS level was observed for every radiation exposure time. After 60 min of exposure, the applied radiation caused significant lipid and protein damage. The highest GSH concentration was detected after 10 minute-exposure. The results of our study showed enhanced susceptibility of SH-SY5Y cells for development of oxidative stress even after short-term RF exposure.

The Role of Hibiscus sabdariffa L. (Roselle) in Maintenance of Ex Vivo Murine Bone Marrow-Derived Hematopoietic Stem Cells

PubMed Central

Abdul Hamid, Zariyantey; Lin Lin, Winnie Hii; Abdalla, Basma Jibril; Bee Yuen, Ong; Latif, Elda Surhaida; Mohamed, Jamaludin; Rajab, Nor Fadilah; Paik Wah, Chow; Budin, Siti Balkis

2014-01-01

Hematopoietic stem cells- (HSCs-) based therapy requires ex vivo expansion of HSCs prior to therapeutic use. However, ex vivo culture was reported to promote excessive production of reactive oxygen species (ROS), exposing HSCs to oxidative damage. Efforts to overcome this limitation include the use of antioxidants. In this study, the role of Hibiscus sabdariffa L. (Roselle) in maintenance of cultured murine bone marrow-derived HSCs was investigated. Aqueous extract of Roselle was added at varying concentrations (0–1000 ng/mL) for 24 hours to the freshly isolated murine bone marrow cells (BMCs) cultures. Effects of Roselle on cell viability, reactive oxygen species (ROS) production, glutathione (GSH) level, superoxide dismutase (SOD) activity, and DNA damage were investigated. Roselle enhanced the survival (P < 0.05) of BMCs at 500 and 1000 ng/mL, increased survival of Sca-1+ cells (HSCs) at 500 ng/mL, and maintained HSCs phenotype as shown from nonremarkable changes of surface marker antigen (Sca-1) expression in all experimental groups. Roselle increased (P < 0.05) the GSH level and SOD activity but the level of reactive oxygen species (ROS) was unaffected. Moreover, Roselle showed significant cellular genoprotective potency against H2O2-induced DNA damage. Conclusively, Roselle shows novel property as potential supplement and genoprotectant against oxidative damage to cultured HSCs. PMID:25405216

Evidence of Dual Mechanisms of Glutathione Uptake in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis

PubMed Central

Whitson, Jeremy A.; Sell, David R.; Goodman, Michael C.; Monnier, Vincent M.; Fan, Xingjun

2016-01-01

Purpose Lens glutathione synthesis knockout (LEGSKO) mouse lenses lack de novo glutathione (GSH) synthesis but still maintain >1 mM GSH. We sought to determine the source of this residual GSH and the mechanism by which it accumulates in the lens. Methods Levels of GSH, glutathione disulfide (GSSG), and GSH-related compounds were measured in vitro and in vivo using isotope standards and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results Wild-type (WT) lenses could accumulate GSH from γ-glutamylcysteine and glycine or from intact GSH, but LEGSKO lenses could only accumulate GSH from intact GSH, indicating that LEGSKO lens GSH content is not due to synthesis by a salvage pathway. Uptake of GSH in cultured lenses occurred at the same rate for LEGSKO and WT lenses, could not be inhibited, and occurred primarily through cortical fiber cells. In contrast, uptake of GSH from aqueous humor could be competitively inhibited and showed an enhanced Km in LEGSKO lenses. Mouse vitreous had >1 mM GSH, whereas aqueous had <20 μM GSH. Testing physiologically relevant GSH concentrations for uptake in vivo, we found that both LEGSKO and WT lenses could obtain GSH from the vitreous but not from the aqueous. Vitreous rapidly accumulated GSH from the circulation, and depletion of circulating GSH reduced vitreous but not aqueous GSH. Conclusions The above data provide, for the first time, evidence for the existence of dual mechanisms of GSH uptake into the lens, one mechanism being a passive, high-flux transport through the vitreous exposed side of the lens versus an active, carrier-mediated uptake mechanism at the anterior of the lens. PMID:27472077

Aminothiol Receptors for Decorporation of Intravenously Administered 60Co in the Rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Levitskaia, Tatiana G.; Morris, James E.; Creim, Jeffrey A.

2010-01-01

The reported investigation provides a comparison of the oral decorporation efficacy of L-glutathione (GSH), L-cysteine (Cys), and a liposomal GSH formulation (ReadiSorb) toward systemic cobalt-60 (60Co) to that observed following intravenous administration of GSH and Cys in F344 rats. L-histidine (His) was tested intravenously to compare in vivo efficacy of the aminothiol GSH and Cys chelators with that of aminoimidazole (His) chelator. 60Co was administered to animals by intravenous injection, followed by intravenous or oral gavage doses of a chelator repeated at 24 hour intervals for a total of 5 doses. The results suggest that GSH and Cys are potentmore » decorporation agents for 60Co in the rat model, although the efficacy of treatment depends largely on systemic availability of a chelator. The intravenous GSH or Cys were most effective in reducing tissue 60Co levels and in increasing excretion of radioactivity compared to control animals. Liposomal encapsulation was found to markedly enhance the oral bioavailability of GSH compared to non-formulated GSH. Oral administration of ReadiSorb reduced 60Co levels in nearly all tissues by 12-43% compared to that observed for non-formulated GSH. Efficacy of oral Cys was only slightly reduced in comparison with intravenous Cys. Further studies to optimize the dosing regimen in order to maximize decorporation efficiency are warranted.« less

Thyroid hormone-induced oxidative damage on lipids, glutathione and DNA in the mouse heart.

PubMed

Gredilla, R; Barja, G; López-Torres, M

2001-10-01

Oxygen radicals of mitochondrial origin are involved in oxidative damage. In order to analyze the possible relationship between metabolic rate, oxidative stress and oxidative damage, OF1 female mice were rendered hyper- and hypothyroid by chronic administration of 0.0012% L-thyroxine (T4) and 0.05% 6-n-propyl-2-thiouracil (PTU), respectively, in their drinking water for 5 weeks. Hyperthyroidism significantly increased the sensitivity to lipid peroxidation in the heart, although the endogenous levels of lipid peroxidation were not altered. Thyroid hormone-induced oxidative stress also resulted in higher levels of GSSG and GSSG/GSH ratio. Oxidative damage to mitochondrial DNA was greater than that to genomic DNA. Hyperthyroidism decreased oxidative damage to genomic DNA. Hypothyroidism did not modify oxidative damage in the lipid fraction but significantly decreased GSSG and GSSG/GSH ratio and oxidative damage to mitochondrial DNA. These results indicate that thyroid hormones modulate oxidative damage to lipids and DNA, and cellular redox potential in the mouse heart. A higher oxidative stress in the hyperthyroid group is presumably neutralized in the case of nuclear DNA by an increase in repair activity, thus protecting this key molecule. Treatment with PTU, a thyroid hormone inhibitor, reduced oxidative damage in the different cell compartments.

Rapamycin alleviates oxidative stress-induced damage in rat erythrocytes.

PubMed

Singh, Abhishek Kumar; Singh, Sandeep; Garg, Geetika; Rizvi, Syed Ibrahim

2016-10-01

An imbalanced cellular redox system promotes the production of reactive oxygen species (ROS) that may lead to oxidative stress-mediated cell death. Erythrocytes are the best-studied model of antioxidant defense mechanism. The present study was undertaken to investigate the effect of the immunosuppressant drug rapamycin, an inducer of autophagy, on redox balance of erythrocytes and blood plasma of oxidatively challenged rats. Male Wistar rats were oxidatively challenged with HgCl 2 (5 mg/kg body mass (b.m.)). A significant (p < 0.05) induction in ROS production, plasma membrane redox system (PMRS), intracellular Ca 2+ influx, lipid peroxidation (LPO), osmotic fragility, plasma protein carbonyl (PCO) content, and plasma advanced oxidation protein products (AOPP) and simultaneously significant reduction in glutathione (GSH) level and ferric reducing ability of plasma (FRAP) were observed in rats exposed to HgCl 2 . Furthermore, rapamycin (0.5 mg/kg b.m.) provided significant protection against HgCl 2 -induced alterations in rat erythrocytes and plasma by reducing ROS production, PMRS activity, intracellular Ca 2+ influx, LPO, osmotic fragility, PCO content, and AOPP and also restored the level of antioxidant GSH and FRAP. Our observations provide evidence that rapamycin improves redox status and attenuates oxidative stress in oxidatively challenged rats. Our data also demonstrate that rapamycin is a comparatively safe immunosuppressant drug.

[Effect of American Ginseng Capsule on the liver oxidative injury and the Nrf2 protein expression in rats exposed by electromagnetic radiation of frequency of cell phone].

PubMed

Luo, Ya-ping; Ma, Hui-Rong; Chen, Jing-Wei; Li, Jing-Jing; Li, Chun-xiang

2014-05-01

To observe the effect of American Ginseng Capsule (AGC) on the liver oxidative injury and the Nrf2 protein expression in the liver tissue of rats exposed by 900 MHz cell phone electromagnetic radiation. Totally 40 male SD rats were randomly divided into the normal control group, the model group, the Shuifei Jibin Capsule (SJC) group, and the AGC group,10 in each group. Rats in the normal control group were not irradiated. Rats in the rest three groups were exposed by imitated 900 MHz cellular phone for 4 h in 12 consecutive days. Meanwhile, rats in the SJC group and the AGC group were intragastrically administrated with suspension of SJC and AGC (1 mL/200 g body weight) respectively. Normal saline was administered to rats in the normal control group and the model group. The histolomorphological changes of the liver tissue were observed by HE staining. Contents of malonic dialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-PX)were detected by colorimetry. The Nrf2 protein expression of hepatocytes was detected by immunohistochemical assay and Western blot. Compared with the normal control group, hepatocyte nucleus was atrophied or partially disappeared, the contents of liver MDA and Nrf2 protein obviously increased (P <0. 05, P <0. 01); contents of liver SOD and GSH decreased (P <0. 05) in the model group. Compared with the model group, karyopyknosis was obviously attenuated and approached to the normal level in the SJC group and the AGC group. The contents of liver MDA and Nrf2 protein expression decreased (P <0. 05), and the contents of liver SOD, GSH, and GSH-PX obviously increased (P < 0.05) in the SJC group. The contents of liver MDA and the Nrf2 protein expression decreased (P < 0.05), and contents of SOD and GSH obviously increased in the AGC group (P <0.01, P <0.05). The electromagnetic radiation induced by 900 MHz cell phone could affect the expression of Nrf2 protein, induce oxidative injury, and induce abnormal morphology of liver cells. SJC and AGC could promote the morphological recovery of the liver cells. Its mechanism might be related to affecting the expression of Nrf2 protein and attenuating oxidative damage of liver cells.

Tumor cytotoxicity by endothelial cells. Impairment of the mitochondrial system for glutathione uptake in mouse B16 melanoma cells that survive after in vitro interaction with the hepatic sinusoidal endothelium.

PubMed

Ortega, Angel L; Carretero, Julian; Obrador, Elena; Gambini, Juan; Asensi, Miguel; Rodilla, Vicente; Estrela, José M

2003-04-18

High GSH content associates with high metastatic activity in B16-F10 melanoma cells cultured to low density (LD B16M). GSH homeostasis was investigated in LD B16M cells that survive after adhesion to the hepatic sinusoidal endothelium (HSE). Invasive B16M (iB16M) cells were isolated using anti-Met-72 monoclonal antibodies and flow cytometry-coupled cell sorting. HSE-derived NO and H(2)O(2) caused GSH depletion and a decrease in gamma-glutamylcysteine synthetase activity in iB16M cells. Overexpression of gamma-glutamylcysteine synthetase heavy and light subunits led to a rapid recovery of cytosolic GSH, whereas mitochondrial GSH (mtGSH) further decreased during the first 18 h of culture. NO and H(2)O(2) damaged the mitochondrial system for GSH uptake (rates in iB16M were approximately 75% lower than in LD B16M cells). iB16M cells also showed a decreased activity of mitochondrial complexes II, III, and IV, less O(2) consumption, lower ATP levels, higher O(2) and H(2)O(2) production, and lower mitochondrial membrane potential. In vitro growing iB16M cells maintained high viability (>98%) and repaired HSE-induced mitochondrial damages within 48 h. However, iB16M cells with low mtGSH levels were highly susceptible to TNF-alpha-induced oxidative stress and death. Therefore depletion of mtGSH levels may represent a critical target to challenge survival of invasive cancer cells.

Probing chromium(III) from chromium(VI) in cells by a fluorescent sensor

NASA Astrophysics Data System (ADS)

Hu, Xiangquan; Chai, Jie; Liu, Yanfei; Liu, Bin; Yang, Binsheng

2016-01-01

Cellular uptake of Cr(VI), followed by its reduction to Cr(III) with the formation of kinetically inert Cr(III) complexes, is a complex process. To better understand its physiological and pathological functions, efficient methods for the monitoring of Cr(VI) are desired. In this paper a selective fluorescent probe L, rhodamine hydrazide bearing a benzo[b]furan-2-carboxaldehyde group, was demonstrated as a red chemosensor for Cr(III) at about 586 nm. This probe has been used to probe Cr(III) which is reduced from Cr(VI) by reductants such as glutathione (GSH), vitamin C, cysteine (Cys), H2O2 and Dithiothreitol (DTT) by fluorescence spectra. Cr(VI) metabolism in vivo is primarily driven by Vc and GSH. Vc could reduce CrO42 - to Cr(III) in a faster rate than GSH. The indirectly detection limit for Cr(VI) by L + GSH system was determined to be 0.06 μM at pH = 6.2. Moreover, the confocal microscopy image experiments indicated that Cr(VI) can be reduced to Cr(III) inside cells rapidly and the resulted Cr(III) can be captured and imaged timely by L.

Blood plasma levels of lipoperoxides, glutathione peroxidase, beta carotene, vitamin A and E in women with habitual abortion.

PubMed

Simşek, M; Naziroğlu, M; Simşek, H; Cay, M; Aksakal, M; Kumru, S

1998-12-01

The plasma levels of lipoperoxides, glutathione peroxidase (GSH-Px), reduced glutathione (GSH), beta carotene, vitamin A, E, some plasma biochemical and blood haematological parameters were investigated in 40 women with habitual abortion (HA) and controls. The levels of GSH, vitamin A, E and beta carotene were significantly lower in women with HA than in controls. However, the plasma levels of lipid peroxidation, alkaline phosphatase (ALP), glucose and blood haemoglobin were significantly higher in HA than in controls. In addition, plasma levels of GSH-Px, AST, ALT, total bilirubin, total protein, albumin, sodium, potassium, calcium and number of white blood cells, red blood cells, platelet and values of packet cell volume showed no significant differences between HA and controls. According to the results of this study, we observed that the levels of lipid peroxidation were increased and plasma levels of vitamin A, E and beta carotene were decreased in HA. The decrease of those antioxidants may play a significant role in women with habitual abortion.

Nitric Oxide Ameliorates Zinc Oxide Nanoparticles Phytotoxicity in Wheat Seedlings: Implication of the Ascorbate–Glutathione Cycle

PubMed Central

Tripathi, Durgesh K.; Mishra, Rohit K.; Singh, Swati; Singh, Samiksha; Vishwakarma, Kanchan; Sharma, Shivesh; Singh, Vijay P.; Singh, Prashant K.; Prasad, Sheo M.; Dubey, Nawal K.; Pandey, Avinash C.; Sahi, Shivendra; Chauhan, Devendra K.

2017-01-01

The present study investigates ameliorative effects of nitric oxide (NO) against zinc oxide nanoparticles (ZnONPs) phytotoxicity in wheat seedlings. ZnONPs exposure hampered growth of wheat seedlings, which coincided with reduced photosynthetic efficiency (Fv/Fm and qP), due to increased accumulation of zinc (Zn) in xylem and phloem saps. However, SNP supplementation partially mitigated the ZnONPs-mediated toxicity through the modulation of photosynthetic activity and Zn accumulation in xylem and phloem saps. Further, the results reveal that ZnONPs treatments enhanced levels of hydrogen peroxide and lipid peroxidation (as malondialdehyde; MDA) due to severely inhibited activities of the following ascorbate–glutatione cycle (AsA–GSH) enzymes: ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase and dehydroascorbate reductase, and its associated metabolites ascorbate and glutathione. In contrast to this, the addition of SNP together with ZnONPs maintained the cellular functioning of the AsA–GSH cycle properly, hence lesser damage was noticed in comparison to ZnONPs treatments alone. The protective effect of SNP against ZnONPs toxicity on fresh weight (growth) can be reversed by 2-(4carboxy-2-phenyl)-4,4,5,5-tetramethyl- imidazoline-1-oxyl-3-oxide, a NO scavenger, and thus suggesting that NO released from SNP ameliorates ZnONPs toxicity. Overall, the results of the present study have shown the role of NO in the reducing of ZnONPs toxicity through the regulation of accumulation of Zn as well as the functioning of the AsA–GSH cycle. PMID:28220127

Accumulation and toxicity of monophenyl arsenicals in rat endothelial cells.

PubMed

Hirano, Seishiro; Kobayashi, Yayoi; Hayakawa, Toru; Cui, Xing; Yamamoto, Megumi; Kanno, Sanae; Shraim, Amjad

2005-01-01

Clark 1 (diphenylarsine chloride) and Clark 2 (diphenylarsine cyanide) were used as chemical weapon agents (CWA), and the soil contamination by these CWA and their degraded products, diphenyl and phenyl arsenicals, has been one of the most serious environmental issues. In a series of comparisons in toxicity between trivalent and pentavalent arsenicals we investigated differences in the accumulation and toxicity of phenylarsine oxide (PAO3+) and phenylarsonic acid (PAA5+) in rat heart microvascular endothelial cells. Both the cellular association and toxicity of PAO3+ were much higher than those of PAA5+, and LC50 values of PAO3+ and PAA5+ were calculated to be 0.295 microM and 1.93 mM, respectively. Buthionine sulfoximine, a glutathione depleter, enhanced the cytotoxicity of both PAO3+ and PAA5+. N-Acetyl-L-cysteine (NAC) reduced the cytotoxicity and induction of heme oxygenase-1 (HO-1) mRNA in PAO3+-exposed cells, while NAC affected neither the cytotoxicity nor the HO-1 mRNA level in PAA5+-exposed cells. The effect of NAC may be due to a strong affinity of PAO3+ to thiol groups because both NAC and GSH inhibited the cellular accumulation of PAO3+, but PAA3+ increased tyrosine phosphorylation levels of cellular proteins. These results indicate that the inhibition of protein phosphatases as well as the high affinity to cellular components may confer PAO3+ the high toxicity.

Gamma-glutamylcysteinylethyl ester attenuates progression of carbon tetrachloride-induced acute liver injury in mice.

PubMed

Nishida, K; Ohta, Y; Ishiguro, I

1998-02-20

We examined the effect of gamma-glutamylcysteinylethyl ester (gamma-GCE), which is readily transported into hepatocytes and increases hepatocellular reduced glutathione (GSH) levels, on the progression of carbon tetrachloride (CCl4)-induced liver injury in mice in comparison with that of GSH. Administration of more than 160 micromol/kg of gamma-GCE, but not GSH, to mice at 3 h after intraperitoneal injection of CCl4 (1 ml/kg) significantly attenuated increases in serum aspartate aminotransferase and alanine aminotransferase activities at 24 h after the CCl4 injection. Increases in hepatic lipid peroxide (LPO) concentrations and decreases in hepatic GSH concentrations after the CCl4 injection were significantly diminished by the gamma-GCE (160 micromol/kg) administration, but not by the same dose of GSH. Gamma-GCE, gamma-glutamylcysteine, and cysteine acted as substrates for glutathione peroxidases much less efficiently than GSH in the post-mitochondrial fraction of normal mouse liver cells. These results indicate that gamma-GCE attenuates the progression of CCl4-induced acute liver injury in mice through the maintenance of hepatic GSH levels, leading to inhibition of hepatic LPO formation, which could be due to an efficient utilization of GSH converted from gamma-GCE in the liver cells.

Oocyte glutathione and fertilisation outcome of Macaca nemestrina and Macaca fascicularis in in vivo- and in vitro-matured oocytes

PubMed Central

Curnow, E. C.; Ryan, J. P.; Saunders, D. M.; Hayes, E. S.

2010-01-01

Fertilisation and development of IVM non-human primate oocytes is limited compared with that of in vivo-matured (IVO) oocytes. The present study describes the IVM of macaque oocytes with reference to oocyte glutathione (GSH). Timing of maturation, comparison of IVM media and cysteamine (CYS) supplementation as a modulator of GSH were investigated. A significantly greater proportion of oocytes reached MII after 30 h compared with 24 h of IVM. Following insemination, IVM oocytes had a significantly lower incidence of normal fertilisation (i.e. 2PN = two pronuclei and at least one polar body) and a higher rate of abnormal fertilisation (1PN = one pronucleus and at least one polar body) compared with IVO oocytes. Immunofluorescence of 1PN zygotes identified incomplete sperm head decondensation and failure of male pronucleus formation as the principal cause of abnormal fertilisation in IVM oocytes. The IVO oocytes had significantly higher GSH content than IVM oocytes. Cumulus-denuded oocytes had significantly lower GSH following IVM compared with immature oocytes at collection. Cysteamine supplementation of the IVM medium significantly increased the GSH level of cumulus-intact oocytes and reduced the incidence of 1PN formation, but did not improve GSH levels of the denuded oocyte. Suboptimal GSH levels in macaque IVM oocytes may be related to reduced fertilisation outcomes. PMID:20591337

Oocyte glutathione and fertilisation outcome of Macaca nemestrina and Macaca fascicularis in in vivo- and in vitro-matured oocytes.

PubMed

Curnow, E C; Ryan, J P; Saunders, D M; Hayes, E S

2010-01-01

Fertilisation and development of IVM non-human primate oocytes is limited compared with that of in vivo-matured (IVO) oocytes. The present study describes the IVM of macaque oocytes with reference to oocyte glutathione (GSH). Timing of maturation, comparison of IVM media and cysteamine (CYS) supplementation as a modulator of GSH were investigated. A significantly greater proportion of oocytes reached MII after 30 h compared with 24 h of IVM. Following insemination, IVM oocytes had a significantly lower incidence of normal fertilisation (i.e. 2PN = two pronuclei and at least one polar body) and a higher rate of abnormal fertilisation (1PN = one pronucleus and at least one polar body) compared with IVO oocytes. Immunofluorescence of 1PN zygotes identified incomplete sperm head decondensation and failure of male pronucleus formation as the principal cause of abnormal fertilisation in IVM oocytes. The IVO oocytes had significantly higher GSH content than IVM oocytes. Cumulus-denuded oocytes had significantly lower GSH following IVM compared with immature oocytes at collection. Cysteamine supplementation of the IVM medium significantly increased the GSH level of cumulus-intact oocytes and reduced the incidence of 1PN formation, but did not improve GSH levels of the denuded oocyte. Suboptimal GSH levels in macaque IVM oocytes may be related to reduced fertilisation outcomes.

Liraglutide attenuates partial warm ischemia-reperfusion injury in rat livers.

PubMed

Abdelsameea, Ahmed A; Abbas, Noha A T; Abdel Raouf, Samar M

2017-03-01

Ischemia-reperfusion (IR) injury constitutes the most important cause of primary dysfunction of liver grafts. In this study, we have addressed the possible hepatoprotective action of liraglutide against partial warm hepatic IR injury in male rats. Rats were randomly assigned into: sham, IR, and liraglutide-pretreated IR groups. Liraglutide was administered 50 μg/kg s.c. twice daily for 14 days, and then, hepatic IR was induced by clamping portal vein and hepatic artery to left and median lobes for 30 min followed by reperfusion for 24 h. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transferase (GGT) activities were determined. Malondialdehyde (MDA) level, reduced glutathione (GSH) content, tumor necrosis factor-α (TNF-α), phosphoralated Akt (p-Akt), and caspase-3 levels of the liver were determined. Hematoxylin and eosin (H&E) stained sections from liver were examined as well as immunohistochemical sections for detection of Bcl-2 expression. IR injury increased ALT, AST, and GGT while decreased GSH and p-Akt with increase in MDA, TNF-α, and caspase-3 levels in the liver with necrosis and inflammatory cellular infiltration with decreased Bcl-2 expression. Pretreatment with liraglutide decreased ALT, AST, and GGT activities while increased glutathione content and Akt activation with decrements in MDA, TNF-α, and caspase-3 levels with attenuation of necrosis and inflammation while enhanced Bcl-2 expression in the liver. Liraglutide protects against IR injury of the liver through antiinflammatory and antioxidant actions as well as inhibition of apoptosis.

Glutathione redox balance in hibernating Chinese soft-shelled turtle Pelodiscus sinensis hatchlings.

PubMed

Zhang, Wenyi; Niu, Cuijuan; Liu, Yukun; Chen, Bojian

2017-05-01

Glutathione (GSH) system is a critical component of antioxidant defense, which is important for hibernating survive of turtle hatchlings. The present work measured changes at the mRNA level of genes involved in GSH synthesis, GSH reduction and GSH utilization, as well as enzyme activity, in Pelodiscus sinensis hatchlings during hibernation. Samples were taken in the field at pre-hibernation (17°C, Mud temperature (MT)), hibernation (5.8°C, MT) and arousal (20.1°C, MT). Cerebral total GSH content decreased during hibernation, recovered after arousal along with a stable ratio of GSH/GSSG. Hepatic total GSH increased after arousal and pushed the ratio of GSH/GSSG to a more reduced status. Cerebral glutathione reductase (GR) mRNA and activity were depressed during hibernation then recovered after arousal. However, hepatic GR mRNA elevated during hibernation but its activity did not change. Tissue-specific changes of GR activity and mRNA may promote these tissue-specific changes of GSH redox. Hibernation caused little effect on mRNA level of glutathione synthetase (GS) while arousal induced them in the brain and liver. Most Glutathione-S-transferase (GST) isoform mRNAs did not change in both brain and liver during hibernation, then induced after arousal. Cerebral and hepatic GST activities kept stable throughout the entire experiment. Our results showed that GSH system may play a more important role in antioxidant defense in the liver while mainly maintaining stable redox balance in the brain of hibernating P. sinensis hatchings. Copyright © 2017 Elsevier Inc. All rights reserved.

N-acetylcysteine prevents the geldanamycin cytotoxicity by forming geldanamycin-N-acetylcysteine adduct.

PubMed

Mlejnek, Petr; Dolezel, Petr

2014-09-05

Geldanamycin (GDN) is a benzoquinone ansamycin antibiotic with anti-proliferative activity on tumor cells. GDN cytotoxicity has been attributed to the disruption of heat shock protein 90 (Hsp90) binding and stabilizing client proteins, and by the induction of oxidative stress with concomitant glutathione (GSH) depletion. The later mechanism of cytotoxicity can be abrogated by N-acetylcysteine (NAC). It was suggested that NAC prevents GDN cytotoxicity mainly by the restoring of glutathione (GSH) level (Clark et al., 2009). Here we argue that NAC does not protect cells from the GDN cytotoxicity by restoring the level of GSH. A detailed LC/MS/MS analysis of cell extracts indicated formation of GDN adducts with GSH. The amount of the GDN-GSH adduct is proportional to the GDN concentration and increases with incubation time. While nanomolar and low micromolar GDN concentrations induce cell death without an apparent GSH decrease, only much higher micromolar GDN concentrations cause a significant GSH decrease. Therefore, only high micromolar GDN concentrations can cause cell death which might be related to GSH depletion. Addition of NAC leads to the formation of adducts with GDN which diminish formation of GDN adducts with GSH. NAC also forms stable adducts with GDN extracellularly. Although NAC induces an increase in the GSH pool, this effect is not crucial for abrogation of GDN cytotoxicity. Indeed, the presence of NAC in the growth medium causes a rapid conversion of GDN into the GDN-NAC adduct, which is the real cause of the abrogated GDN cytotoxicity. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Combining reduced glutathione and ascorbic acid has supplementary beneficial effects on boar sperm cryotolerance.

PubMed

Giaretta, Elisa; Estrada, Efrén; Bucci, Diego; Spinaci, Marcella; Rodríguez-Gil, Joan E; Yeste, Marc

2015-02-01

The main aim of this work was to evaluate how supplementing freezing and thawing media with reduced glutathione (GSH) and l-ascorbic acid (AA) affected the quality parameters of frozen-thawed boar spermatozoa. With this purpose, semen samples of 12 ejaculates coming from 12 boars were used. Each ejaculate was split into seven aliquots to which 5 mM of GSH and 100 μM of AA were added separately or together at two different steps of freeze-thawing. Various sperm parameters (levels of free cysteine residues in sperm nucleoproteins, sperm viability, acrosome membrane integrity, intracellular peroxide and superoxide levels [ROS], and total and progressive motility) were evaluated before freezing and at 30 and 240 minutes after thawing. Both GSH and AA significantly improved boar sperm cryotolerance when they were separately added to freezing and thawing media. However, the highest improvement was recorded when both freezing and thawing media were supplemented with 5 mM of GSH plus 100 μM of AA. This improvement was observed in sperm viability and acrosome integrity, sperm motility, and nucleoprotein structure. Although ROS levels were not much increased by freeze-thawing procedures, the addition of GSH and AA to both freezing and thawing extenders significantly decreased intracellular peroxide levels and had no impact on superoxide levels. According to our results, we can conclude that supplementation of freezing and thawing media with both GSH and AA has a combined, beneficial effect on frozen-thawed boar sperm, which is greater than that obtained with the separate addition of either GSH or AA. Copyright © 2015 Elsevier Inc. All rights reserved.

The prospective protective effect of selenium nanoparticles against chromium-induced oxidative and cellular damage in rat thyroid.

PubMed

Hassanin, Kamel M A; Abd El-Kawi, Samraa H; Hashem, Khalid S

2013-01-01

Nanotechnology has enabled researchers to synthesize nanosize particles that possess increased surface areas. Compared to conventional microparticles, it has resulted in increased interactions with biological targets. The objective of this study was to determine the protective ability of selenium nanoparticles against hexavalent chromium-induced thyrotoxicity. Twenty male rats were used in the study, and arbitrarily assigned to four groups. Group 1 was the control group, and was given phosphate-buffered saline. Group 2 was the chromium-treated group and was given K2Cr2O7 60 μg/kg body weight intraperitoneally as a single dose on the third day of administration. Group 3 was the nano-selenium-treated group and was given selenium nanoparticles (size 3-20 nm) 0.5 mg/kg body weight intraperitoneally daily for 5 consecutive days. Group 4 was the nano-selenium chromium-treated group, which received selenium nanoparticles for 5 days and a single dose of K2Cr2O7 on the third day of administration. Blood samples were collected from rats for measuring thyroid hormones (free triiodothyronine [T3] and free thyroxine [T4]) and oxidative and antioxidant parameters (malondialdehyde [MDA], reduced glutathione [GSH], catalase, and superoxide dismutase [SOD]). Upon dissection, thyroid glands were taken for histopathological examination by using paraffin preparations stained with hematoxylin and eosin (H&E) and Masson's trichrome. Immunohistochemical staining was performed for detecting cellular proliferation using Ki67 antibodies. The present study shows that K2Cr2O7 has a toxic effect on the thyroid gland as a result of inducing a marked oxidative damage and release of reactive oxygen species. This was shown by the significant decrease in free T3 and T4 and GSH levels, which was accompanied by significant increases in catalase, SOD, and MDA in the chromium-treated group compared to the control group. Se nanoparticles have a protective effect on K2Cr2O7-induced thyroid damage, as a result of correcting the free T3 and T4 levels and GSH, catalase, SOD, and MDA compared to the K2Cr2O7-treated group. Administration of nano-selenium alone in the nano-selenium-treated group had no toxic effect on rats' thyroid compared to the control group. The biochemical results were confirmed by histopathological, immunohistochemical and pathomorphological studies.

The diverse roles of glutathione-associated cell resistance against hypericin photodynamic therapy.

PubMed

Theodossiou, Theodossis A; Olsen, Cathrine E; Jonsson, Marte; Kubin, Andreas; Hothersall, John S; Berg, Kristian

2017-08-01

The diverse responses of different cancers to treatments such as photodynamic therapy of cancer (PDT) have fueled a growing need for reliable predictive markers for treatment outcome. In the present work we have studied the differential response of two phenotypically and genotypically different breast adenocarcinoma cell lines, MCF7 and MDA-MB-231, to hypericin PDT (HYP-PDT). MDA-MB-231 cells were 70% more sensitive to HYP PDT than MCF7 cells at LD 50 . MCF7 were found to express a substantially higher level of glutathione peroxidase (GPX4) than MDA-MB-231, while MDA-MB-231 differentially expressed glutathione-S-transferase (GSTP1), mainly used for xenobiotic detoxification. Eighty % reduction of intracellular glutathione (GSH) by buthionine sulfoximine (BSO), largely enhanced the sensitivity of the GSTP1 expressing MDA-MB-231 cells to HYP-PDT, but not in MCF7 cells. Further inhibition of the GSH reduction however by carmustine (BCNU) resulted in an enhanced sensitivity of MCF7 to HYP-PDT. HYP loading studies suggested that HYP can be a substrate of GSTP for GSH conjugation as BSO enhanced the cellular HYP accumulation by 20% in MDA-MB-231 cells, but not in MCF7 cells. Studies in solutions showed that L-cysteine can bind the GSTP substrate CDNB in the absence of GSTP. This means that the GSTP-lacking MCF7 may use L-cysteine for xenobiotic detoxification, especially during GSH synthesis inhibition, which leads to L-cysteine build-up. This was confirmed by the lowered accumulation of HYP in both cell lines in the presence of BSO and the L-cysteine source NAC. NAC reduced the sensitivity of MCF7, but not MDA-MB-231, cells to HYP PDT which is in accordance with the antioxidant effects of L-cysteine and its potential as a GSTP substrate. As a conclusion we have herein shown that the different GSH based cell defense mechanisms can be utilized as predictive markers for the outcome of PDT and as a guide for selecting optimal combination strategies. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Glutathione level after long-term occupational elemental mercury exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kobal, Alfred Bogomir; Prezelj, Marija; Horvat, Milena

2008-05-15

Many in vitro and in vivo studies have elucidated the interaction of inorganic mercury (Hg) and glutathione. However, human studies are limited. In this study, we investigated the potential effects of remote long-term intermittent occupational elemental Hg vapour (Hg{sup o}) exposure on erythrocyte glutathione levels and some antioxidative enzyme activities in ex-mercury miners in the period after exposure. The study included 49 ex-mercury miners divided into subgroups of 28 still active, Hg{sup o}-not-exposed miners and 21 elderly retired miners, and 41 controls, age-matched to the miners subgroup. The control workers were taken from 'mercury-free works'. Reduced glutathione (GSH) and oxidizedmore » disulphide glutathione (GSSG) concentrations in haemolysed erythrocytes were determined by capillary electrophoresis, while total glutathione (total GSH) and the GSH/GSSG ratio were calculated from the determined values. Catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) activities in erythrocytes were measured using commercially available reagent kits, while urine Hg (U-Hg) concentrations were determined by cold vapour atomic absorption (CVAAS). No correlation of present U-Hg levels, GSH, GSSG, and antioxidative enzymes with remote occupational biological exposure indices were found. The mean CAT activity in miners and retired miners was significantly higher (p<0.05) than in the controls. No differences in mean GPx activity among the three groups were found, whereas the mean GR activity was significantly higher (p<0.05) in miners than in retired miners. The mean concentrations of GSH (mmol/g Hb) in miners (13.03{+-}3.71) were significantly higher (p<0.05) than in the control group (11.68{+-}2.66). No differences in mean total GSH, GSSG levels, and GSH/GSSG ratio between miners and controls were found. A positive correlation between GSSG and present U-Hg excretion (r=0.41, p=0.001) in the whole group of ex-mercury miners was observed. The significantly lower GSH level (p<0.05) determined in the group of retired miners (9.64{+-}1.45) seems to be age-related (r=-0.39, p=0.001). Thus, the moderate but significantly increased GSH level, GR and CAT activity in erythrocytes in the subgroup of miners observed in the period after exposure to Hg{sup o} could be an inductive and additive response to maintain the balance between GSH and antioxidative enzymes in interaction with the Hg body burden accumulated during remote occupational exposure, which does not represent a severely increased oxidative stress.« less

l-Cysteine supplementation increases insulin sensitivity mediated by upregulation of GSH and adiponectin in high glucose treated 3T3-L1 adipocytes.

PubMed

Achari, Arunkumar E; Jain, Sushil K

2017-09-15

Diabetic patients have lower blood levels of l-cysteine (LC) and glutathione (GSH). This study examined the hypothesis that LC supplementation positively up regulates the effects of insulin on GSH and glucose metabolism in 3T3-L1 adipocyte model. 3T3L1 adipocytes were treated with LC (250 μM, 2 h) and/or insulin (15 or 30 nM, 2 h), and high glucose (HG, 25 mM, 20 h). Results showed that HG caused significant increase (95%) in ROS and reduction in the protein levels of DsbA-L (43%), adiponectin (64%), GCLC (20%), GCLM (21%), GSH (50%), and GLUT-4 (23%) in adipocytes. Furthermore, HG caused a reduction in total (35%) and HMW adiponectin (30%) secretion. Treatment with insulin alone significantly (p < 0.05) reduced ROS levels as well as increased DsbA-L, adiponectin, GCLC, GCLM, GSH, and GLUT-4 protein levels, glucose utilization, and improved total and HMW adiponectin secretion in HG treated adipocytes compared to HG alone. Interestingly, LC supplementation along with insulin caused greater reduction in ROS levels and significantly (p < 0.05) boosted the DsbA-L (41% vs LC, 29% vs Insulin), adiponectin (92% Vs LC, 84% Vs insulin) protein levels and total (32% Vs LC, 22% Vs insulin) and HMW adiponectin (75% Vs LC, 39% Vs insulin) secretion compared with the either insulin or LC alone in HG-treated cells. In addition, LC supplementation along with insulin increased GCLC (21% Vs LC, 14% insulin), GCLM (28% Vs LC, 16% insulin) and GSH (25% Vs LC and insulin) levels compared with the either insulin or LC alone in HG-treated cells. Furthermore, LC and insulin increases GLUT-4 protein expression (65% Vs LC, 18% Vs Insulin), glucose utilization (57% Vs LC, 27% Vs insulin) compared with the either insulin or LC alone in HG-treated cells. Similarly, LC supplementation increased insulin action significantly in cells maintained in medium contained control glucose. To explore the beneficial effect of LC is mediated by the upregulation of GCLC, we knocked down GCLC using siRNA in adipoctyes. There was a significant decrease in DsbA-L and GLUT-4 mRNA levels and GSH levels in GCLC knockdown adipocytes and LC supplementation up regulates GCLC, DsbA-L and GLUT-4 mRNA expression and GSH levels in GCLC knockdown cells. These results demonstrated that LC along with insulin increases GSH levels thereby improving adiponectin secretion and glucose utilization in adipocytes. This suggests that LC supplementation can increase insulin sensitivity and can be used as an adjuvant therapy for diabetes. Copyright © 2017. Published by Elsevier Inc.

Competitive reactions among glutathione, cisplatin and copper-phenanthroline complexes.

PubMed

Cadoni, Enzo; Valletta, Elisa; Caddeo, Graziano; Isaia, Francesco; Cabiddu, Maria Grazia; Vascellari, Sarah; Pivetta, Tiziana

2017-08-01

A large number of cancers are treated with cisplatin (CDDP). However, its use is limited by drug resistance, which is often related to intracellular levels of thiol-containing molecules such as glutathione (GSH). The role of GSH in cisplatin-resistant cancer cells is still unclear. GSH may form adducts with CDDP which results in the deactivation of the drug, and, actually, a high intracellular level of GSH was observed in some cisplatin-resistant cancers. To overcome drug resistance, CDDP is often administered in combination with one or more drugs to exploit a possible synergistic effect. In previous studies, we observed that the sensitivity to CDDP of leukemic and ovarian cisplatin-resistant cancer cells was restored in the presence of [Cu(phen) 2 (H 2 O)](ClO 4 ) 2 (C0) (phen is 1,10-phenathroline). In order to clarify the possible interactions between GSH and CDDP, the reactivity and competitive reactions among CDDP, C0 and GSH in binary and ternary mixtures were studied. The investigation was extended also to [Cu(phen)(H 2 O) 2 (ClO 4 ) 2 ] (C10) and GSSG, the oxidized form of GSH. It was observed that CDDP was able to react with the studied copper complexes and with GSH or GSSG. However, in mixtures containing CDDP, GSH or GSSG and C0 or C10, only copper-glutathione complexes were detected, while no platinum-glutathione adducts were found. Copyright © 2017 Elsevier Inc. All rights reserved.

The fine-tuning of TRAF2–GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex

PubMed Central

De Luca, A; Mei, G; Rosato, N; Nicolai, E; Federici, L; Palumbo, C; Pastore, A; Serra, M; Caccuri, A M

2014-01-01

We provide the first biochemical evidence of a direct interaction between the glutathione transferase P1-1 (GSTP1-1) and the TRAF domain of TNF receptor-associated factor 2 (TRAF2), and describe how ligand binding modulates such an equilibrium. The dissociation constant of the heterocomplex is Kd=0.3 μM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (Kd≥2.6 μM) or is inactivated by oxidation (Kd=1.7 μM). This indicates that GSTP1-1's TRAF2-binding region involves the GSH-binding site. The GSTP1-1 inhibitor NBDHEX further decreases the complex's binding affinity, as compared with when GSH is the only ligand; this suggests that the hydrophobic portion of the GSTP1-1 active site also contributes to the interaction. We therefore hypothesize that TRAF2 binding inactivates GSTP1-1; however, analysis of the data, using a model taking into account the dimeric nature of GSTP1-1, suggests that GSTP1-1 engages only one subunit in the complex, whereas the second subunit maintains the catalytic activity or binds to other proteins. We also analyzed GSTP1-1's association with TRAF2 at the cellular level. The TRAF2–GSTP1-1 complex was constitutively present in U-2OS cells, but strongly decreased in S, G2 and M phases. Thus the interaction appears regulated in a cell cycle-dependent manner. The variations in the levels of individual proteins seem too limited to explain the complex's drastic decline observed in cells progressing from the G0/G1 to the S–G2–M phases. Moreover, GSH's intracellular content was so high that it always saturated GSTP1-1. Interestingly, the addition of NBDHEX maintains the TRAF2–GSTP1-1 complex at low levels, thus causing a prolonged cell cycle arrest in the G2/M phase. Overall, these findings suggest that a reversible sequestration of TRAF2 into the complex may be crucial for cell cycle progression and that multiple factors are involved in the fine-tuning of this interaction. PMID:24457959

Long-wavelength Low-intensity Photon Therapy (LLPT) for Traumatic Brain Injuries

DTIC Science & Technology

2010-09-08

analysis . These estimates are based on the variability in previously published experiments. Design: For each in vivo model of TBI, rats will be...ATP, GSH, apoptotic indicators, mitochondrial function, and changes in the levels of NSE and S100B proteins will be examined. Analysis of ATP, GSH...or to be run with the sample size intended. In particular, the weight drop assays were limited to the analysis of GSH levels. The cortical contusion

Increased endogenous DNA oxidation correlates to increased iron levels in melanocytes relative to keratinocytes.

PubMed

Pelle, Edward; Huang, Xi; Zhang, Qi; Pernodet, Nadine; Yarosh, Daniel B; Frenkel, Krystyna

2014-01-01

The endogenous oxidative state of normal human epidermal melanocytes was investigated and compared to normal human epidermal keratinocytes (NHEKs) in order to gain new insight into melanocyte biology. Previously, we showed that NHEKs contain higher levels of hydrogen peroxide (H2O2) than melanocytes and that it can migrate from NHEKs to melanocytes by passive permeation. Nevertheless, despite lower concentrations of H2O2, we now report higher levels of oxidative DNA in melanocytes as indicated by increased levels of 8-oxo-2'-deoxyguanosine (8-oxo-dG): 4.49 (±0.55 SEM) 8-oxo-dG/10(6) dG compared to 1.49 (±0.11 SEM) 8-oxo-dG/10(6) dG for NHEKs. An antioxidant biomarker, glutathione (GSH), was also lower in melanocytes (3.14 nmoles (±0.15 SEM)/cell) in comparison to NHEKs (5.98 nmoles (±0.33 SEM)/cell). Intriguingly, cellular bioavailable iron as measured in ferritin was found to be nearly fourfold higher in melanocytes than in NHEKs. Further, ferritin levels in melanocytes were also higher than in hepatocarcinoma cells, an iron-rich cell, and it indicates that higher relative iron levels may be characteristic of melanocytes. To account for the increased oxidative DNA and lower GSH and H2O2 levels that we observe, we propose that iron may contribute to higher levels of oxidation by reacting with H2O2 through a Fenton reaction leading to the generation of DNA-reactive hydroxyl radicals. In conclusion, our data support the concept of elevated oxidation and high iron levels as normal parameters of melanocytic activity. We present new evidence that may contribute to our understanding of the melanogenic process and lead to the development of new skin care products.

Effects of mitochondrial poisons on glutathione redox potential and carotid body chemoreceptor activity.

PubMed

Gomez-Niño, A; Agapito, M T; Obeso, A; Gonzalez, C

2009-01-01

Low oxygen sensing in chemoreceptor cells involves the inhibition of specific plasma membrane K(+) channels, suggesting that mitochondria-derived reactive oxygen species (ROS) link hypoxia to K(+) channel inhibition, subsequent cell depolarization and activation of neurotransmitter release. We have used several mitochondrial poisons, alone and in combination with the antioxidant N-acetylcysteine (NAC), and quantify their capacity to alter GSH/GSSG levels and glutathione redox potential (E(GSH)) in rat diaphragm. Selected concentrations of mitochondrial poisons with or without NAC were tested for their capacity to activate neurotransmitter release in chemoreceptor cells and to alter ATP levels in intact rat carotid body (CB). We found that rotenone (1 microM), antimycin A (0.2 microg/ml) and sodium azide (5mM) decreased E(GSH); NAC restored E(GSH) to control values. At those concentrations mitochondrial poisons activated neurotransmitter release from CB chemoreceptor cells and decreased CB ATP levels, NAC being ineffective to modify these responses. Additional experiments with 3-nitroprionate (5mM), lower concentrations of rotenone and dinitrophenol revealed variable relationships between E(GSH) and chemoreceptor cell neurotransmitter release responses and ATP levels. These findings indicate a lack of correlation between mitochondrial-generated modifications of E(GSH) and chemoreceptor cells activity. This lack of correlation renders unlikely that alteration of mitochondrial production of ROS is the physiological pathway chemoreceptor cells use to signal hypoxia.

Effects of polyphenols from seed shells of Japanese horse chestnut (Aesculus turbinata BLUME) on methotrexate-induced intestinal injury in rats.

PubMed

Sugiyama, Akihiko; Kimura, Hideto; Ogawa, Satoshi; Yokota, Kazushige; Takeuchi, Takashi

2011-05-01

The purpose of this study was to evaluate the effects of polyphenols from seed shells of Japanese horse chestnut (JHP) on methotrexate (MTX)-induced intestinal injury in rats. MTX application caused intestinal morphological injury and increase in malondialdehyde (MDA) levels, decrease in levels of glutathione (GSH) and glutathione peroxidase (GSH-Px) activities in small intestine. However, oral administration of JHP ameliorated MTX-induced intestinal injury and inhibited the increase in MDA and the decrease in GSH and GSH-Px activity in small intestine. In conclusion, our results indicated that oral administration of JHP alleviated MTX-induced intestinal injury through its antioxidant properties.

Cocaine-Induced Adaptations in Cellular Redox Balance Contributes to Enduring Behavioral Plasticity

PubMed Central

Uys, Joachim D; Knackstedt, Lori; Hurt, Phelipe; Tew, Kenneth D; Manevich, Yefim; Hutchens, Steven; Townsend, Danyelle M; Kalivas, Peter W

2011-01-01

Impaired glutamate homeostasis in the nucleus accumbens has been linked to cocaine relapse in animal models, and results in part from cocaine-induced downregulation of the cystine–glutamate exchanger. In addition to regulating extracellular glutamate, the uptake of cystine by the exchanger is a rate-limiting step in the synthesis of glutathione (GSH). GSH is critical for balancing cellular redox in response to oxidative stress. Cocaine administration induces oxidative stress, and we first determined if downregulated cystine–glutamate exchange alters redox homeostasis in rats withdrawn from daily cocaine injections and then challenged with acute cocaine. Among the daily cocaine-induced changes in redox homeostasis were an increase in protein S-glutathionylation and a decrease in expression of GSH-S-transferase pi (GSTpi). To mimic reduced GSTpi, a genetic mouse model of GSTpi deletion or pharmacological inhibition of GSTpi by administering ketoprofen during daily cocaine administration was used. The capacity of cocaine to induce conditioned place preference or locomotor sensitization was augmented, indicating that reducing GSTpi may contribute to cocaine-induced behavioral neuroplasticity. Conversely, an acute cocaine challenge after withdrawal from daily cocaine elicited a marked increase in accumbens GSTpi, and the expression of behavioral sensitization to a cocaine challenge injection was inhibited by ketoprofen pretreatment; supporting a protective effect by the acute cocaine-induced rise in GSTpi. Together, these data indicate that cocaine-induced oxidative stress induces changes in GSTpi that contribute to cocaine-induced behavioral plasticity. PMID:21796101

[Cyclosporin A causes oxidative stress and mitochondrial dysfunction in renal tubular cells].

PubMed

Pérez de Hornedo, J; de Arriba, G; Calvino, M; Benito, S; Parra, T

2007-01-01

Reactive oxygen species (ROS) have been implicated in cyclosporin A (CsA) nephrotoxicity. As mitochondria are one of the main sources of ROS in cells, we evaluated the role of CsA in mitochondrial structure and function in LLC-PK1 cells. We incubated cells with CsA 1 microM for 24 hours and studies were performed with flow citometry and confocal microscopy. We studied mitochondrial NAD(P)H content, superoxide anion (O2.-) production (MitoSOX Red), oxidation of cardiolipin of inner mitochondrial membrane (NAO) and mitochondrial membrane potential (DIOC2(3)). Also we analyzed the intracellular ROS synthesis (H2DCF-DA) and reduced glutation (GSH) of cells. Our results showed that CsA decreased NAD(P)H and membrane potential, and increased O2.- in mitochondria. CsA also provoked oxidation of cardiolipin. Furthermore, CsA increased intracellular ROS production and decreased GSH content. These results suggest that CsA has crucial effects in mitochondria. CsA modified mitochondrial physiology through the decrease of antioxidant mitochondrial compounds as NAD(P)H and the dissipation of mitochondrial membrane potential and increase of oxidants as O2.-. Also, CsA alters lipidic structure of inner mitochondrial membrane through the oxidation of cardiolipin. These effects trigger a chain of events that favour intracellular synthesis of ROS and depletion of GSH that can compromise cellular viability. Nephrotoxic cellular effects of CsA can be explained, at least in part, through its influence on mitochondrial functionalism.

Maneb and Paraquat-Mediated Neurotoxicity: Involvement of Peroxiredoxin/Thioredoxin System

PubMed Central

Roede, James R.; Hansen, Jason M.; Go, Young-Mi; Jones, Dean P.

2011-01-01

Epidemiological and in vivo studies have demonstrated that exposure to the pesticides paraquat (PQ) and maneb (MB) increase the risk of developing Parkinson’s disease (PD) and cause dopaminergic cell loss, respectively. PQ is a well-recognized cause of oxidative toxicity; therefore, the purpose of this study was to determine if MB potentiates oxidative stress caused by PQ, thus providing a mechanism for enhanced neurotoxicity by the combination. The results show that PQ alone at a moderately toxic dose (20–30% cell death in 24 h) caused increased reactive oxygen species (ROS) generation, oxidation of mitochondrial thioredoxin-2 and peroxiredoxin-3, lesser oxidation of cytoplasmic thioredoxin-1 and peroxiredoxin-1, and no oxidation of cellular GSH/GSSG. In contrast, MB alone at a similar toxic dose resulted in no ROS generation, no oxidation of thioredoxin and peroxiredoxin, and an increase in cellular GSH after 24 h. Together, MB increased GSH and inhibited ROS production and thioredoxin/peroxiredoxin oxidation observed with PQ alone, yet resulted in more extensive (> 50%) cell death. MB treatment resulted in increased abundance of nuclear Nrf2 and mRNA for phase II enzymes under the control of Nrf2, indicating activation of cell protective responses. The results show that MB potentiation of PQ neurotoxicity does not occur by enhancing oxidative stress and suggests that increased toxicity occurs by a combination of divergent mechanisms, perhaps involving alkylation by MB and oxidation by PQ. PMID:21402726

The glutathione redox system is essential to prevent ferroptosis caused by impaired lipid metabolism in clear cell renal cell carcinoma.

PubMed

Miess, Heike; Dankworth, Beatrice; Gouw, Arvin M; Rosenfeldt, Mathias; Schmitz, Werner; Jiang, Ming; Saunders, Becky; Howell, Michael; Downward, Julian; Felsher, Dean W; Peck, Barrie; Schulze, Almut

2018-06-05

Metabolic reprogramming is a prominent feature of clear cell renal cell carcinoma (ccRCC). Here we investigated metabolic dependencies in a panel of ccRCC cell lines using nutrient depletion, functional RNAi screening and inhibitor treatment. We found that ccRCC cells are highly sensitive to the depletion of glutamine or cystine, two amino acids required for glutathione (GSH) synthesis. Moreover, silencing of enzymes of the GSH biosynthesis pathway or glutathione peroxidases, which depend on GSH for the removal of cellular hydroperoxides, selectively reduced viability of ccRCC cells but did not affect the growth of non-malignant renal epithelial cells. Inhibition of GSH synthesis triggered ferroptosis, an iron-dependent form of cell death associated with enhanced lipid peroxidation. VHL is a major tumour suppressor in ccRCC and loss of VHL leads to stabilisation of hypoxia inducible factors HIF-1α and HIF-2α. Restoration of functional VHL via exogenous expression of pVHL reverted ccRCC cells to an oxidative metabolism and rendered them insensitive to the induction of ferroptosis. VHL reconstituted cells also exhibited reduced lipid storage and higher expression of genes associated with oxidiative phosphorylation and fatty acid metabolism. Importantly, inhibition of β-oxidation or mitochondrial ATP-synthesis restored ferroptosis sensitivity in VHL reconstituted cells. We also found that inhibition of GSH synthesis blocked tumour growth in a MYC-dependent mouse model of renal cancer. Together, our data suggest that reduced fatty acid metabolism due to inhibition of β-oxidation renders renal cancer cells highly dependent on the GSH/GPX pathway to prevent lipid peroxidation and ferroptotic cell death.

Post-fermentative production of glutathione by baker's yeast (S. cerevisiae) in compressed and dried forms.

PubMed

Musatti, Alida; Manzoni, Matilde; Rollini, Manuela

2013-01-25

The study was aimed at investigating the best biotransformation conditions to increase intracellular glutathione (GSH) levels in samples of baker's yeast (Saccharomyces cerevisiae) employing either the commercially available compressed and dried forms. Glucose, GSH precursors amino acids, as well as other cofactors, were dissolved in a biotransformation solution and yeast cells were added (5%dcw). Two response surface central composite designs (RSCCDs) were performed in sequence: in the first step the influence of amino acid composition (cysteine, glycine, glutamic acid and serine) on GSH accumulation was investigated; once their formulation was set up, the influence of other components was studied. Initial GSH content was found 0.53 and 0.47%dcw for compressed and dried forms. GSH accumulation ability of baker's yeast in compressed form was higher at the beginning of shelf life, that is, in the first week, and a maximum of 2.04%dcw was obtained. Performance of yeast in dried form was not found satisfactory, as the maximum GSH level was 1.18%dcw. When cysteine lacks from the reaction solution, yeast cells do not accumulate GSH. With dried yeast, the highest GSH yields occurred when cysteine was set at 3 g/L, glycine and glutamic acid at least at 4 g/L, without serine. Employing compressed yeast, the highest GSH yields occurred when cysteine and glutamic acid were set at 2-3 g/L, while glycine and serine higher than 2 g/L. Results allowed to set up an optimal and feasible procedure to obtain GSH-enriched yeast biomass, with up to threefold increase with respect to initial content. Copyright © 2012 Elsevier B.V. All rights reserved.

Relative importance of redox buffers GSH and NAD(P)H in age-related neurodegeneration and Alzheimer disease-like mouse neurons.

PubMed

Ghosh, Debolina; Levault, Kelsey R; Brewer, Gregory J

2014-08-01

Aging, a major risk factor in Alzheimer's disease (AD), is associated with an oxidative redox shift, decreased redox buffer protection, and increased free radical reactive oxygen species (ROS) generation, probably linked to mitochondrial dysfunction. While NADH is the ultimate electron donor for many redox reactions, including oxidative phosphorylation, glutathione (GSH) is the major ROS detoxifying redox buffer in the cell. Here, we explored the relative importance of NADH and GSH to neurodegeneration in aging and AD neurons from nontransgenic and 3xTg-AD mice by inhibiting their synthesis to determine whether NADH can compensate for the GSH loss to maintain redox balance. Neurons stressed by either depleting NAD(P)H or GSH indicated that NADH redox control is upstream of GSH levels. Further, although depletion of NAD(P)H or GSH correlated linearly with neuron death, compared with GSH depletion, higher neurodegeneration was observed when NAD(P)H was extrapolated to zero, especially in old age, and in the 3xTg-AD neurons. We also observed an age-dependent loss of gene expression of key redox-dependent biosynthetic enzymes, NAMPT (nicotinamide phosphoribosyltransferase), and NNT (nicotinamide nucleotide transhydrogenase). Moreover, age-related correlations between brain NNT or NAMPT gene expression and NADPH levels suggest that these genes contribute to the age-related declines in NAD(P)H. Our data indicate that in aging and more so in AD-like neurons, NAD(P)H redox control is upstream of GSH and an oxidative redox shift that promotes neurodegeneration. Thus, NAD(P)H generation may be a more efficacious therapeutic target upstream of GSH and ROS. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Binding of Glutathione to Enterovirus Capsids Is Essential for Virion Morphogenesis

PubMed Central

Thibaut, Hendrik Jan; Thys, Bert; Canela, María-Dolores; Aguado, Leire; Wimmer, Eckard; Paul, Aniko; Pérez-Pérez, María-Jesús; van Kuppeveld, Frank J. M.; Neyts, Johan

2014-01-01

Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis. PMID:24722756

Effects of captopril on the cysteamine-induced duodenal ulcer in the rat.

PubMed

Saghaei, Firoozeh; Karimi, Iraj; Jouyban, Abolghasem; Samini, Morteza

2012-05-01

Oxidative stress is important factor underlying in a variety of diseases. Antioxidative enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) are part of the physiological defenses against oxidative stress. Malondialdehyde (MDA) is a lipid peroxidation biomarker and its elevated level in various diseases is related to free radical damage. Cysteamine is a cytotoxic agent, acting through generation of reactive oxygen species (ROS) and may decrease defense activity of antioxidative enzymes against ROS and induce duodenal ulcer. Captopril, acts as free radical scavengers and protect against injuries from oxidative damage to tissues.The aim of this study was the evaluation of the effect of captopril against cysteamine-induced duodenal ulcer by determining duodenal damage, duodenal tissue SOD and GSH-PX activities and plasma MAD level. This study was performed on 3 groups of 7 rats each: saline, cysteamine and cysteamine plus captopril treated groups. The effect of captopril against cysteamine-induced duodenal ulcer is determined by evaluating the duodenal damage, duodenal tissue SOD and GSH-PX activities and plasma MDA level. All animals were euthanized 24h after the last treatment and 2 ml blood and duodena samples were collected for calculation of ulcer index, histopathological assessment and measurement of tissue SOD, GSH-PX activities and plasma MDA level. Cysteamine produced severe duodenal damage, decreased the activity of duodenal tissue SOD and GSH-PX and increased the plasma MDA level compared with saline pretreated rats. Pretreatment with captopril decreased the cysteamine-induced duodenal damage and plasma level of MDA and increased the activities of SOD and GSH-PX in duodenal tissue compared with cysteamine pretreated animal. Our results suggest that captopril protects against cysteamine-induced duodenal ulcer and inhibits the decrease in SOD and GSH-PX activities and lipid peroxidation by increasing antioxidant defenses. Copyright © 2010 Elsevier GmbH. All rights reserved.

The Amelioration of N-Acetyl-p-Benzoquinone Imine Toxicity by Ginsenoside Rg3: The Role of Nrf2-Mediated Detoxification and Mrp1/Mrp3 Transports

PubMed Central

Gum, Sang Il; Cho, Min Kyung

2013-01-01

Previously, we found that Korean red ginseng suppressed acetaminophen (APAP)-induced hepatotoxicity via alteration of its metabolic profile involving GSTA2 induction and that ginsenoside Rg3 was a major component of this gene induction. In the present study, therefore, we assessed the protective effect of Rg3 against N-acetyl-p-benzoquinone imine (NAPQI), a toxic metabolic intermediate of APAP. Excess NAPQI resulted in GSH depletion with increases in the ALT and AST activities in H4IIE cells. Rg3 pretreatment reversed GSH depletion by NAPQI. Rg3 resulted in increased mRNA levels of the catalytic and modulatory subunit of glutamate cysteine ligase (GCL), the rate-limiting steps in GSH synthesis and subsequently increased GSH content. Rg3 increased levels of nuclear Nrf2, an essential transcriptional factor of these genes. The knockdown or knockout of the Nrf2 gene abrogated the inductions of mRNA and protein by Rg3. Abolishment of the reversal of GSH depletion by Rg3 against NAPQI was observed in Nrf2-deficient cells. Rg3 induced multidrug resistance-associated protein (Mrp) 1 and Mrp3 mRNA levels, but not in Nrf2-deficient cells. Taken together, these results demonstrate that Rg3 is efficacious in protecting hepatocytes against NAPQI insult, due to GSH repletion and coordinated gene regulations of GSH synthesis and Mrp family genes by Nrf2. PMID:23766864

Methylenedioxy designer drugs: mass spectrometric characterization of their glutathione conjugates by means of liquid chromatography-high-resolution mass spectrometry/mass spectrometry and studies on their glutathionyl transferase inhibition potency.

PubMed

Meyer, Markus R; Richter, Lilian H J; Maurer, Hans H

2014-04-25

Methylenedioxy designer drugs of abuse such as 3,4-methylenedioxymethamphetamine (MDMA) can be selectively toxic to serotonergic neurons and glutathione (GSH) adducts have been implicated in its neurotoxicity. The catecholic demethylenyl metabolites of MDMA, 3,4-dihydroxymethamphetamine and 3,4-dihydroxyamphetamine, are metabolically oxidized to the corresponding ortho-quinones, which are highly reactive intermediates. These intermediates can then be conjugated with GSH preventing cellular damage. Furthermore, glutathionyl transferase (GST) activity was described to be irreversibly inhibited by the catechols dopamine, α-methyldopa and their GSH conjugates. Therefore, the aims of the present work were the detection and characterization of GSH conjugates of ten methylenedioxy drugs of abuse and their phase I metabolites as well as to assess their inhibition potency on GST activity. The substrates were incubated using human placental GST with or without preincubation by cytochrome P450 enzymes preparations. GST inhibition was tested using chlorodinitrobenzene GSH conjugation as marker reaction. GSH conjugates were analyzed and characterized using LC-high-resolution-MS/MS. For confirmation of postulated fragmentation patterns, formation of GSH conjugates of selected deuterated analogs (deuterated analogue approach, DAA) of the investigated drugs was explored. For the methylenedioxy amphetamines the following steps could be identified: conjugation of the parent compounds at position 2, 5, 6, of the demethylenyl metabolites at position 2 and 5, and of the further deaminated demethylenyl metabolites at position 2. For the β-keto-phenylalkylamine and pyrrolidinophenone, conjugation of the demethylenyl metabolites and of the deaminated demethylenyl metabolites at position 2 could be identified. The DAA allowed the differentiation of the 2 and 5/6 isomers by confirmation of the postulated mass spectral fragments. Finally, the tested drugs and phase I metabolites showed no inhibition potency on GST activity. Copyright © 2014 Elsevier B.V. All rights reserved.

Glutathione redox regulates airway hyperresponsiveness and airway inflammation in mice.

PubMed

Koike, Yoko; Hisada, Takeshi; Utsugi, Mitsuyoshi; Ishizuka, Tamotsu; Shimizu, Yasuo; Ono, Akihiro; Murata, Yukie; Hamuro, Junji; Mori, Masatomo; Dobashi, Kunio

2007-09-01

Glutathione is the major intracellular redox buffer. We have shown that glutathione redox status, which is the balance between intracellular reduced (GSH) and oxidized (GSSG) glutathione, in antigen-presenting cells (APC) regulates the helper T cell type 1 (Th1)/Th2 balance due to the production of IL-12. Bronchial asthma is a typical Th2 disease. Th2 cells and Th2 cytokines are characteristic of asthma and trigger off an inflammation. Accordingly, we studied the effects of the intracellular glutathione redox status on airway hyperresponsiveness (AHR) and allergen-induced airway inflammation in a mouse model of asthma. We used gamma-Glutamylcysteinylethyl ester (gamma-GCE), which is a membrane-permeating GSH precursor, to elevate the intracellular GSH level and GSH/GSSG ratio of mice. In vitro, gamma-GCE pretreatment of human monocytic THP-1 cells elevated the GSH/GSSG ratio and enhanced IL-12(p70) production induced by LPS. In the mouse asthma model, intraperitoneal injection of gamma-GCE elevated the GSH/GSSG ratio of lung tissue and reduced AHR. gamma-GCE reduced levels of IL-4, IL-5, IL-10, and the chemokines eotaxin and RANTES (regulated on activation, normal T cell expressed and secreted) in bronchoalveolar lavage fluid, whereas it enhanced the production of IL-12 and IFN-gamma. Histologically, gamma-GCE suppressed eosinophils infiltration. Interestingly, we also found that gamma-GCE directly inhibited chemokine-induced eosinophil chemotaxis without affecting eotaxin receptor chemokine receptor 3 (CCR3) expressions. Taken together, these findings suggest that changing glutathione redox balance, increase in GSH level, and the GSH/GSSG ratio by gamma-GCE, ameliorate bronchial asthma by altering the Th1/Th2 imbalance through IL-12 production from APC and suppressing chemokine production and eosinophil migration itself.

Development of Smart Phthalocyanine-based Photosensitizers for Photodynamic Therapy

NASA Astrophysics Data System (ADS)

Chow, Yun Sang

Phthalocyanines are versatile functional dyes that have shown great potential in cancer theranostics, especially in photodynamic therapy (PDT). This research work aims to develop "smart" phthalocyanine-based photosensitizers for targeted PDT. This thesis describes the synthesis, spectroscopic characterization, photophysical properties, and in vitro photodynamic activities of several series of carefully designed phthalocyanine-based photosensitizers. Chapter 1 presents an overview of PDT, including its historical development, photophysical mechanisms, and biological mechanisms. Various classes of photosensitizers are introduced with emphasis putting on phthalocyanines, which exhibit ideal characteristics of photosensitizers for PDT. In recent years, several approaches have been used to develop photosensitizers with higher tumor selectivity and minimal skin photosensitivity after PDT. Activatable photosensitizers can provide a "turn on" mechanism to offer an additional control of the specificity of treatment. Photosensitizers can also work cooperatively with the tumor-targeting groups or anticancer drugs so as to achieve targeted or dual therapy, which can enhance the efficacy of PDT. The novel approaches mentioned above have been widely used and combined to form multi-functional photosensitizing agents. These novel concepts and development of PDT are discussed and illustrated with relevant examples at the end of this chapter. To minimize the prolonged skin photosensitivity, photosensitizers that can only be activated by tumor-associated stimuli have been developed. Due to the abnormal metabolism in tumor tissues, their surface usually exhibits a lower pH compared to that of the normal tissues. Also, the pH difference between the intracellular and the physiological environment provides a pH-activation mechanism. Chapter 2 presents the synthesis and spectroscopic characterization of a pH-responsive zinc(II) phthalocyanine tetramer, in which the phthalocyanine units are self-quenched, but separated and activated after cleavage of the acetal linkages. The basic photophysical properties, activation behavior in fluorescence emission, singlet oxygen generation, and in vitro photodynamic activities of this compound have been investigated and compared with those of the non-cleavable analogue. The activation of this tetramer has been demonstrated in the aqueous medium and at the cellular level, in which the fluorescence emission and photosensitizing ability can be greatly enhanced. This tetramer exhibits low dark toxicity and enhanced cytotoxicity upon illumination toward the HT29 human colon adenocarcinoma cells. Glutathione (GSH) is another well-documented stimulus for the design of activatable photosensitizers. It is the most abundant biological thiol which acts as the reducing agent in many biochemical processes. The much higher intracellular GSH concentration compared with the extracellular GSH level provides an activation mechanism. By using this feature, we have developed a series of GSH-responsive phthalocyanine dendrons in order to optimize the molecular design of self-quenched activatable photosensitizers. The aggregation tendency and the activation behavior in fluorescence emission and singlet oxygen generation in aqueous media have been investigated. The in vitro properties of the phthalocyanine dendrons including relative intracellular fluorescence and photocytotoxicity are also examined and presented in Chapter 3.. Chapter 4 reports the molecular design and synthesis of a biotin-decorated GSH-responsive zinc(II) phthalocyanine trimer, in which the phthalocyanine units are severely self-quenched, but are separated and then activated upon cleavage of the disulfide bondings by GSH. The basic photophysical properties, cleavage kinetics, and activation behavior in fluorescence and photosensitizing ability of this compound have been investigated. Its in vitro photodynamic activity, cellular uptake by different cell lines, and intracellular fluorescence emission have also been examined. Apart from targeted PDT, dual therapy is another novel approach to improve the efficacy of PDT treatment. Chapter 5 describes the molecular design and synthesis of a novel doxorubicin-conjugated phthalocyanine trimer. Doxorubicin is a well-known drug for the treatment of a range of cancers. By conjugating this unit to a GSH-responsive zinc(II) phthalocyanine trimer, it is expected that the resulting hybrid can exhibit controlled toxicity. The basic photophysical properties and effects of GSH on the fluorescence emission and photosensitizing ability of this compound have been investigated. Its cytotoxicity in the presence and absence of light, cellular uptake, and intracellular fluorescence emission have been examined and are reported in this chapter. Chapter 6 describes a novel strategy to prepare low-symmetry phthalocyanines, which can serve as versatile platforms for the development of multi-functional phthalocyanine-based photosensitizers. By using an intracellular cyclization reaction of the pre-linked tetrakisphthalonitrile, the first ABCD-type zinc(II) phthalocyanine has been synthesized and characterized. By optimizing the reaction conditions, this compound can be prepared in 7.2 % yield. Chapter 7 concludes the present study while Chapter 8 reports the experimental details. All references mentioned in this thesis are provided in Chapter 9. 1H and 13C{1H} NMR spectra of all the new compounds are given in the Appendix. (Abstract shortened by ProQuest.).

Glutathione metabolism in the HaCaT cell line as a model for the detoxification of the model sensitisers 2,4-dinitrohalobenzenes in human skin.

PubMed

Jacquoilleot, Sandrine; Sheffield, David; Olayanju, Adedamola; Sison-Young, Rowena; Kitteringham, Neil R; Naisbitt, Dean J; Aleksic, Maja

2015-08-19

Glutathione (GSH) is the most prominent antioxidant in cells and the co-factor of an important set of enzymes involved in the skin metabolic clearance system, glutathione S-transferases (GST). Here, we describe an LC-MS (liquid chromatography-mass spectroscopy) method to measure GSH and its disulfide form (GSSG) in HaCaT cells and a 3D Reconstructed Human Epidermis (RHE) model. In our assay, the basal level of GSH in both systems was in the low nmol/mg soluble protein range, while the level of GSSG was systematically below our limit of quantification (0.1 μM). We found that 2,4-dinitrohalobenzenes deplete the GSH present in HaCaT cells within the first hour of exposure, in a dose dependent manner. The level of GSH in HaCaT cells treated with a single non-toxic dose of 10 μM of dinitrohalobenzene was also shown to increase after two hours. While cells treated with 1-chloro-2,4-dinitrobenzene (DNCB) and 1-fluoro-2,4-dinitrobenzene (DNFB) repleted GSH to levels similar to untreated control cells within 24h, 1-bromo-2,4-dinitrobenzene (DNBB) seemed to prevent such a repletion and appeared to be the most toxic compound in all assays. A mathematical modelling of experimental results was performed to further rationalise the differences observed between test chemicals. For this purpose the biological phenomena observed were simplified into two sequential events: the initial depletion of the GSH stock after chemical treatment followed by the repletion of the GSH once the chemical was cleared. Activation of the nuclear factor E2-related factor 2 (Nrf2) pathway was observed with all compounds within two hours, and at concentrations less than 10 μM. These data show that GSH depletion and repletion occur rapidly in skin cells and emphasize the importance of conducting kinetic studies when performing in vitro experiments exploring skin sensitization. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Neurochemistry of major depression: a study using magnetic resonance spectroscopy.

PubMed

Godlewska, Beata R; Near, Jamie; Cowen, Philip J

2015-02-01

Magnetic resonance spectroscopy (MRS) is an acceptable non-invasive means of studying brain neurochemistry in depression. Previous studies in depressed patients have focused on measurement of the amino acid neurotransmitters, γ-aminobutyric acid (GABA) and glutamate. The aim of this study is to use MRS in conjunction with the ultrashort echo time 'SPECIAL' technique to measure cortical levels of GABA, glutamate and glutathione (GSH) levels in unmedicated patients with major depression. We also examined the effect of 6-week treatment with the selective serotonin re-uptake inhibitor, escitalopram. We studied patients with DSM-IV major depression and healthy age-matched controls using proton MRS. GABA, glutamate and GSH were measured relative to creatine in a voxel placed in occipital cortex. There was no difference in GABA or glutamate levels between depressed participants and controls; however, depressed patients had lower GSH levels. Six-week escitalopram treatment, which resulted in significant clinical responses in some patients, did not alter concentrations of GABA, glutamate or GSH. The sources of variability of GABA and glutamate measures in different studies of depressed patients require further study. Our results suggest that concomitant treatment with selective serotonin re-uptake inhibitors (SSRIs) is unlikely to be an important confounding factor. If lowered GSH levels can be confirmed, they may represent the presence of oxidative stress in some depressed patients.

Effects of exogenous vitamins A, C, and E and NADH supplementation on proliferation, cytokines release, and cell redox status of lymphocytes from healthy aged subjects.

PubMed

Bouamama, Samia; Merzouk, Hafida; Medjdoub, Amel; Merzouk-Saidi, Amel; Merzouk, Sid Ahmed

2017-06-01

Aging is an inevitable biological event that is associated with immune alterations. These alterations are related to increased cellular oxidative stress and micronutrient deficiency. Antioxidant supplementation could improve these age-related abnormalities. The aim of this study was to determine in vitro effects of vitamin A, vitamin C, vitamin E, and nicotinamide adenine dinucleotide (NADH) on T cell proliferation, cytokine release, and cell redox status in the elderly compared with young adults. Peripheral blood lymphocytes were isolated using a density gradient of Histopaque. They were cultured in vitro and stimulated with concanavalin A in the presence or absence of vitamins. Cell proliferation was determined by conducting MTT assays, and based on interleukin-2 and interleukin-4 secretions. Cell oxidant/antioxidant balance was assessed by assaying reduced glutathione (GSH), malondialdehyde, carbonyl protein levels, and catalase activity. The present study demonstrated that T-lymphocyte proliferation was decreased with aging and was associated with cytokine secretion alterations, GSH depletion, and intracellular oxidative stress. In the elderly, vitamin C, vitamin E, and NADH significantly improved lymphocyte proliferation and mitigated cellular oxidative stress, whereas vitamin A did not affect cell proliferation or cell redox status. In conclusion, vitamin C, vitamin E, and NADH supplementation improved T-lymphocytes response in the elderly, and could contribute to the prevention of age-related immune alterations. Consumption of food items containing these vitamins is recommended, and further investigation is necessary to evaluate the effect of vitamin supplementation in vivo.

Role of Oxidative Stress in the Induction of Metallothionein-2A and Heme Oxygenase-1 Gene Expression by the Antineoplastic Agent Gallium Nitrate in Human Lymphoma Cells

PubMed Central

Yang, Meiying; Chitambar, Christopher R.

2008-01-01

The mechanisms of action of gallium nitrate, an antineoplastic drug, are only partly understood. Using a DNA microarray to examine genes induced by gallium nitrate in CCRF-CEM cells, we found that gallium increased metallothionein-2A (MT2A) and heme oxygenase-1 (HO-1) gene expression and altered the levels of other stress-related genes. MT2A and HO-1 were increased after 6 and 16 h of incubation with gallium nitrate. An increase in oxidative stress, evidenced by a decrease in cellular GSH and GSH/GSSG ratio, and an increase in dichlorodihydrofluoroscein (DCF) fluorescence, was seen after 1 – 4 h incubation of cells with gallium nitrate. DCF fluorescence was blocked by the mitochondria-targeted antioxidant mitoquinone. N-acetyl-L-cysteine blocked gallium-induced MT2A and HO-1 expression and increased gallium’s cytotoxicity. Studies with a zinc-specific fluoroprobe suggested that gallium produced an expansion of an intracellular labile zinc pool, suggesting an action of gallium on zinc homeostasis. Gallium nitrate increased the phosphorylation of p38 mitogen-activated protein kinase and activated Nrf-2, a regulator of HO-1 gene transcription. Gallium-induced Nrf-2 activation and HO-1 expression were diminished by a p38 MAP kinase inhibitor. We conclude that gallium nitrate induces cellular oxidative stress as an early event which then triggers the expression of HO-1 and MT2A through different pathways. PMID:18586083

Different roles of glutathione in copper and zinc chelation in Brassica napus roots.

PubMed

Zlobin, Ilya E; Kartashov, Alexander V; Shpakovski, George V

2017-09-01

We investigated the specific features of copper and zinc excess action on the roots of canola (Brassica napus L.) plants. Copper rapidly accumulated in canola root cells and reached saturation during several hours of treatment, whereas the root zinc content increased relatively slowly. Excessive copper and zinc entry inside the cell resulted in significant cell damage, as evidenced by alterations in plasmalemma permeability and decreases in cellular enzymatic activity. Zinc excess specifically damaged root hair cells, which correlated with a pronounced elevation of their labile zinc level. In vitro, we showed that reduced glutathione (GSH) readily reacted with copper ions to form complexes with blocked sulfhydryl groups. In contrast, zinc ions were ineffective as glutathione blockers, and glutathione molecules did not lose their specific chemical activity in the presence of Zn 2+ ions. The effect of copper and zinc excess on the glutathione pool in canola root cells was analysed by a combination of biochemical determination of total and oxidized glutathione contents and fluorescent staining of free reduced glutathione with monochlorobimane dye. Excess copper led to dose-dependent diminution of free reduced glutathione contents in the root cells, which could not be explained by the loss of total cellular glutathione or its oxidation. In contrast, we observed little effect of much higher intracellular zinc concentrations on the free reduced glutathione content. We concluded that GSH plays an important role in copper excess, but not zinc excess chelation, in canola root cells. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Effects of N-acetyl-cysteine treatment on glutathione depletion and a short-term spatial memory deficit in 2-cyclohexene-1-one-treated rats.

PubMed

Choy, Kwok Ho Christopher; Dean, Olivia; Berk, Michael; Bush, Ashley I; van den Buuse, Maarten

2010-12-15

Glutathione (GSH) is the primary antioxidant in the body and is present in high levels in the brain. Levels of GSH and other antioxidants are significantly altered in major psychiatric illnesses, such as schizophrenia. Recent clinical trials have demonstrated that chronic treatment with N-acetyl-l-cysteine (NAC), a GSH precursor, improved symptoms in individuals with this illness. We previously showed in rats and mice that depletion of GSH by treatment with 2-cyclohexene-1-one (CHX) induced short-term spatial memory deficits in the Y-maze test. The aim of present study was to characterise the effect of NAC in this CHX-induced glutathione depletion model. Consistent with our previous studies, CHX treatment induced approximately 50% reduction of GSH levels in striatum, hippocampus and frontal cortex tissue. GSH depletion was significantly rescued by either 1.2 g/kg or 1.6 g/kg of NAC administration, with a full recovery observed in the frontal cortex after the high dose of NAC. CHX treatment also induced a disruption in short-term spatial recognition memory in Y-maze test, as measured by the duration of time spent in the novel arm. This disruption was reversed by treatment with 1.6 g/kg of NAC. In conclusion, this study suggests that rescue of depleted levels of GSH in the brain restores cognitive deficits, as measured by the Y-maze. These effects appear to be dose-dependent and region-specific. These results may be relevant to the understanding and management of the cognitive symptoms of schizophrenia and bipolar disorder. Copyright © 2010 Elsevier B.V. All rights reserved.

A γ-Glutamyl Transpeptidase-Independent Pathway of Glutathione Catabolism to Glutamate via 5-Oxoproline in Arabidopsis1[W][OA

PubMed Central

Ohkama-Ohtsu, Naoko; Oikawa, Akira; Zhao, Ping; Xiang, Chengbin; Saito, Kazuki; Oliver, David J.

2008-01-01

The degradation pathway of glutathione (GSH) in plants is not well understood. In mammals, GSH is predominantly metabolized through the γ-glutamyl cycle, where GSH is degraded by the sequential reaction of γ-glutamyl transpeptidase (GGT), γ-glutamyl cyclotransferase, and 5-oxoprolinase to yield glutamate (Glu) and dipeptides that are subject to peptidase action. In this study, we examined if GSH is degraded through the same pathway in Arabidopsis (Arabidopsis thaliana) as occurs in mammals. In Arabidopsis, the oxoprolinase knockout mutants (oxp1-1 and oxp1-2) accumulate more 5-oxoproline (5OP) and less Glu than wild-type plants, suggesting substantial metabolite flux though 5OP and that 5OP is a major contributor to Glu steady-state levels. In the ggt1-1/ggt4-1/oxp1-1 triple mutant with no GGT activity in any organs except young siliques, the 5OP concentration in leaves was not different from that in oxp1-1, suggesting that GGTs are not major contributors to 5OP production in Arabidopsis. 5OP formation strongly tracked the level of GSH in Arabidopsis plants, suggesting that GSH is the precursor of 5OP in a GGT-independent reaction. Kinetics analysis suggests that γ-glutamyl cyclotransferase is the major source of GSH degradation and 5OP formation in Arabidopsis. This discovery led us to propose a new pathway for GSH turnover in plants where GSH is converted to 5OP and then to Glu by the combined action of γ-glutamyl cyclotransferase and 5-oxoprolinase in the cytoplasm. PMID:18768907

A gamma-glutamyl transpeptidase-independent pathway of glutathione catabolism to glutamate via 5-oxoproline in Arabidopsis.

PubMed

Ohkama-Ohtsu, Naoko; Oikawa, Akira; Zhao, Ping; Xiang, Chengbin; Saito, Kazuki; Oliver, David J

2008-11-01

The degradation pathway of glutathione (GSH) in plants is not well understood. In mammals, GSH is predominantly metabolized through the gamma-glutamyl cycle, where GSH is degraded by the sequential reaction of gamma-glutamyl transpeptidase (GGT), gamma-glutamyl cyclotransferase, and 5-oxoprolinase to yield glutamate (Glu) and dipeptides that are subject to peptidase action. In this study, we examined if GSH is degraded through the same pathway in Arabidopsis (Arabidopsis thaliana) as occurs in mammals. In Arabidopsis, the oxoprolinase knockout mutants (oxp1-1 and oxp1-2) accumulate more 5-oxoproline (5OP) and less Glu than wild-type plants, suggesting substantial metabolite flux though 5OP and that 5OP is a major contributor to Glu steady-state levels. In the ggt1-1/ggt4-1/oxp1-1 triple mutant with no GGT activity in any organs except young siliques, the 5OP concentration in leaves was not different from that in oxp1-1, suggesting that GGTs are not major contributors to 5OP production in Arabidopsis. 5OP formation strongly tracked the level of GSH in Arabidopsis plants, suggesting that GSH is the precursor of 5OP in a GGT-independent reaction. Kinetics analysis suggests that gamma-glutamyl cyclotransferase is the major source of GSH degradation and 5OP formation in Arabidopsis. This discovery led us to propose a new pathway for GSH turnover in plants where GSH is converted to 5OP and then to Glu by the combined action of gamma-glutamyl cyclotransferase and 5-oxoprolinase in the cytoplasm.

Diesel exhaust particles (DEP) induce an early redox imbalance followed by an IL-6 mediated inflammatory response on human conjunctival epithelial cells.

PubMed

Lasagni Vitar, Romina M; Tau, Julia; Janezic, Natasha S; Tesone, Agustina I; Hvozda Arana, Ailen G; Reides, Claudia G; Berra, Alejandro; Ferreira, Sandra M; Llesuy, Susana F

2018-06-01

The aim of this study was to evaluate the time course of oxidative stress markers and inflammatory mediators in human conjunctival epithelial cells (IOBA-NHC) exposed to diesel exhaust particles (DEP) for 1, 3, and 24 h. Reactive oxygen species (ROS) production, lipid and protein oxidation, Nrf2 pathway activation, enzymatic antioxidants, glutathione (GSH) levels and synthesis, as well as cytokine release and cell proliferation were analyzed. Cells exposed to DEP showed an increase in ROS at all time points. The induction of NADPH oxidase-4 appeared later than mitochondrial superoxide anion production, when the cell also underwent a proinflammatory response mediated by IL-6. DEP exposure triggered the activation of Nrf2 in IOBA-NHC, as a strategy for increasing cellular antioxidant capacity. Antioxidant enzyme activities were significantly increased at early stages except for glutathione reductase (GR) that showed a significant decrease after a 3-h-incubation. GSH levels were found increased after 1 and 3 h of incubation with DEP, despite the increase in its consumption by the antioxidant enzymes as it works as a cofactor. GSH recycling and the de novo synthesis were responsible for the maintenance of its content at these time points, respectively. After 24 h, the decrease in GR and glutamate cysteine ligase as wells as the enhanced activity of glutathione peroxidase and glutathione S-transferase produced a depletion in the GSH pool. Lipid-peroxidation was found increased in cells exposed to DEP after 1-h-incubation, whereas protein oxidation was found increased in cells exposed to DEP after a 3-h-incubation that persisted after a longer exposure. Furthermore, DEP lead IOBA-NHC cells to hyperplasia after 1 and 3 h of incubation, but a decrease in cell proliferation was found after longer exposure. ROS production seems to be an earlier event triggered by DEP on IOBA-NHC, comparing to the proinflammatory response mediated by IL-6. Despite the fact that under short periods of exposure to DEP lipids and then proteins are targets of oxidative damage, the viability of the cells is not affected at early stages, since cell hyperplasia was detected as compensatory mechanism. Although after 24 h Nrf2 pathway is still enhanced, the epithelial cell capacity to maintain redox balance is exceeded. The antioxidant enzymes activation and the depleted GSH pool are not capable of counteracting the increased ROS production, leading to oxidative damage. Copyright © 2018 Elsevier Ltd. All rights reserved.

Glutathione as a promising anti-hydrophobicity agent against Malassezia spp.

PubMed

Sivasankar, Chandran; Ponmalar, Ayyappan; Bhaskar, James P; Pandian, Shunmugiah K

2015-10-01

The genus Malassezia has recently attracted wide attention in medical microbiology and dermatology as a pathogen. They are lipophilic yeasts possessing high level of cell surface hydrophobicity (CSH). L-glutathione (GSH) is a ubiquitous antioxidant which offers protection against microbial infections. This study is intended to investigate the role of GSH as a potential anti-hydrophobicity agent against Malazessia spp. Microbial adherence to hydrocarbon assay was performed to assess the anti-hydrophobicity activity (AHA) of GSH against four Malassezia spp. The assay revealed that GSH at 400 μg ml(-1) concentration inhibited CSH, ranging from 84% to 95% in M. furfur, M. globosa, M. restricta and M. sympodialis without killing the cells. The AHA of GSH was corroborated by auto-aggregation assay and zeta-potential measurement, through which delayed cell aggregation was observed due to reduction in CSH level and not by modification in cell surface charge. In addition, colony-forming unit assay was performed in which 62-93% of CSH reduction was observed in Malassezia spp. tested. Furthermore, GSH treatment enhanced the sensitivity of Malassezia spp. towards human blood at the rate of 64-72%. The AHA was further confirmed through Fourier transform infrared analysis. Thus, this study portrays GSH as a prospective therapeutic alternative for Malassezia-mediated infections. © 2015 Blackwell Verlag GmbH.

Cellular Uptake and Tissue Biodistribution of Functionalized Gold Nanoparticles and Nanoclusters.

PubMed

Escudero-Francos, María A; Cepas, Vanesa; González-Menédez, Pedro; Badía-Laíño, Rosana; Díaz-García, Marta E; Sainz, Rosa M; Mayo, Juan C; Hevia, David

2017-02-01

In this study, the in vitro uptake by fibroblasts and in vivo biodistribution of 15 nm 11-mercaptoundecanoicacid-protected gold nanoparticles (AuNPs-MUA) and 3 nm glutathione- and 3 nm bovine serum albumin-protected gold nanoclusters (AuNCs@GSH and AuNCs@BSA, respectively) were evaluated. In vitro cell viability was examined after gold nanoparticle treatment for 48 h, based on MTT assays and analyses of morphological structure, the cycle cell, cellular doubling time, and the gold concentration in cells. No potential toxicity was observed at any studied concentration (up to 10 ppm) for AuNCs@GSH and AuNCs@BSA, whereas lower cell viability was observed for AuNPs-MUA at 10 ppm than for other treatments. Neither morphological damage nor modifications to the cell cycle and doubling time were detected after contact with nanoparticles. Associations between cells and AuNPs and AuNCs were demonstrated by inductively coupled plasma mass spectrometry (ICP-MS). AuNCs@GSH exhibited fluorescence emission at 611 nm, whereas AuNCs@BSA showed a band at 640 nm. These properties were employed to confirm their associations with cells by fluorescence confocal microscopy; both clusters were observed in cells and maintained their original fluorescence. In vivo assays were performed using 9 male mice treated with 1.70 μg Au/g body weight gold nanoparticles for 24 h. ICP-MS measurements showed a different biodistribution for each type of nanoparticle; AuNPs-MUA mainly accumulated in the brain, AuNCs@GSH in the kidney, and AuNCs@BSA in the liver and spleen. Spleen indexes were not affected by nanoparticle treatment; however, AuNCs@BSA increased the thymus index significantly from 1.28 to 1.79, indicating an immune response. These nanoparticles have great potential as organ-specific drug carriers and for diagnosis, photothermal therapy, and imaging.

Label-free Raman spectroscopy for accessing intracellular anticancer drug release on gold nanoparticles.

PubMed

Ock, Kwang-Su; Ganbold, Erdene Ochir; Park, Jin; Cho, Keunchang; Joo, Sang-Woo; Lee, So Yeong

2012-06-21

We investigated glutathione (GSH)-induced purine or pyrimidine anticancer drug release on gold nanoparticle (AuNP) surfaces by means of label-free Raman spectroscopy. GSH-triggered releases of 6-thioguanine (6TG), gemcitabine (GEM), acycloguanosine (ACY), and fadrozole (FAD) were examined in a comparative way by means of surface-enhanced Raman scattering (SERS). The GSH-induced dissociation constant of GEM (or ACY/FAD) from AuNPs was estimated to be larger by more than 38 times than that of 6TG from the kinetic relationship. Tripeptide control experiments were presented to check the turn-off Raman signalling mechanism. Dark-field microscopy (DFM) and transmission electron microscopy (TEM) indicated the intracellular AuNP loads. After their cellular uptake, GEM, ACY, and FAD would not show SERS intensities as strong as 6TG. This may be due to easier release of GEM, ACY, and FAD than 6TG by intracellular reducing species including GSH. We observed fairly strong SERS signals of GEM and 6TG in cell culture media solution. Our CCK-8 cytotoxicity assay data support that 6TG-AuNPs did not exhibit a substantial decrease in cell viability presumably due to strong binding. Label-free confocal Raman spectroscopy can be utilized as an effective tool to access intracellular anticancer drug release.

Decreasing GSH and increasing ROS in chemosensitivity gliomas with IDH1 mutation.

PubMed

Shi, Jinlong; Sun, Baolan; Shi, Wei; Zuo, Hao; Cui, Daming; Ni, Lanchun; Chen, Jian

2015-02-01

Gliomas are the most malignant and aggressive primary brain tumor in adults. Despite concerted efforts to improve therapies, their prognosis remains very poor. Isocitrate dehydrogenase 1 (IDH1) mutations have been discovered frequently in glioma patients and are strongly correlated with improved survival. However, the effect of IDH1 mutations on the chemosensitivity of gliomas remains unclear. In this study, we generated clonal U87 and U251 glioma cell lines overexpressing the R132H mutant protein (IDH1-R132H). Compared with control cells and cells overexpressing IDH wild type (IDH1-WT), both types of IDH1-R132H cells were more sensitive to temozolomide (TMZ) and cis-diamminedichloroplatinum (CDDP) in a time- and dose-dependent manner. The IDH1-R132H-induced higher chemosensitivity was associated with nicotine adenine disphosphonucleotide (NADPH), glutathione (GSH) depletion, and reactive oxygen species (ROS) generation. Accordingly, this IDH1-R132H-induced growth inhibition was effectively abrogated by GSH in vitro and in vivo. Our study provides direct evidence that the improved survival in patients with IDH1-R132H tumors may partly result from the effects of the IDH1-R132H protein on chemosensitivity. The primary cellular events associated with improved survival are the GSH depletion and increased ROS generation.

Redox-responsive nanoparticles with Aggregation-Induced Emission (AIE) characteristic for fluorescence imaging.

PubMed

Cheng, Weiren; Wang, Guan; Pan, Xiaoyong; Zhang, Yong; Tang, Ben Zhong; Liu, Ye

2014-08-01

The redox environment between intracellular compartments and extracellular matrix is significantly different, and the cellular redox homeostasis determines many physiological functions. Here, redox-responsive nanoparticles with aggregation-induced emission (AIE) characteristic for fluorescence imaging are developed by encapsulation of fluorophore with redox "turn-on" AIE characteristic, TPE-MI, into the micelles of poly(ethylene glycol) (PEG)- and cholesterol (CE)-conjugated disulfide containing poly(amido amine)s. The redox-responsive fluorescence profiles of the nanoparticles are investigated after reaction with glutathione (GSH). The encapsulation of TPE-MI in micelles leads to a higher efficiency and red shift in emission, and the fluorescence intensity of the nanoparticles increases with the concentration of GSH. Confocal microscopy imaging shows that the nanoparticles can provide obvious contrast between the intracellular compartments and the extracellular matrix in MCF-7 and HepG2 cells. So the nanoparticles with PEG shells and low cytotoxicity are promising to provide fluorescence bioimaging with a high contrast and for differentiation of cellular redox environment. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Effects of Electroacupuncture Intervention on Oxygen Free Radicals and Expression of Apoptosis-related Proteins in Rats with Ischemic Learning and Memory Disorder].

PubMed

Hou, Zhi-tao; Sun, Zhong-ren; Liu, Song-tao; Xiong, Sheng-biao; Liu, Yi-tian; Han, Xiao-xia; Sun, Hong-fang; Han, Yu-sheng; Yin, Hong-na; Xu, Jin-qiao; Li, Dong-dong

2015-12-01

To observe the effect of electroacupuncture (EA) therapy on levels of oxygen free radicals (OFR) and hippocampal apoptosis-related protein expression in ischemic learning-memory disorder rats so as to investigate its mechanisms underlying improvement of ischemic learning-memory impairment. A total of 60 SD rats were randomly divided into sham operation (sham), model, medication, and EA groups, with 15 rats in each group. The learning-memory disorder model was made by occlusion of bilateral carotid arteries. EA (2- 3 Hz, 2 mA) was applied to "Zhi San Zhen" ["Shenting" (GV 24) and bilateral "Benshen" (GB 13)] for 30 min, once a day for 3 weeks. The rats of the medication group were treated by lavage of Aricept (0.03 mg . kg(-1) . d(-1)), once daily for 3 weeks. The rats' learning-memory ability was detected by Morris water maze tests and the state of hippocampal apoptosis cells was observed by light microscope after TUNEL staining and the expression of hippocampal Bcl-2, Bax and Caspase-3 proteins was detected by immunohistochemistry. Serum and hippocampal superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) contents were detected by chemical colorimetric analysis. Compared with the sham group, the escape latencies (place-navigation) after modeling were evidently prolonged, and the times of target-platform crossing in 90 sec (spatial probe test) considerably reduced in the model group (P<0.01), suggesting an impairment of learning-memory ability. After the treatment for 21 d, the increased escape latency and the reduced target-platform crossing time in both EA and medication groups were reversed in comparison with the model group (P<0.01), suggesting an improvement of memory ability, and the effect of the EA group was significantly superior to that of the medication group (P<0.05). Compared with the sham group, the number of apoptotic cells in hippocampal CA 1- CA 3 regions, and the expression levels of hippocampal Bcl-2, Bax and Caspase-3 proteins, and serum and hippocampal MDA contents were significantly increased in the model group (P<0.01), while serum and hippocampal SOD and GSH-Px levels obviously decreased in the model group (P<0.01). After the treatment for 21 days, compared to the model group, the number of the apoptotic cells, the expression levels of hippocampal Bax and Caspase--3 proteins, and the contents of serum and hippocampal MDA were notably decreased in the EA and medication groups (P<0.01), whereas, Bcl-2 protein expression levels, and serum and hippocampal SOD and GSH-Px activity were notably up-regulated in the EA and medication groups (P<0.01). The effects of EA group were obviously superior to those of medication group in increasing hippocampal Bcl-2 immunoactivity, serum SOD and GSH-Px and hippocampal GSH-Px activity and in down-regulating serum MDA level (P<0.01, P<0.05). Electroacupuncture intervention can improve learning-memory ability in ischemic learning-memory disorder rats which may be associated with its effects in reducing blood and hippocampal OFR contents and hippocampal cellular apoptosis.

[Effect of DNA polymerase beta on apoptosis and mitochondrial membrane potential induced by hydroquinone, a metabolite of benzene].

PubMed

Chen, Chen; Yang, Mo; Zhang, Zun-zhen; Wu, Mei; Deng, Wen-wen

2011-12-01

To explore the effect and mechanism of DNA polymerase β expression level on cell apoptosis and mitochondrial membrane potential induced by hydroquinone. Polβ wild-type cells (polβ+/+), polβ overexpressed cells (polβ oe) and polβ null cells (polβ-/-) were applied as a model cell system, The effect of cell apoptosis and mitochondrial membrane potential induced by different doses of hydroquinone were analyzed by flow cytometry. The ROS and ·OH assay kit were used to examine the cellular ROS and ·OH level. The activity of cellular SOD and GSH-Px were tested by Chemiluminescence method after exposed to different concentrations of hydroquinone. With the dose of hydroquinone increased, the rate of apoptosis and falling of mitochondrial membrane potential (ΔΨm) in cells were increased compared with the control. When compared with polβ+/+ cells, the rate of apoptosis in polβ-/- cells exposed to 20.00, 40.00, 80.00 µmol/L hydroquinone increased and the rate of apoptosis in polβ oe cells exposed to 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone decreased (P < 0.05). Compared with polβ+/+ cells (20.60% ± 0.57%, 37.95% ± 0.64%, 44.50% ± 1.27%, 57.55% ± 1.06%), the rate of cell which undergone mitochondrial depolarization in polβ-/- cells treated with 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone (33.60% ± 1.55%, 46.05% ± 1.77%, 52.75% ± 2.05%, 75.20% ± 0.56%) increased. The rate of cell which undergone mitochondrial depolarization in polβ oe cells exposed to 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone (16.05% ± 1.20%, 29.80% ± 1.21%, 35.15% ± 1.06%, 53.80% ± 0.85%) decreased (P < 0.05). When compared with polβ+/+ cells, fluorescent intensity of polβ-/- cells treated with different dosages of hydroquinone increased, while which of polβ oe cells decreased (P < 0.05). Compared with polβ+/+ cells, ·OH level of polβ-/- cells treated with 20.00, 40.00 µmol/L hydroquinone significantly enhanced, while which of polβ oe cells decreased sharply (P < 0.05). Under the same concentrations of hydroquinone, the activity of SOD and GSH-Px were decreased most rapidly in polβ-/- cells. The activity of SOD and GSH-Px in polβ oe cells decreased slower than in the polβ-/- cells. Hydroquinone could induced apoptosis by the generation of ROS and decrease of ΔΨm; polβ could protect cells from apoptosis induced by hydroquinone through decrease of ROS level and depolarization of mitochondria.

Protective role of hesperidin against γ-radiation-induced oxidative stress and apoptosis in rat testis.

PubMed

Shaban, Nadia Z; Ahmed Zahran, Ahmed M; El-Rashidy, Fatma H; Abdo Kodous, Ahmad S

2017-12-01

Gamma (γ) ray, an electromagnetic radiation, is occasionally accompanying the emission of an alpha or beta particle. Exposure to such radiation can cause cellular changes such as mutations, chromosome aberration and cellular damage which depend upon the total amount of energy, duration of exposure and the dose. Ionizing radiation can impair spermatogenesis and can cause mutations in germ cells. In general, type B spermatogonia are sensitive to this type of radiation. The current study was carried out to evaluate the protective role of hesperidin (H), as a polyphenolic compound, on rat testis injury induced by γ-radiation. Rats were divided into groups including C group (control rats), R (irradiated) group (rats irradiated with γ-radiation), Vehicle (V) group (rats administered with dimethylsulfoxide "DMSO"), H group (rats administered with H only), HR and RH groups (rats treated with H before and after exposure to γ-radiation, respectively). Malondialdehyde (MDA: the end product of lipid peroxidation "LPO") and xanthine oxidase (XO: it generates reactive oxygen species "ROS") in testes homogenate as well as nitric oxide (NO: as ROS) in mitochondrial matrix were determined. The apoptotic markers including DNA-fragmentation (DNAF) in testes homogenate and calcium ions (Ca 2+ ) in mitochondrial matrix were determined. Superoxide dismutase (SOD) and catalase (CAT) activities in testes homogenate, while reduced glutathione "GSH" in nuclear matrix were determined. Also histopathological examination for testes tissues through electron microscope was studied. Exposure of rats to γ-radiation (R group) increased the levels of MDA, NO, DNAF, Ca 2+ and XO activity, while it decreased GSH level, SOD and CAT activities as compared to the C groups; γ-radiation increased oxidative stress (OS), LPO, apoptosis and induced testes injuries. These results are in agreement with the histopathological examination. In contrast, treatment with H before or after exposure to γ-radiation (HR and RH groups, respectively) decreased the levels of MDA, NO, DNAF and Ca 2+ but increased GSH level and the activities of SOD, CAT and XO as compared to R group and this indicates that H decreased OS, LPO and apoptosis. Also, the histopathological results showed that H improved testis architecture and this is related to the antioxidant and anti-apoptotic activities of H contents. Protection is more effective when H is given before rather than after exposure. Finally, administration of H to healthy rats for a short period had no adverse affect on testes cells. Hesperidin showed antioxidant and anti-apoptotic activities. It has a protective role against OS, injury and apoptosis induced by γ-radiation in testes. Protection is more effective when H is given before rather than after exposure.Graphical Abstract.

Yeast aquaporin regulation by 4-hydroxynonenal is implicated in oxidative stress response.

PubMed

Rodrigues, Claudia; Tartaro Bujak, Ivana; Mihaljević, Branka; Soveral, Graça; Cipak Gasparovic, Ana

2017-05-01

Reactive oxygen species, especially hydrogen peroxide (H 2 O 2 ), contribute to functional molecular impairment and cellular damage, but also are necessary in normal cellular metabolism, and in low doses play stimulatory role in cell proliferation and stress resistance. In parallel, reactive aldehydes such as 4-hydroxynonenal (HNE), are lipid peroxidation breakdown products which also contribute to regulation of numerous cellular processes. Recently, channeling of H 2 O 2 by some mammalian aquaporin isoforms has been reported and suggested to contribute to aquaporin involvement in cancer malignancies, although the mechanism by which these membrane water channels are implicated in oxidative stress is not clear. In this study, two yeast models with increased levels of membrane polyunsaturated fatty acids (PUFAs) and aquaporin AQY1 overexpression, respectively, were used to evaluate their interplay in cell's oxidative status. In particular, the aim of the study was to investigate if HNE accumulation could affect aquaporin function with an outcome in oxidative stress response. The data showed that induction of aquaporin expression by PUFAs results in increased water permeability in yeast membranes and that AQY1 activity is impaired by HNE. Moreover, AQY1 expression increases cellular sensitivity to oxidative stress by facilitating H 2 O 2 influx. On the other hand, AQY1 expression has no influence on the cellular antioxidant GSH levels and catalase activity. These results strongly suggest that aquaporins are important players in oxidative stress response and could contribute to regulation of cellular processes by regulation of H 2 O 2 influx. © 2017 IUBMB Life, 69(5):355-362, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Abnormal transsulfuration metabolism and reduced antioxidant capacity in Chinese children with autism spectrum disorders.

PubMed

Han, Yu; Xi, Qian-qian; Dai, Wei; Yang, Shu-han; Gao, Lei; Su, Yuan-yuan; Zhang, Xin

2015-11-01

Autism spectrum disorder (ASD) is a neurological disorder that presents a spectrum of qualitative impairments in social interaction, communication, as well as restricted and stereotyped behavioral patterns, interests, and activities. Several studies have suggested that the etiology of ASD can be partly explained by oxidative stress. However, the implications of abnormal transsulfuration metabolism and oxidative stress, and their relation with ASD are still unclear. The purpose of this study was to evaluate several transsulfuration pathway metabolites in Chinese participants diagnosed with ASD, to better understand their role in the etiology of this disorder. Fifty children (39 male, 11 female) diagnosed with ASD and 50 age- and gender-matched non-ASD children (i.e., control group) were included in this study. This prospective blinded study was undertaken to assess transsulfuration and oxidative metabolites, including levels of homocysteine (Hcy), cysteine (Cys), total glutathione (tGSH), reduced glutathione (GSH), oxidized glutathione (GSSG), and glutathione ratio (GSH/GSSG). The clinical severity of ASD was evaluated with the Childhood Autism Rating Scale (CARS), and the autistic children's present behavior was measured by the Autism Behavior Checklist (ABC). The results indicated that Hcy and GSSG levels were significantly higher in children diagnosed with ASD, Cys, tGSH and GSH levels as well as the GSH/GSSG ratio showed remarkably lower values in ASD children compared to control subjects. Hcy levels correlated significantly with increasing CARS scores and GSSG levels in children with ASD. Our results suggest that an abnormal transsulfuration metabolism and reduced antioxidant capacity (i.e., hyperhomocysteinemia and increased oxidative stress), and Hcy level appears to have a potentially negative impact on clinical severity of autistic disorder. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ajoene restored behavioral patterns and liver glutathione level in morphine treated C57BL6 mice.

PubMed

Yun, Jaesuk; Oliynyk, Sergiy; Lee, Yeonju; Kim, Jieun; Yun, Kyunghwa; Jeon, Raok; Ryu, Jae-Ha; Oh, Seikwan

2017-01-01

Oxidative stress exacerbates drug dependence induced by administration of opiate analgesics such as morphine-induced tolerance and physical dependence associated with the reduction in hepatic glutathione (GSH) level. Ajoene obtained from garlic (Allium sativum L.) has been reported for anti-tumorigenic, anti-oxidative and neuroprotective properties, however, little is known about its effect on morphine-induced dependence. Therefore, this study aimed at the effect of ajoene on physical and/or psychological dependence and liver GSH content in morphine-treated mice. Conditioned place preference (CPP) test and measurement of morphine withdrawal syndrome were performed in C57BL6 mice for behavioral experiments. Thereafter, mice were sacrificed for measurement of serum and liver GSH levels. Ajoene restored CPP and naloxone-precipitated jumping behavior in mice exposed to morphine. Moreover, the reduced level of liver GSH content in morphine treated mice was back to normal after ajoene administration. Taken together, ajoene improved behavioral patterns in mice exposed to morphine suggesting its potential therapeutic benefit against morphine-induced dependence.

Dexpanthenol therapy reduces lung damage in a hyperoxic lung injury in neonatal rats.

PubMed

Ozdemir, Ramazan; Demirtas, Gulsum; Parlakpinar, Hakan; Polat, Alaadin; Tanbag, Kevser; Taslidere, Elif; Karadag, Ahmet

2016-01-01

Dexpanthenol (Dxp) plays a major role in cellular defense and in repair systems against oxidative stress and inflammatory response and it has not yet been evaluated in treatment of bronchopulmonary dysplasia (BPD). We tested the hypothesis that proposes whether Dxp decreases the severity of lung injury in an animal model of BPD. Forty rat pups were divided into four groups: control, control + Dxp, hyperoxia and hyperoxia + Dxp. All animals were processed for lung histology and tissue analysis. The degree of lung inflammation, oxidative and antioxidant capacity was assessed from lung homogenates. Lung injury score and alveol diameter increased in the hyperoxia group (p < 0.001). Median level of malondialdehyde, total oxidant status and oxidative stress indexes was significantly higher in the hyperoxia group compared to the other groups. The median superoxide dismutase activity in the hyperoxia group was notably less than those of control + Dxp and hyperoxia + Dxp groups (p < 0.01). Similarly, lung catalase, glutathione (GSH) peroxidase and reduced GSH activities in the hyperoxia group were significantly lower than other groups. Furthermore, the hyperoxia + Dxp group had lower tumor necrosis factor-α and interleukin-1β median levels compared to the hyperoxia group (p = 0.007). Dxp treatment results in less emphysematous change as well as decrease in inflammation and oxidative stress markers in an animal model of BPD.

The effect of melatonin on eye lens of rats exposed to ultraviolet radiation.

PubMed

Anwar, M M; Moustafa, M A

2001-05-01

We investigated the influence of exogenously administered melatonin on adult rats eye lenses exposed to ultraviolet radiation (UV) A and B ranging from 356-254 nm irradiation at 8 microW/cm(2). Rats exposed to this range of UV for 15 min for one week showed a significant (P<0.05) reduction in antioxidant enzymes activities; superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and elevated (P<0.001) lipid peroxidation served as an index of cellular damage by free radicals. UV-radiation significantly (P<0.001) elevated calcium ions (Ca(2+)) and lactate dehydrogenase (LDH) activity in lenses. Depleting animals of their stores of important intracellular antioxidant and elevating lenticular Ca(2+) by UV irradiation, may be the main cause of lens opacification. Melatonin injection with radiation significantly reduced (P<0.05) lipid peroxidation, Ca(2+) and (P<0.001) for LDH. When melatonin was injected after radiation, SOD and GSH-Px enzyme activities increased significantly (P<0.01), and lipid peroxidation, Ca(2+) levels and LDH activities were reduced significantly. Melatonin injection after UV radiation was as effective as melatonin treatment concurrent with UV irradiation. We conclude that melatonin may protect the eye lens from the damaging effects of UV exposure, and its actions protect lens from oxidative stress, elevating Ca(2+) levels, which are considered as an important causes of cataractogenesis.

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells.

PubMed

Takahashi, Kyohei; Shibata, Tomohito; Oba, Tatsuya; Ishikawa, Tetsuya; Yoshikawa, Masahito; Tatsunami, Ryosuke; Takahashi, Kazuhiko; Tampo, Yoshiko

2009-02-13

Menadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs). BAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody. Intracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells. We conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.

Protective properties of ginsenoside Rb1 against UV-B radiation-induced oxidative stress in human dermal keratinocytes.

PubMed

Oh, Sun-Joo; Kim, Kyunghoon; Lim, Chang-Jin

2015-06-01

Ginsenosides, also known as ginseng saponins, are responsible for most pharmacological effect of ginseng. Ginsenoside Rb1 (Rb1) exerts a variety of pharmacological properties, including anti-inflammatory, antistress, anti-aging and anti-neurodegenerative activities. The aim of the present work was to assess the skin anti-photoaging properties of Rb1 in human dermal keratinocyte HaCaT cells. The anti-photoaging activity was evaluated by analyzing the levels of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) as well as cell viability for HaCaT cells under UV-B irradiation. Rb1 was able to suppress the ROS levels which were elevated under UV-B irradiation, and unable to influence cellular survival in UV-B-irradiated HaCaT cells. Rb1 diminished the enhancement of MMP-2 gelatinolytic activity in conditioned medium, which corresponded with the decreased MMP-2 protein levels in both conditioned medium and cellular lysate prepared from UV-B-irradiated HaCaT cultures. Rb1 could restore the total glutathione (GSH) and superoxide dismutase (SOD) activity diminished in UV-B-irradiated HaCaT cells. Ginsenoside Rb1 possesses skin anti-photoaging properties through scavenging ROS and decreasing MMP-2 levels possibly by enhancing antioxidant activity in keratinocytes under UV-B irradiation.

The Effects of Ibogaine on Uterine Smooth Muscle Contractions: Relation to the Activity of Antioxidant Enzymes.

PubMed

Oreščanin-Dušić, Zorana; Tatalović, Nikola; Vidonja-Uzelac, Teodora; Nestorov, Jelena; Nikolić-Kokić, Aleksandra; Mijušković, Ana; Spasić, Mihajlo; Paškulin, Roman; Bresjanac, Mara; Blagojević, Duško

2018-01-01

Ibogaine is an indole alkaloid originally extracted from the root bark of the African rainforest shrub Tabernanthe iboga . It has been explored as a treatment for substance abuse because it interrupts drug addiction and relieves withdrawal symptoms. However, it has been shown that ibogaine treatment leads to a sharp and transient fall in cellular ATP level followed by an increase of cellular respiration and ROS production. Since contractile tissues are sensitive to changes in the levels of ATP and ROS, here we investigated an ibogaine-mediated link between altered redox homeostasis and uterine contractile activity. We found that low concentrations of ibogaine stimulated contractile activity in spontaneously active uteri, but incremental increase of doses inhibited it. Inhibitory concentrations of ibogaine led to decreased SOD1 and elevated GSH-Px activity, but doses that completely inhibited contractions increased CAT activity. Western blot analyses showed that changes in enzyme activities were not due to elevated enzyme protein concentrations but posttranslational modifications. Changes in antioxidant enzyme activities point to a vast concentration-dependent increase in H 2 O 2 level. Knowing that extracellular ATP stimulates isolated uterus contractility, while H 2 O 2 has an inhibitory effect, this concentration-dependent stimulation/inhibition could be linked to ibogaine-related alterations in ATP level and redox homeostasis.

The Effects of Ibogaine on Uterine Smooth Muscle Contractions: Relation to the Activity of Antioxidant Enzymes

PubMed Central

Paškulin, Roman

2018-01-01

Ibogaine is an indole alkaloid originally extracted from the root bark of the African rainforest shrub Tabernanthe iboga. It has been explored as a treatment for substance abuse because it interrupts drug addiction and relieves withdrawal symptoms. However, it has been shown that ibogaine treatment leads to a sharp and transient fall in cellular ATP level followed by an increase of cellular respiration and ROS production. Since contractile tissues are sensitive to changes in the levels of ATP and ROS, here we investigated an ibogaine-mediated link between altered redox homeostasis and uterine contractile activity. We found that low concentrations of ibogaine stimulated contractile activity in spontaneously active uteri, but incremental increase of doses inhibited it. Inhibitory concentrations of ibogaine led to decreased SOD1 and elevated GSH-Px activity, but doses that completely inhibited contractions increased CAT activity. Western blot analyses showed that changes in enzyme activities were not due to elevated enzyme protein concentrations but posttranslational modifications. Changes in antioxidant enzyme activities point to a vast concentration-dependent increase in H2O2 level. Knowing that extracellular ATP stimulates isolated uterus contractility, while H2O2 has an inhibitory effect, this concentration-dependent stimulation/inhibition could be linked to ibogaine-related alterations in ATP level and redox homeostasis. PMID:29599898

Glutathione aerosol suppresses lung epithelial surface inflammatory cell-derived oxidants in cystic fibrosis.

PubMed

Roum, J H; Borok, Z; McElvaney, N G; Grimes, G J; Bokser, A D; Buhl, R; Crystal, R G

1999-07-01

Cystic fibrosis (CF) is characterized by accumulation of activated neutrophils and macrophages on the respiratory epithelial surface (RES); these cells release toxic oxidants, which contribute to the marked epithelial derangements seen in CF. These deleterious consequences are magnified, since reduced glutathione (GSH), an antioxidant present in high concentrations in normal respiratory epithelial lining fluid (ELF), is deficient in CF ELF. To evaluate the feasibility of increasing ELF GSH levels and enhancing RES antioxidant protection, GSH aerosol was delivered (600 mg twice daily for 3 days) to seven patients with CF. ELF total, reduced, and oxidized GSH increased (P < 0.05, all compared with before GSH therapy), suggesting adequate RES delivery and utilization of GSH. Phorbol 12-myristate 13-acetate-stimulated superoxide anion (O2-.) release by ELF inflammatory cells decreased after GSH therapy (P < 0.002). This paralleled observations that GSH added in vitro to CF ELF inflammatory cells suppressed O2-. release (P < 0.001). No adverse effects were noted during treatment. Together, these observations demonstrate the feasibility of using GSH aerosol to restore RES oxidant-antioxidant balance in CF and support the rationale for further clinical evaluation.

Copper accumulation in senescent cells: Interplay between copper transporters and impaired autophagy.

PubMed

Masaldan, Shashank; Clatworthy, Sharnel A S; Gamell, Cristina; Smith, Zoe M; Francis, Paul S; Denoyer, Delphine; Meggyesy, Peter M; Fontaine, Sharon La; Cater, Michael A

2018-06-01

Cellular senescence is characterized by irreversible growth arrest incurred through either replicative exhaustion or by pro-oncogenic cellular stressors (radioactivity, oxidative stress, oncogenic activation). The enrichment of senescent cells in tissues with age has been associated with tissue dyshomeostasis and age-related pathologies including cancers, neurodegenerative disorders (e.g. Alzheimer's, Parkinson's, etc.) and metabolic disorders (e.g. diabetes). We identified copper accumulation as being a universal feature of senescent cells [mouse embryonic fibroblasts (MEF), human prostate epithelial cells and human diploid fibroblasts] in vitro. Elevated copper in senescent MEFs was accompanied by elevated levels of high-affinity copper uptake protein 1 (Ctr1), diminished levels of copper-transporting ATPase 1 (Atp7a) (copper export) and enhanced antioxidant defence reflected by elevated levels of glutathione (GSH), superoxide dismutase 1 (SOD1) and glutaredoxin 1 (Grx1). The levels of intracellular copper were further increased in senescent MEFs cultured in copper supplemented medium and in senescent Mottled Brindled (Mo br ) MEFs lacking functional Atp7a. Finally, we demonstrated that the restoration/preservation of autophagic-lysosomal degradation in senescent MEFs following rapamycin treatment correlated with attenuation of copper accumulation in these cells despite a further decrease in Atp7a levels. This study for the first time establishes a link between Atp7a and the autophagic-lysosomal pathway, and a requirement for both to effect efficient copper export. Such a connection between cellular autophagy and copper homeostasis is significant, as both have emerged as important facets of age-associated degenerative disease. Copyright © 2018. Published by Elsevier B.V.

Assessment of the antioxidant activity of an olive oil total polyphenolic fraction and hydroxytyrosol from a Greek Olea europea variety in endothelial cells and myoblasts

PubMed Central

Kouka, Paraskevi; Priftis, Alexandros; Stagos, Dimitrios; Angelis, Apostolis; Stathopoulos, Panagiotis; Xinos, Nikos; Skaltsounis, Alexios-Léandros; Mamoulakis, Charalampos; Tsatsakis, Aristides M.; Spandidos, Demetrios A.; Kouretas, Demetrios

2017-01-01

Olive oil (OO) constitutes the basis of the Mediterranean diet, and it seems that its biophenols, such as hydroxytyrosol (HT) may scavenge free radicals, attracting distinct attention due to their beneficial effects in many pathological conditions, such as cancer. To the best of our knowedge, this is the first study in which the functional properties of an OO total polyphenolic fraction (TPF) and pure HT were examined in order to determine their antioxidant effects at a cellular level in endothelial cells and myoblasts. The test compounds were isolated using a green gradient-elution centrifugal partition chromatography-based method that allows the isolation of large volumes of OO in a continuous extraction procedure and with extremely low solvent consumption. For the isolation of HT, a combination of two chromatographic techniques was used, which is effective for the recovery of pure compounds from complex natural extracts. Moreover, TPF and HT exhibited potent free radical scavenging activity in vitro. The cells were treated with non-cytotoxic concentrations and their redox status [in terms of glutathione (GSH) and reactive oxygen species (ROS) levels] was assessed. TPF extract was less cytotoxic than HT, and the observed differences between the two cell lines used suggest a tissue-specific activity. Finally, flow cytometric analysis revealed that both TPF and HT improved the redox status by increasing the levels of GSH, one of the most important antioxidant molecules, in both endothelial cells and myoblasts, while the ROS levels were not significantly affected. PMID:28731131

Effects of coarse chalk dust particles (2.5-10 μm) on respiratory burst and oxidative stress in alveolar macrophages.

PubMed

Zhang, Yuexia; Yang, Zhenhua; Feng, Yan; Li, Ruijin; Zhang, Quanxi; Geng, Hong; Dong, Chuan

2015-08-01

The main aim of the present study was to examine in vitro responses of rat alveolar macrophages (AMs) exposed to coarse chalk dust particles (particulate matter in the size range 2.5-10 μm, PM(coarse)) by respiratory burst and oxidative stress. Chalk PM(coarse)-induced respiratory burst in AMs was measured by using a luminol-dependent chemiluminescence (CL) method. Also, the cell viability; lactate dehydrogenase (LDH) release; levels of cellular superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), and acid phosphatase (ACP); plasma membrane ATPase; and extracellular nitric oxide (NO) level were determined 4 h following the treatment with the different dosages of chalk PM(coarse). The results showed that chalk PM(coarse) initiated the respiratory burst of AMs as indicated by strong CL, which was inhibited by diphenyleneiodonium chloride and L-N-nitro-L-arginine methyl ester hydrochloride. It suggested that chalk PM(coarse) induced the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in AMs. This hypothesis was confirmed by the fact that chalk PM(coarse) resulted in a significant decrease of intracellular SOD, GSH, ACP, and ATPase levels and a notable increase of intracellular CAT, MDA content, and extracellular NO level, consequently leading to a decrease of the cell viability and a increase of LDH release. It was concluded that AMs exposed to chalk PM(coarse) can suffer from cytotoxicity which may be mediated by generation of excessive ROS/RNS. Graphical Abstract The possible mechanism of coarse chalk particles-induced adverse effects in AMs.

Assessment of the antioxidant activity of an olive oil total polyphenolic fraction and hydroxytyrosol from a Greek Olea europea variety in endothelial cells and myoblasts.

PubMed

Kouka, Paraskevi; Priftis, Alexandros; Stagos, Dimitrios; Angelis, Apostolis; Stathopoulos, Panagiotis; Xinos, Nikos; Skaltsounis, Alexios-Léandros; Mamoulakis, Charalampos; Tsatsakis, Aristides M; Spandidos, Demetrios A; Kouretas, Demetrios

2017-09-01

Olive oil (OO) constitutes the basis of the Mediterranean diet, and it seems that its biophenols, such as hydroxytyrosol (HT) may scavenge free radicals, attracting distinct attention due to their beneficial effects in many pathological conditions, such as cancer. To the best of our knowedge, this is the first study in which the functional properties of an OO total polyphenolic fraction (TPF) and pure HT were examined in order to determine their antioxidant effects at a cellular level in endothelial cells and myoblasts. The test compounds were isolated using a green gradient‑elution centrifugal partition chromatography‑based method that allows the isolation of large volumes of OO in a continuous extraction procedure and with extremely low solvent consumption. For the isolation of HT, a combination of two chromatographic techniques was used, which is effective for the recovery of pure compounds from complex natural extracts. Moreover, TPF and HT exhibited potent free radical scavenging activity in vitro. The cells were treated with non‑cytotoxic concentrations and their redox status [in terms of glutathione (GSH) and reactive oxygen species (ROS) levels] was assessed. TPF extract was less cytotoxic than HT, and the observed differences between the two cell lines used suggest a tissue‑specific activity. Finally, flow cytometric analysis revealed that both TPF and HT improved the redox status by increasing the levels of GSH, one of the most important antioxidant molecules, in both endothelial cells and myoblasts, while the ROS levels were not significantly affected.

Alterations of GSH and MDA levels and their association with bee venom-induced DNA damage in human peripheral blood leukocytes.

PubMed

Gajski, Goran; Domijan, Ana-Marija; Garaj-Vrhovac, Vera

2012-07-01

Bee venom (BV) has toxic effects in a variety of cell systems and oxidative stress has been proposed as a possible mechanism of its toxicity. This study investigated the in vitro effect of BV on glutathione (GSH) and malondialdehyde (MDA) levels, and their association with BV-induced DNA strand breaks and oxidative DNA damage in human peripheral blood leukocytes (HPBLs). Blood samples were treated with BV at concentrations ranging from 0.1 to 10 μg/ml over different lengths of time, and DNA damage in HPBLs was monitored with the alkaline and formamidopyrimidine glycoslyase (FPG)-modified comet assays, while GSH and MDA levels were determined in whole blood. Results showed a significant increase in overall DNA damage and FPG-sensitive sites in DNA of HPBLs exposed to BV compared with HPBLs from controls. An increase in DNA damage (assessed with both comet assays) was significantly associated with changes in MDA and GSH levels. When pretreated with N-acetyl-L-cysteine, a source of cysteine for the synthesis of the endogenous antioxidant GSH, a significant reduction of the DNA damaging effects of BV in HPBLs was noted. This suggests that oxidative stress is at least partly responsible for the DNA damaging effects of BV. Copyright © 2012 Wiley Periodicals, Inc.

Genetic Polymorphism Associated Prefrontal Glutathione and Its Coupling With Brain Glutamate and Peripheral Redox Status in Early Psychosis.

PubMed

Xin, Lijing; Mekle, Ralf; Fournier, Margot; Baumann, Philipp S; Ferrari, Carina; Alameda, Luis; Jenni, Raoul; Lu, Huanxiang; Schaller, Benoit; Cuenod, Michel; Conus, Philippe; Gruetter, Rolf; Do, Kim Q

2016-09-01

Oxidative stress and glutathione (GSH) metabolism dysregulation has been implicated in the pathophysiology of schizophrenia. GAG-trinucleotide repeat (TNR) polymorphisms in the glutamate-cysteine ligase catalytic gene (GCLC), the rate-limiting enzyme for GSH synthesis, are associated with schizophrenia. In addition, GSH may serve as a reserve pool for neuronal glutamate (Glu) through the γ-glutamyl cycle. The aim of this study is to investigate brain [GSH] and its association with GCLC polymorphism, peripheral redox indices and brain Glu. Magnetic resonance spectroscopy was used to measure [GSH] and [Glu] in the medial prefrontal cortex (mPFC) of 25 early-psychosis patients and 33 controls. GCLC polymorphism was genotyped, glutathione peroxidases (GPx) and glutathione reductase (GR) activities were determined in blood cells. Significantly lower [GSHmPFC] in GCLC high-risk genotype subjects were revealed as compared to low-risk genotype subjects independent of disease status. In male subjects, [GSHmPFC] and blood GPx activities correlate positively in controls (P = .021), but negatively in patients (P = .039). In GCLC low-risk genotypes, [GlumPFC] are lower in patients, while it is not the case for high-risk genotypes. GCLC high-risk genotypes are associated with low [GSHmPFC], highlighting that GCLC polymorphisms should be considered in pathology studies of cerebral GSH. Low brain GSH levels are related to low peripheral oxidation status in controls but with high oxidation status in patients, pointing to a dysregulated GSH homeostasis in early psychosis patients. GCLC polymorphisms and disease associated correlations between brain GSH and Glu levels may allow patients stratification. © The Author 2016. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The effect of hemodialysis and dialyzer biocompatibility on erythrocyte glutathione-defense system in chronic hemodialysis patients.

PubMed

Alhamdani, M S; Al-Najjar, A F; Al-Kassir, A H

2005-06-01

Uremic patients, especially those receiving regular hemodialysis (HD) treatment, are at high risk of oxidative damage by noxious free radicals and reactive oxygen species (ROS). The erythrocyte glutathione-defense system (GSH-DS) is one of the major enzymatic means of scavenging and detoxifying ROS. This study aimed to elucidate the effect of HD and dialyzer biocompatibility on erythrocyte GSH-DS in uremic patients on maintenance HD treatment. Twenty-five healthy volunteers and 42 HD patients were enrolled in this study. Blood samples were drawn immediately before and after HD session, and erythrocyte glutathione (GSH) level as well as the activities of the enzymes glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-Rd), and glutathione S-transferase (GST) were measured. To evaluate the effect of dialyzer type on the studied parameters the patients were were subdivided into two groups: those who had dialysis with cuprophane (CU) membranes (n=23) and those who received dialysis with the aid of polysulfone (PS) membranes (n=19). The activities of G6PD and GSH-Px as well as GSH level were significantly decreased in HD patients as compared with controls. On the other hand, the activities of GSSG-Rd and GST were significantly elevated among HD patients in comparison with control values. A single HD session, regardless of the type of dialyzer, did not induce any significant effect on any of the measured parameters, although G6PD activity increased significantly after dialysis. CU membrane did not result in any change in GSH or its metabolizing enzymes, while PS dialyzers exerted a minor but significant restoration in GSH-DS. The antioxidant pool, as represented by GSH-DS, is significantly affected by dialyzer type in HD patients being significantly corrected with polysulfone dialyzer.

The evolution of glutathione metabolism in phototrophic microorganisms

NASA Technical Reports Server (NTRS)

Fahey, R. C.; Buschbacher, R. M.; Newton, G. L.

1987-01-01

Of the many roles ascribed to glutathione (GSH) the one most clearly established is its role in the protection of higher eucaryotes against oxygen toxicity through destruction of thiol-reactive oxygen byproducts. If this is the primary function of GSH then GSH metabolism should have evolved during or after the evolution of oxygenic photosynthesis. That many bacteria do not produce GSH is consistent with this view. In the present study we have examined the low-molecular-weight thiol composition of a variety of phototrophic microorganisms to ascertain how evolution of GSH production is related to evolution of oxygenic photosynthesis. Cells were extracted in the presence of monobromobimane (mBBr) to convert thiols to fluorescent derivatives, which were analyzed by high-pressure liquid chromatography. Significant levels of GSH were not found in the green bacteria (Chlorobium thiosulfatophilum and Chloroflexus aurantiacus). Substantial levels of GSH were present in the purple bacteria (Chromatium vinosum, Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodocyclus gelatinosa), the cyanobacteria [Anacystis nidulans, Microcoleus chthonoplastes S.G., Nostoc muscorum, Oscillatoria amphigranulata, Oscillatoria limnetica, Oscillatoria sp. (Stinky Spring, Utah), Oscillatoria terebriformis, Plectonema boryanum, and Synechococcus lividus], and eucaryotic algae (Chlorella pyrenoidsa, Chlorella vulgaris, Euglena gracilis, Scenedesmus obliquus, and Chlamydomonas reinhardtii). Other thiols measured included cysteine, gamma-glutamylcysteine, thiosulfate, coenzyme A, and sulfide; several unidentified thiols were also detected. Many of the organisms examined also exhibited a marked ability to reduce mBBr to syn-(methyl,methyl)bimane, an ability that was quenched by treatment with 2-pyridyl disulfide or 5,5'-bisdithio-(2-nitrobenzoic acid) prior to reaction with mBBr. These observations indicate the presence of a reducing system capable of electron transfer to mBBr and reduction of reactive disulfides. The distribution of GSH in phototrophic eubacteria indicates that GSH synthesis evolved at or around the time that oxygenic photosynthesis evolved.

Permeation enhancing polymers in oral delivery of hydrophilic macromolecules: thiomer/GSH systems.

PubMed

Bernkop-Schnürch, A; Kast, C E; Guggi, D

2003-12-05

Thiolated polymers (= thiomers) in combination with reduced glutathione (GSH) were shown to improve the uptake of hydrophilic macromolecules from the GI tract. The mechanism responsible for this permeation enhancing effect seems to be based on the thiol groups of the polymer. These groups inhibit protein tyrosine phosphatase, being involved in the closing process of tight junctions, via a GSH-mediated mechanism. The strong permeation enhancing effect of various thiomer/GSH systems such as poly(acrylic acid)-cysteine/GSH or chitosan-4-thio-butylamidine (chitosan-TBA)/GSH could be shown via permeation studies on freshly excised intestinal mucosa in Ussing-type chambers. Furthermore, the efficacy of the system was also shown in vivo. By utilizing poly(acrylic acid)-cysteine/GSH as carrier matrix, an absolute oral bioavailability for low molecular weight heparin of 19.9 +/- 9.3% and a pharmacological efficacy--calculated on the basis of the areas under the reduction in serum glucose levels of the oral formulation versus subcutaneous (s.c.) injection-for orally given insulin of 7% could be achieved. The incorporation of salmon calcitonin in chitosan-TBA/GSH led on the other hand to a pharmacological efficacy based on the areas under the reduction in plasma calcium levels of the oral thiomer formulation versus intravenous (i.v.) injection of 1.3%. Because of this high efficacy (i), the possibility to combine thiomer/GSH systems with additional low molecular weight permeation enhancers acting in other ways (ii) and minimal toxicological risks as these polymers are not absorbed from the GI tract (iii), thiolated polymers represent a promising novel tool for the oral administration of hydrophilic macromolecules.

Physical activity and lung cancer among non-smokers: A pilot molecular epidemiologic study within EPIC

PubMed Central

RUNDLE, ANDREW; RICHIE, JOHN; STEINDORF, KAREN; PELUSO, MARCO; OVERVAD, KIM; RAASCHOU-NIELSEN, OLE; CLAVEL-CHAPELON, FRANCOISE; LINSEISEN, JACOB P.; BOEING, HEINER; TRICHOPOULOU, ANTONIA; PALLI, DOMENICO; KROGH, VITTORIO; TUMINO, ROSARIO; PANICO, SALVATORE; BUENO-DE-MESQUITA, HENDRIK B.; PEETERS, PETRA H.; LUND, EILIV; GONZALEZ, CARLOS A.; MARTINEZ, CARMEN; DORRONSORO, MIREN; BARRICARTE, AURELIO; TORMO, M. JOSE; QUIROS, JOSÈ R.; AGUDO, ANTONIO; BERGLUND, GORAN; JARVHOLM, BENGT; BINGHAM, SHEILA; KEY, TIMOTHY J.; GORMALLY, EMMANUELLE; SARACCI, RODOLFO; KAAKS, RUDOLF; RIBOLI, ELIO; VINEIS, PAOLO

2013-01-01

The association between physical activity, potential intermediate biomarkers and lung cancer risk was investigated in a study of 230 cases and 648 controls nested within the European Prospective Investigation of Cancer and Nutrition. Data on white blood cell aromatic-DNA adducts by 32P-postlabeling and glutathione (GSH) in red blood cells were available from a subset of cases and controls. Compared to the first quartile, the fourth quartile of recreational physical activity was associated with lower lung cancer risk [odds ratio=0.56 (0.35–0.90)], higher GSH levels [+1.87 micro mole GSH/gram haemoglobin, p=0.04] but not with the presence of high levels of adducts [odds ratio=1.05 (0.38–2.86)]. Despite being associated with recreational physical activity, in these small scale pilot analyses GSH levels were not associated with lung cancer risk, [odds ratio=0.95 (0.84 – 1.07) per unit increase in glutathione levels]. Household and occupational activity was not associated with lung cancer risk or biomarker levels. PMID:20050820

Oxidative Stress Level in the Testes of Mice and Rats during Nickel Intoxication

PubMed Central

Murawska-Ciałowicz, Eugenia; Bal, Wojciech; Januszewska, Lidia; Zawadzki, Marcin; Rychel, Joanna; Zuwała-Jagiełło, Jolanta

2012-01-01

The genotioxic and carcinogenic effect of nickel probably results from its capacity to produce reactive oxygen species (ROS) and disturb the redox balance. The aim of the study was to find out if rats lacking spermatic protamine 2 are less susceptible to Ni(II) than mice. Consequently, the levels of malondialdehyde + 4 hydroxynonenal (MDA+4HDA) − markers of lipid peroxidation, as well as the level of reduced glutathione (GSH) were measured within the rat and mouse testes. Our results showed that the levels of lipid peroxidation markers were elevated in testicular homogenates of intoxicated mice without any changes in rats. GSH level was lower in the group of intoxicated mice comparing to the control without statistically significant changes in rats' homogenates. Moreover, the level of GSH in the testes of intoxicated mice was lower than in rats. On the basis of our results, it appears that Ni(II) can initiate oxidative stress in the testes of mice but not of rats and can reduce GSH level. Consequently, the antioxidative defense of the testes is reduced. Ni(II) that causes oxidative stress in the testes may also contribute to infertility. PMID:22448131

Prenatal methylmercury exposure hampers glutathione antioxidant system ontogenesis and causes long-lasting oxidative stress in the mouse brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stringari, James; Nunes, Adriana K.C.; Franco, Jeferson L.

2008-02-15

During the perinatal period, the central nervous system (CNS) is extremely sensitive to metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-induced developmental neurotoxicity remains obscure, several studies point to the glutathione (GSH) antioxidant system as an important molecular target for this toxicant. To extend our recent findings of MeHg-induced GSH dyshomeostasis, the present study was designed to assess the developmental profile of the GSH antioxidant system in the mouse brain during the early postnatal period after in utero exposure to MeHg. Pregnant mice were exposed to different doses of MeHg (1, 3 and 10 mg/l, diluted in drinkingmore » water, ad libitum) during the gestational period. After delivery, pups were killed at different time points - postnatal days (PND) 1, 11 and 21 - and the whole brain was used for determining biochemical parameters related to the antioxidant GSH system, as well as mercury content and the levels of F{sub 2}-isoprostane. In control animals, cerebral GSH levels significantly increased over time during the early postnatal period; gestational exposure to MeHg caused a dose-dependent inhibition of this developmental event. Cerebral glutathione peroxidase (GPx) and glutathione reductase (GR) activities significantly increased over time during the early postnatal period in control animals; gestational MeHg exposure induced a dose-dependent inhibitory effect on both developmental phenomena. These adverse effects of prenatal MeHg exposure were corroborated by marked increases in cerebral F{sub 2}-isoprostanes levels at all time points. Significant negative correlations were found between F{sub 2}-isoprostanes and GSH, as well as between F{sub 2}-isoprostanes and GPx activity, suggesting that MeHg-induced disruption of the GSH system maturation is related to MeHg-induced increased lipid peroxidation in the pup brain. In utero MeHg exposure also caused a dose-dependent increase in the cerebral levels of mercury at birth. Even though the cerebral mercury concentration decreased to nearly basal levels at postnatal day 21, GSH levels, GPx and GR activities remained decreased in MeHg-exposed mice, indicating that prenatal exposure to MeHg affects the cerebral GSH antioxidant systems by inducing biochemical alterations that endure even when mercury tissue levels decrease and become indistinguishable from those noted in pups born to control dams. This study is the first to show that prenatal exposure to MeHg disrupts the postnatal development of the glutathione antioxidant system in the mouse brain, pointing to an additional molecular mechanism by which MeHg induces pro-oxidative damage in the developing CNS. Moreover, our experimental observation corroborates previous reports on the permanent functional deficits observed after prenatal MeHg exposure.« less

Prenatal methylmercury exposure hampers glutathione antioxidant system ontogenesis and causes long-lasting oxidative stress in the mouse brain

PubMed Central

Stringari, James; Nunes, Adriana KC; Franco, Jeferson L; Bohrer, Denise; Garcia, Solange C; Dafre, Alcir L; Milatovic, Dejan; Souza, Diogo O; Rocha, João BT; Aschner, Michael; Farina, Marcelo

2010-01-01

During the perinatal period, the central nervous system (CNS) is extremely sensitive to metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-induced developmental neurotoxicity remains obscure, several studies point to the glutathione (GSH) antioxidant system as an important molecular target for this toxicant. To extend our recent findings of MeHg-induced GSH dyshomeostasis, the present study was designed to assess the developmental profile of the GSH antioxidant system in the mouse brain during the early postnatal period after in utero exposure to MeHg. Pregnant mice were exposed to different doses of MeHg (1, 3 and 10 mg/L, diluted in drinking water, ad libitum) during the gestational period. After delivery, pups were killed at different time points - postnatal days (PNDs) 1, 11 and 21 - and the whole brain was used for determining biochemical parameters related to the antioxidant GSH system, as well as mercury content and the levels of F2-isoprostane. In control animals, cerebral GSH levels significantly increased over time during the early postnatal period; gestational exposure to MeHg caused a dose-dependent inhibition of this developmental event. Cerebral glutathione peroxidase (GPx) and glutathione reductase (GR) activities significantly increased over time during the early postnatal period in control animals; gestational MeHg exposure induced a dose-dependent inhibitory effect on both developmental phenomena. These adverse effects of prenatal MeHg exposure were corroborated by marked increases in cerebral F2-isoprostanes levels at all time points. Significant negative correlations were found between F2-isoprostanes and GSH, as well as between F2-isoprostanes and GPx activity, suggesting that MeHg-induced disruption of the GSH system maturation is related to MeHg-induced increased lipid peroxidation in the pup brain. In utero MeHg exposure also caused a dose-dependent increase in the cerebral levels of mercury at birth. Even though the cerebral mercury concentration decreased to nearly basal levels at postnatal day 21, GSH levels, GPx and GR activities remained decreased in MeHg-exposed mice, indicating that prenatal exposure to MeHg affects the cerebral GSH antioxidant systems by inducing biochemical alterations that endure even when mercury tissue levels decrease and become indistinguishable from those noted in pups born to control dams. This study is the first to show that prenatal exposure to MeHg disrupts the postnatal development of the glutathione antioxidant system in the mouse brain, pointing to an additional molecular mechanism by which MeHg induces pro-oxidative damage in the developing CNS. Moreover, our experimental observation corroborates previous reports on the permanent functional deficits observed after prenatal MeHg exposure. PMID:18023834

Degraded protein adducts of cis-2-butene-1,4-dial are urinary and hepatocyte metabolites of furan.

PubMed

Lu, Ding; Sullivan, Mathilde M; Phillips, Martin B; Peterson, Lisa A

2009-06-01

Furan is a liver toxicant and carcinogen in rodents. On the basis of these observations and the large potential for human exposure, furan has been classified as a possible human carcinogen. The mechanism of tumor induction by furan is unknown. However, the toxicity requires cytochrome P450-catalyzed oxidation of furan. The product of this oxidation, cis-2-butene-1,4-dial (BDA), reacts readily with glutathione, amino acids, and DNA and is a bacterial mutagen in Ames assay strain TA104. Characterization of the urinary metabolites of furan is expected to provide information regarding the structure(s) of the reactive metabolite(s). Recently, several urinary metabolites have been identified. We reported the presence of a monoglutathione-BDA reaction product, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-l-cysteinylglycine cyclic sulfide. Three additional urinary metabolites of furan were also characterized as follows: R-2-acetylamino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid, N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine, and its sulfoxide. It was postulated that these three metabolites are derived from degraded protein adducts. However, the possibility that these metabolites result from the reaction of BDA with free lysine and/or cysteine was not ruled out. In this latter case, one might predict that the reaction of thiol-BDA with free lysine would not occur exclusively on the epsilon-amino group. Reaction of BDA with N-acetylcysteine or GSH in the presence of lysine indicated that both the alpha- and the epsilon-amino groups of lysine can be modified by thiol-BDA. The N-acetylcysteine-BDA-N-acetyllysine urinary metabolites were solely linked through the epsilon-amino group of lysine. A GSH-BDA-lysine cross-link was a significant hepatocyte metabolite of furan. In this case, the major product resulted from reaction with the epsilon-amino group of lysine; however, small amounts of the alpha-amino reaction product were also observed. Western analysis of liver and hepatocyte protein extracts using anti-GSH antibody indicated that GSH was covalently linked to proteins in tissues or cells exposed to furan. Our data support the hypothesis that GSH-BDA can react with either free lysine or protein lysine groups. These data suggest that there are multiple pathways by which furan can modify cellular nucleophiles. In one pathway, BDA reacts directly with proteins to form cysteine-lysine reaction products. In another, BDA reacts with GSH to form GSH-BDA conjugates, which then react with cellular nucleophiles like free lysine or lysine moieties in proteins. Both pathways will give rise to N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine. Given the abundance of these metabolites in urine of furan-treated rats, these pathways appear to be major pathways of furan biotransformation in vivo.

Degraded protein adducts of cis-2-butene-1,4-dial are urinary and hepatocyte metabolites of furan

PubMed Central

Lu, Ding; Sullivan, Mathilde M.; Phillips, Martin B.; Peterson, Lisa A.

2009-01-01

Furan is a liver toxicant and carcinogen in rodents. Based on these observations and the large potential for human exposure, furan has been classified as a possible human carcinogen. The mechanism of tumor induction by furan is unknown. However, the toxicity requires cytochrome P450 catalyzed oxidation of furan. The product of this oxidation, cis-2-butene-1,4-dial (BDA), reacts readily with glutathione, amino acids and DNA and is a bacterial mutagen in Ames assay strain TA104. Characterization of the urinary metabolites of furan is expected to provide information regarding the structure(s) of the reactive metabolite(s). Recently, several urinary metabolites have been identified. We reported the presence of a mono-glutathione-BDA reaction product, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine cyclic sulfide. Three additional urinary metabolites of furan were also characterized: R-2-acetylamino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid, N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-L-cysteine and its sulfoxide. It was postulated that these three metabolites are derived from degraded protein adducts. However, the possibility that these metabolites result from reaction of BDA with free lysine and/or cysteine was not ruled out. In this latter case, one might predict that the reaction of thiol-BDA with free lysine would not occur exclusively on the ε-amino group. Reaction of BDA with N-acetylcysteine or GSH in the presence of lysine indicated that both the α- and ε-amino groups of lysine can be modified by thiol-BDA. The N-acetylcysteine-BDA-N-acetyllysine urinary metabolites were solely linked through the ε-amino group of lysine. A GSH-BDA-lysine crosslink was a significant hepatocyte metabolite of furan. In this case, the major product resulted from reaction with the ε-amino group of lysine, however, small amounts of the α-amino reaction product were also observed. Western analysis of liver and hepatocyte protein extracts using anti-GSH antibody indicated that GSH was covalently linked to proteins in tissues or cells exposed to furan. Our data support the hypothesis that GSH-BDA can react with either free lysine or protein lysine groups. These data suggest that there are multiple pathways by which furan can modify cellular nucleophiles. In one pathway, BDA reacts directly with proteins to form cysteine-lysine reaction products. In another, BDA reacts with GSH to form GSH-BDA conjugates which then reacts with cellular nucleophiles like free lysine or lysine moieties in proteins. Both pathways will give rise to N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-L-cysteine. Given the abundance of these metabolites in urine of furan-treated rats, these pathways appear to be major pathways of furan biotransformation in vivo. PMID:19441776

Age-related alterations of plasma glutathione and oxidation of redox potentials in chimpanzee (Pan troglodytes) and rhesus monkey (Macaca mulatta).

PubMed

Paredes, Jamespaul; Jones, Dean P; Wilson, Mark E; Herndon, James G

2014-04-01

Chimpanzee (Pan troglodytes) and rhesus macaque (Macaca mulatta) and humans (Homo sapiens) share physiological and genetic characteristics, but have remarkably different life spans, with chimpanzees living 50-60 % and the rhesus living 35-40 % of maximum human survival. Since oxidative processes are associated with aging and longevity, we might expect to see species differences in age-related oxidative processes. Blood and extracellular fluid contain two major thiol redox nodes, glutathione (GSH)/glutathione-disulfide (GSSG) and cysteine (Cys)/cystine (CySS), which are subject to reversible oxidation-reduction reactions and are maintained in a dynamic non-equilibrium state. Disruption of these thiol redox nodes leads to oxidation of their redox potentials (EhGSSG and EhCySS) which affects cellular physiology and is associated with aging and the development of chronic diseases in humans. The purpose of this study was to measure age-related changes in these redox thiols and their corresponding redox potentials (Eh) in chimpanzees and rhesus monkeys. Our results show similar age-related decreases in the concentration of plasma GSH and Total GSH as well as oxidation of the EhGSSG in male and female chimpanzees. Female chimpanzees and female rhesus monkeys also were similar in several outcome measures. For example, similar age-related decreases in the concentration of plasma GSH and Total GSH, as well as age-related oxidation of the EhGSSG were observed. The data collected from chimpanzees and rhesus monkeys corroborates previous reports on oxidative changes in humans and confirms their value as a comparative reference for primate aging.

Relationship of antioxidant and oxidative stress markers in different organs following copper toxicity in a rat model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Vijay; Kalita, Jayantee, E-mail: jayanteek@yahoo.com; Bora, Himangsu K.

Copper (Cu) at a higher level becomes toxic and it can catalyze the formation of highly reactive hydroxyl radical. We report the vulnerability of liver, kidney and brain to different dose of copper sulfate (CuSO{sub 4}) induced oxidative stress at different time duration. Fifty-four male Wistar rats (weight range = 205 ± 10 g) were equally divided into three groups. CuSO{sub 4} was administered orally to the experimental groups (Group-II and III) up to 90 days in a dose of 100 and 200 mg/Kg body weight per day. Saline water was given to the control group (Group-I). At the endmore » of 30, 60 and 90 days of administration, neurobehavioral studies were done and six rats from each group were sacrificed. Their liver, kidney and brain tissues were subjected for Cu, glutathione (GSH), malondialdehyde (MDA) and total antioxidant capacity (TAC) assay. Blood urea nitrogen (BUN), serum creatinine, bilirubin and transaminases were measured. GSH, TAC and MDA levels were correlated with the markers of respective organ dysfunction. Administration of CuSO{sub 4} resulted in increased free Cu and MDA level, and decrease GSH and TAC levels in group-II and III compared with group-I. In experimental groups, the reduction in TAC and GSH levels was maximum in liver tissue followed by brain and kidney; whereas increase in MDA level was highest in liver followed by brain and kidney at 30, 60 and 90 days. TAC and GSH levels in the liver inversely correlated with serum transaminases and bilirubin, and tissue free Cu, and positively correlated with MDA levels. Free Cu level in kidney tissue and BUN inversely correlated with TAC and GSH, and positively with MDA level. Grip-strength, rotarod and Y-maze findings were inversely correlated with brain free Cu and MDA levels and positively with GSH and TAC levels. The oxidative stress was highest in liver followed by brain and kidney after oral CuSO{sub 4} exposure in a rat model. These levels correlated with the respective organ dysfunction and tissue free Cu concentration. - Highlights: • Oral dosing of CuSO{sub 4} leads to oxidative stress in liver, brain and kidney. • Liver has maximum oxidative stress followed by brain and kidney. • Oxidative stress correlated with the respective organ dysfunction and tissue Cu concentration.« less

SA19. N-Acetyl-Cysteine in a Double-Blind Randomized Placebo-Controlled Trial: Toward Biomarker-Guided Treatment in Early Psychosis

PubMed Central

Do, Kim; Seidman, Larry J.; Fournier, Margot; Xin, Lijing; Cleusix, Martine; Baumann, Philipp S.; Ferrari, Carina; Cousins, Ann; Alameda, Luis; Gholam-Rezaee, Mehdi; Golay, Philippe; Jenni, Raoul; Woo, T-U Wilson; Keshavan, Matcheri S.; Eap, Chin B.; Wojcik, Joanne; Cuenod, Michel; Buclin, Thierry; Gruetter, Rolf; Conus, Philippe

2017-01-01

Abstract Background: Biomarker-guided treatments are needed in psychiatry and previous data suggest redox dysregulation / oxidative stress may be a target in schizophrenia (1,2). A previous add-on trial with the antioxidant N-Acetyl-Cysteine (NAC) led to negative symptoms reductions in chronic patients (3). We aim to study NAC impact on symptoms and neurocognition in early psychosis (EP) and to explore whether glutathione (GSH)/redox markers could represent valid biomarkers to guide treatment. Methods: In a double-blind, randomized, placebo-controlled trial in 63 EP patients, we assessed the effect of NAC supplementation (2700 mg/day, 6 months) on PANSS, neurocognition (MATRICS Consensus Cognitive Battery [MCCB]), and redox markers (brain GSH [GSH-mPFC], blood cells GSH [GSH-BC] levels, and GSH peroxidase activity [GPx-BC]). Results: No changes in negative, positive symptoms, or functional outcome were observed with NAC, but significant improvements were found in favor of NAC on the MCCB Processing Speed factor and two of its components: Trail Making and Verbal Fluency. NAC leads to increases in GSH-mPFC by 23% (P = .005) and GSH-BC by 19% (P = .05). In patients with high-baseline GPx-BC (>22.3U/gHb), subgroup explorations revealed an improvement with NAC of positive symptoms when compared to patients with low-baseline GPx (P = .02), with an improvement of positive symptoms in parallel with that of the redox status. Conclusion: In conclusion, NAC supplementation in a limited sample of EP patients did not improve negative symptoms, which were at modest levels at baseline. However, NAC leads to neurocognition improvement as well as to brain GSH levels increases, pointing to good target engagement. Blood GPx activity, a redox peripheral index associated with brain GSH levels, could help to identify a subgroup of patients who improve their positive symptoms with NAC. Future trials with antioxidant in EP should consider biomarker-guided treatment. References 1. Steullet P et al. 2016, Redox dysregulation, neuroinflammation, and NMDA receptor hypofunction: A “central hub” in schizophrenia pathophysiology? Schizophr Res. 176:41. 2. Hardingham GE, Do KQ, 2016, Linking early-life NMDAR hypofunction and oxidative stress in schizophrenia pathogenesis. Nat Rev Neurosci. 17:125. 3. Berk M et al. 2008, N-acetyl cysteine as a glutathione precursor for schizophrenia--a double-blind, randomized, placebo-controlled trial. Biol Psychiatry, 64:361.

A perillyl alcohol-conjugated analog of 3-bromopyruvate without cellular uptake dependency on monocarboxylate transporter 1 and with activity in 3-BP-resistant tumor cells.

PubMed

Chen, Thomas C; Yu, Jiali; Nouri Nigjeh, Eslam; Wang, Weijun; Myint, Phyo Thazin; Zandi, Ebrahim; Hofman, Florence M; Schönthal, Axel H

2017-08-01

The anticancer agent 3-bromopyruvate (3-BP) is viewed as a glycolytic inhibitor that preferentially kills glycolytic cancer cells through energy depletion. However, its cytotoxic activity is dependent on cellular drug import through transmembrane monocarboxylate transporter 1 (MCT-1), which restricts its anticancer potential to MCT-1-positive tumor cells. We created and characterized an MCT-1-independent analog of 3-BP, called NEO218. NEO218 was synthesized by covalently conjugating 3-BP to perillyl alcohol (POH), a natural monoterpene. The responses of various tumor cell lines to treatment with either compound were characterized in the presence or absence of supplemental pyruvate or antioxidants N-acetyl-cysteine (NAC) and glutathione (GSH). Drug effects on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme activity were investigated by mass spectrometric analysis. The development of 3-BP resistance was investigated in MCT-1-positive HCT116 colon carcinoma cells in vitro. Our results show that NEO218: (i) pyruvylated GAPDH on all 4 of its cysteine residues and shut down enzymatic activity; (ii) severely lowered cellular ATP content below life-sustaining levels, and (iii) triggered rapid necrosis. Intriguingly, supplemental antioxidants effectively prevented cytotoxic activity of NEO218 as well as 3-BP, but supplemental pyruvate powerfully protected cells only from 3-BP, not from NEO218. Unlike 3-BP, NEO218 exerted its potent cytotoxic activity irrespective of cellular MCT-1 status. Treatment of HCT116 cells with 3-BP resulted in prompt development of resistance, based on the emergence of MCT-1-negative cells. This was not the case with NEO218, and highly 3-BP-resistant cells remained exquisitely sensitive to NEO218. Thus, our study identifies a mechanism by which tumor cells develop rapid resistance to 3-BP, and presents NEO218 as a superior agent not subject to this cellular defense. Furthermore, our results offer alternative interpretations of previously published models on the role of supplemental antioxidants: Rather than quenching reactive oxygen species (ROS), supplemental NAC or GSH directly interact with 3-BP, thereby neutralizing the drug's cytotoxic potential before it can trigger ROS production. Altogether, our study introduces new aspects of the cytotoxic mechanism of 3-BP, and characterizes NEO218 as an analog able to overcome a key cellular defense mechanism towards this drug. Copyright © 2017 Elsevier B.V. All rights reserved.

Selenium uptake through cystine transporter mediated by glutathione conjugation.

PubMed

Tobe, Takao; Ueda, Koji; Aoki, Akira; Okamoto, Yoshinori; Kojima, Nakao; Jinno, Hideto

2017-01-01

Selenium (Se) is an essential trace element and is regarded as a protective agent against cancer. In particular, antioxidant effects of selenoenzymes contribute to cancer prevention. Se can also produce reactive oxygen species and, thereby, exert cancer-selective cytotoxicity. Selenodiglutathione (SDG) is a primary Se metabolite conjugated to two glutathione (GSH) moieties. SDG increases intracellular Se accumulation and is more toxic than selenous acid (H 2 SeO 3 ), but the mechanisms for importing Se compounds into cells are not fully understood. Here, we propose a novel mechanism for importing Se, in the form of SDG. Cellular intake of Se compounds was assessed based on Se accumulation, as detected by ICP-MS. SDG incorporation was decreased in the presence of thiols (GSH, cysteine or their oxidized forms, GSSG and cystine), whereas H 2 SeO 3 uptake was increased by addition of GSH or cysteine. Cellular SDG uptake was decreased by pretreatment with specific inhibitors against gamma-glutamyl transpeptidase (GGT) or the cystine/glutamate antiporter (system x c - ). Furthermore, siRNA against xCT, which is the light chain component of system x c - , significantly decreased SDG incorporation. These data suggest an involvement of SDG in Se incorporation, with SDG processed at the cell surface by GGT, leading to formation of selenodicysteine which, in turn, is likely to be imported via xCT. Because GGT and xCT are highly expressed in cancer cells, these mechanisms mediated by the cystine transporter might underlie the cancer-selective toxicity of Se. In addition, the system described in our study appears to represent a physiological transport mechanism for the essential element Se.

Antioxidative effects of fermented sesame sauce against hydrogen peroxide-induced oxidative damage in LLC-PK1 porcine renal tubule cells

PubMed Central

Song, Jia-Le; Choi, Jung-Ho; Seo, Jae-Hoon; Kil, Jeung-Ha

2014-01-01

BACKGROUND/OBJECTIVES This study was performed to investigate the in vitro antioxidant and cytoprotective effects of fermented sesame sauce (FSeS) against hydrogen peroxide (H2O2)-induced oxidative damage in renal proximal tubule LLC-PK1 cells. MATERIALS/METHODS 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (•OH), and H2O2 scavenging assay was used to evaluate the in vitro antioxidant activity of FSeS. To investigate the cytoprotective effect of FSeS against H2O2-induced oxidative damage in LLC-PK1 cells, the cellular levels of reactive oxygen species (ROS), lipid peroxidation, and endogenous antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) were measured. RESULTS The ability of FSeS to scavenge DPPH, •OH and H2O2 was greater than that of FSS and AHSS. FSeS also significantly inhibited H2O2-induced (500 µM) oxidative damage in the LLC-PK1 cells compared to FSS and AHSS (P < 0.05). Following treatment with 100 µg/mL of FSeS and FSS to prevent H2O2-induced oxidation, cell viability increased from 56.7% (control) to 83.7% and 75.6%, respectively. However, AHSS was not able to reduce H2O2-induced cell damage (viability of the AHSS-treated cells was 54.6%). FSeS more effectively suppressed H2O2-induced ROS generation and lipid peroxidation compared to FSS and AHSS (P < 0.05). Compared to the other sauces, FSeS also significantly increased cellular CAT, SOD, and GSH-px activities and mRNA expression (P < 0.05). CONCULUSIONS These results from the present study suggest that FSeS is an effective radical scavenger and protects against H2O2-induced oxidative damage in LLC-PK1 cells by reducing ROS levels, inhibiting lipid peroxidation, and stimulating antioxidant enzyme activity. PMID:24741396

Characterization of the Vibrio vulnificus 1-Cys peroxiredoxin Prx3 and regulation of its expression by the Fe-S cluster regulator IscR in response to oxidative stress and iron starvation.

PubMed

Lim, Jong Gyu; Bang, Ye-Ji; Choi, Sang Ho

2014-12-26

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes that reduce toxic peroxides. A new Vibrio vulnificus Prx, named Prx3, was identified and characterized in this study. Biochemical and mutational analyses revealed that Prx3 reduces H2O2, utilizing glutaredoxin 3 (Grx3) and glutathione (GSH) as reductants, and requires only N-terminal peroxidatic cysteine for its catalysis. These results, combined with the monomeric size of Prx3 observed under non-reducing conditions, suggested that Prx3 is a Grx3/GSH-dependent 1-Cys Prx and oxidized without forming intermolecular disulfide bonds. The prx3 mutation impaired growth in the medium containing peroxides and reduced virulence in mice, indicating that Prx3 is essential for survival under oxidative stress and pathogenesis of V. vulnificus. The Fe-S cluster regulator IscR activates prx3 by direct binding to a specific binding sequence centered at -44 from the transcription start site. The binding sequence was homologous to the Type 2 IscR-binding sequence, most likely recognized by the Fe-S clusterless apo-IscR in Escherichia coli. The iscR3CA mutant, chromosomally encoding the apo-locked IscR, exhibited 3-fold higher levels of activation of prx3 than the wild type and accumulated more IscR3CA protein in cells. The IscR-dependent activation of prx3 by aerobic growth and iron starvation was also associated with the increase in cellular levels of IscR protein. Taken together, the results suggested that IscR senses iron starvation as well as reactive oxygen species and shifts to the apo-form, which leads to the increase of cellular IscR and in turn prx3 expression, contributing to the survival and virulence of V. vulnificus during pathogenesis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Respiratory syncytial virus increases lung cellular bioenergetics in neonatal C57BL/6 mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alsuwaidi, Ahmed R., E-mail: alsuwaidia@uaeu.ac.ae; Albawardi, Alia, E-mail: alia.albawardi@uaeu.ac.ae; Almarzooqi, Saeeda, E-mail: saeeda.almarzooqi@uaeu.ac.ae

2014-04-15

We have previously reported that lung cellular bioenergetics (cellular respiration and ATP) increased in 4–10 week-old BALB/c mice infected with respiratory syncytial virus (RSV). This study examined the kinetics and changes in cellular bioenergetics in ≤2-week-old C57BL/6 mice following RSV infection. Mice (5–14 days old) were inoculated intranasally with RSV and the lungs were examined on days 1–10 post-infection. Histopathology and electron microscopy revealed preserved pneumocyte architectures and organelles. Increased lung cellular bioenergetics was noted from days 1–10 post-infection. Cellular GSH remained unchanged. These results indicate that the increased lung cellular respiration (measured by mitochondrial O{sub 2} consumption) and ATPmore » following RSV infection is independent of either age or genetic background of the host. - Highlights: • RSV infection increases lung cellular respiration and ATP in neonatal C57BL/6 mice. • Increased lung cellular bioenergetics is a biomarker of RSV infection. • Lung cellular glutathione remains unchanged in RSV infection.« less

Differential modulatory effects of morphine on acute and chronic stress induced neurobehavioral and cellular markers in rats.

PubMed

Joshi, Jagdish C; Ray, Arunabha; Gulati, Kavita

2014-04-15

The present study evaluated the effects of morphine treatments on elevated plus maze test parameters, oxidative stress markers and Hsp70 expression in normal and stressed rats. Acute and chronic stress caused neurobehavioral suppression, altered prooxidant-antioxidant balance and increased Hsp70 expression in brain homogenates in a differential manner. Morphine (1 and 5mg/kg) attenuated RS induced anxiogenesis, changes in MDA and GSH but further enhanced Hsp70 expression. Similar anxiolytic and Hsp70 enhancing effects were seen after morphine in normal rats (no RS). Exposure to chronic RS did not elicit any appreciable neurobehavioral response in EPM but enhanced MDA, lowered GSH and exaggerated the Hsp70 expression. Pretreatment with morphine did not affect the neurobehavioral response to chronic RS, but reverted the GSH and Hsp70 expression. The results suggest that morphine differentially influences acute and chronic stress induced changes in anxiety behavior and complex interactions between oxidative stress markers and Hsp70 expression which may contribute to these effects. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of methylmercury exposure on glutathione metabolism, oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks

USGS Publications Warehouse

Kenow, K.P.; Hoffman, D.J.; Hines, R.K.; Meyer, M.W.; Bickham, J.W.; Matson, C.W.; Stebbins, K.R.; Montagna, P.; Elfessi, A.

2008-01-01

We quantified the level of dietary mercury (Hg), delivered as methylmercury chloride (CH3HgCl), associated with negative effects on organ and plasma biochemistries related to glutathione (GSH) metabolism and oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks reared from hatch to 105 days. Mercury-associated effects related to oxidative stress and altered glutathione metabolism occurred at 1.2 :g Hg/g and 0.4 :g Hg/g, an ecologically relevant dietary mercury level, but not at 0.08 :g Hg/g. Among the variables that contributed most to dissimilarities in tissue chemistries between control and treatment groups were increased levels of oxidized glutathione (GSSG), GSH peroxidase, and the ratio of GSSG to GSH in brain tissue; increased levels of hepatic GSH; and decreased levels of hepatic glucose-6-phosphate dehydrogenase (G-6-PDH). Our results also suggest that chronic exposure to environmentally relevant dietary Hg levels did not result in statistically significant somatic chromosomal damage in common loon chicks. Oxidative stress and altered glutathione metabolism were evident in common loon chicks exposed to >0.4 :g Hg as CH3HgCl per gram wet food intake.

The improving effect of reduced glutathione on boar sperm cryotolerance is related with the intrinsic ejaculate freezability.

PubMed

Yeste, Marc; Estrada, Efrén; Pinart, Elisabeth; Bonet, Sergi; Miró, Jordi; Rodríguez-Gil, Joan E

2014-04-01

Reduced glutathione (GSH) improves boar sperm cryosurvival and fertilising ability when added to freezing extenders. Poor freezability ejaculates (PFE) are known to present lower resistance than good freezability ejaculates (GFE) to cryopreservation procedures. So far, no study has evaluated whether the ability of GSH to counteract the cryopreservation-induced injuries depends on ejaculate freezability (i.e. GFE vs. PFE). For this reason, thirty boar ejaculates were divided into three equal volume fractions and cryopreserved with or without GSH at a final concentration of either 2 or 5mM in freezing media. Before and after freeze-thawing, sperm quality was evaluated through analysis of viability, motility, integrity of outer acrosome membrane, ROS levels, integrity of nucleoprotein structure, and DNA fragmentation. Ejaculates were classified into two groups (GFE or PFE) according to their post-thaw sperm motility and viability assessments in negative control (GSH 0mM), after running cluster analyses. Values of each sperm parameter were then compared between treatments (GSH 0mM, GSH 2mM, GSH 5mM) and freezability groups (GFE, PFE). In the case of GFE, GSH significantly improved boar sperm cryotolerance, without differences between 2 and 5mM. In contrast, PFE freezability was significantly increased when supplemented with 5mM GSH, but not when supplemented with 2mM GSH. In conclusion, PFE need a higher concentration of GSH than GFE to improve their cryotolerance. Copyright © 2014 Elsevier Inc. All rights reserved.

Investigation of the effects of local glutathione and chitosan administration on incisional oral mucosal wound healing in rabbits.

PubMed

Kılıç, Ciğdem; Güleç Peker, Emine Gülçeri; Acartürk, Füsun; Kılıçaslan, Seda M Sarı; Çoşkun Cevher, Şule

2013-12-01

The aim of the present study was to investigate the effects of local glutathione (GSH) and chitosan applications on the oxidant events and histological changes that occur, during healing processes in rabbits with incisional intraoral mucosal wounds. For this purpose, discs containing glutathione and chitosan (1:1) were prepared and their physicochemical characteristics were evaluated. New Zealand white rabbits were used in in vivo studies. A standard incision was applied to the oral mucosa of rabbits. The rabbits were divided into four groups, being: an untreated incisional group (n=6), a group treated with discs containing GSH+chitosan (n=6), a group treated with discs containing solely chitosan (n=5) and a group treated with discs containing solely GSH (n=5). The levels of malondialdehyde (MDA), glutathione and nitric oxide (NOx) in the oral wound tissues were measured on the fifth day after the injury. Histological changes in the wound tissues were also investigated. The tissue MDA levels in the group treated with the disc containing GSH+chitosan were found to be lower than those in the other groups. There were no statistically significant differences in terms of tissue GSH and NOx levels between the group treated with the disc comprising GSH+chitosan and the control group that had untreated incision wounds. According to the histological findings, wound healing in the group treated with the disc containing solely chitosan was found to be better than in the other groups. The results of the experiments showed that the local application to the intraoral incision wounds of chitosan+GSH, and chitosan alone, can be effective in the wound healing processes of soft tissues and dental implants. Copyright © 2013 Elsevier B.V. All rights reserved.

Oxidative stress in elite athletes training at moderate altitude and at sea level.

PubMed

León-López, Josefa; Calderón-Soto, Carmen; Pérez-Sánchez, Matías; Feriche, Belén; Iglesias, Xavier; Chaverri, Diego; Rodréguez, Ferran A

2018-03-24

Using a controlled parallel group longitudinal trial design, we investigated the effects of different training interventions on the prooxidant/antioxidant status of elite athletes: living and training at moderate altitude for 3 (Hi-Hi3) and 4 weeks (Hi-Hi), and for 4 weeks too, living high and training high and low (Hi-HiLo) and living and training at sea level (Lo-Lo). From 61 swimmers, 54 completed the study. Nitrites, carbonyls, and lipid peroxidation (LPO) levels were assessed in plasma. Enzymatic antioxidants glutathione peroxidase (GPx) and glutathione reductase (GRd), and non-enzymatic antioxidants total glutathione (GST), reduced glutathione (GSH) and oxidized glutathione (GSSG) were analysed in the erythrocyte fraction. At the end of the intervention, nitrites levels were similar in all altitude groups but higher than in the Lo-Lo controls (P = .02). Hi-HiLo had greater GPx activity than Hi-Hi and Hi-Hi3 during most of the intervention (P ≤ .001). GRd activity was higher in Lo-Lo than in Hi-Hi at the end of the training camp (P ≤ .001). All groups showed increased levels of LPO, except Lo-Lo, and carbonyls at the end of the study (P ≤ .001). Training at altitude for 3 or 4 weeks drives oxidative stress leading to cellular damage mainly by worsening the antioxidant capacities. The GSSG/GSH ratio appears to be related to perceived exertion and fatigue. The stronger antioxidant defence showed by the Hi-HiLo group suggests an inverse relationship between redox alterations and performance. Further studies are required to investigate the role of oxidative stress in acclimatization, performance, and health.

N-acetyl cysteine, L-cysteine, and beta-mercaptoethanol augment selenium-glutathione peroxidase activity in glucose-6-phosphate dehydrogenase-deficient human erythrocytes.

PubMed

Alicigüzel, Y; Aslan, M

2004-09-01

In glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes, failure to maintain normal levels of reduced glutathione (GSH) due to decreased NADPH regeneration in the hexose monophosphate pathway results in acute hemolytic anemia following exposure to oxidative insults, such as ingestion of Vicia fava beans or use of certain drugs. GSH is a source of protection against oxidative attack, used by the selenium-dependent glutathione peroxidase (Se-GSH-Px)/reductase (GR) system to detoxify hydrogen peroxide and organic peroxides, provided that sufficient GSH is made available. In this study, Se-GSH-Px activity was analyzed in G6PD-deficient patients in the presence of reducing agents such as N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol. Se-GSH-Px activity was decreased in G6PD-deficient red blood cells (RBCs). N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol increased Se-GSH-Px activity in G6PD-deficient human erythrocytes, indicating that other reducing agents can be utilized to complement Se-GSH-Px activity in G6PD deficiency. Based on the increased susceptibility of G6PD-deficient patients to oxidative stress, the reported increase in Se-GSH-Px activity can facilitate the detoxification of reactive oxygen species.

Regeneration of glutathione by α-lipoic acid via Nrf2/ARE signaling pathway alleviates cadmium-induced HepG2 cell toxicity.

PubMed

Zhang, Jiayu; Zhou, Xue; Wu, Wenbo; Wang, Jiachun; Xie, Hong; Wu, Zhigang

2017-04-01

Alpha-lipoic acid (α-LA) is an important antioxidant that is capable of regenerating other antioxidants, such as glutathione (GSH). However, the underlying molecular mechanism by which α-LA regenerates GSH remains poorly understood. The current study aimed to investigate whether α-LA regenerates GSH by activation of Nrf2 to alleviate cadmium-induced cytotoxicity in HepG2 cells. In the present study, we found that cadmium induced cell death by depletion of GSH through inactivation of Nrf2. Addition of α-LA to cadmium-treated cells reactivated Nrf2 and regenerated GSH through elevating the Nrf2-downstream genes γ-glutamate-cysteine ligase (γ-GCL) and GR, both of which are key enzymes for GSH synthesis. However, blocking Nrf2 with brusatol in the cells co-treated with α-LA and cadmium reduced the mRNA and the protein levels of γ-GCL and GR, thus suppressed GSH regeneration by α-LA. Our results indicated that α-LA activated Nrf2 signaling pathway, which upregulated the transcription of the enzymes for GSH synthesis and therefore GSH contents to alleviate cadmium-induced cytotoxicity in HepG2 cells. Copyright © 2017. Published by Elsevier B.V.

The prospective protective effect of selenium nanoparticles against chromium-induced oxidative and cellular damage in rat thyroid

PubMed Central

Hassanin, Kamel MA; El-Kawi, Samraa H Abd; Hashem, Khalid S

2013-01-01

Background Nanotechnology has enabled researchers to synthesize nanosize particles that possess increased surface areas. Compared to conventional microparticles, it has resulted in increased interactions with biological targets. Objective The objective of this study was to determine the protective ability of selenium nanoparticles against hexavalent chromium-induced thyrotoxicity. Design Twenty male rats were used in the study, and arbitrarily assigned to four groups. Group 1 was the control group, and was given phosphate-buffered saline. Group 2 was the chromium-treated group and was given K2Cr2O7 60 μg/kg body weight intraperitoneally as a single dose on the third day of administration. Group 3 was the nano-selenium-treated group and was given selenium nanoparticles (size 3–20 nm) 0.5 mg/kg body weight intraperitoneally daily for 5 consecutive days. Group 4 was the nano-selenium chromium-treated group, which received selenium nanoparticles for 5 days and a single dose of K2Cr2O7 on the third day of administration. Materials and methods Blood samples were collected from rats for measuring thyroid hormones (free triiodothyronine [T3] and free thyroxine [T4]) and oxidative and antioxidant parameters (malondialdehyde [MDA], reduced glutathione [GSH], catalase, and superoxide dismutase [SOD]). Upon dissection, thyroid glands were taken for histopathological examination by using paraffin preparations stained with hematoxylin and eosin (H&E) and Masson’s trichrome. Immunohistochemical staining was performed for detecting cellular proliferation using Ki67 antibodies. Results The present study shows that K2Cr2O7 has a toxic effect on the thyroid gland as a result of inducing a marked oxidative damage and release of reactive oxygen species. This was shown by the significant decrease in free T3 and T4 and GSH levels, which was accompanied by significant increases in catalase, SOD, and MDA in the chromium-treated group compared to the control group. Se nanoparticles have a protective effect on K2Cr2O7-induced thyroid damage, as a result of correcting the free T3 and T4 levels and GSH, catalase, SOD, and MDA compared to the K2Cr2O7-treated group. Administration of nano-selenium alone in the nano-selenium-treated group had no toxic effect on rats’ thyroid compared to the control group. The biochemical results were confirmed by histopathological, immunohistochemical and pathomorphological studies. PMID:23658489

Effect of all-trans retinoic acid treatment on prohibitin and renin-angiotensin-aldosterone system expression in hypoxia-induced renal tubular epithelial cell injury.

PubMed

Zhou, Tian-Biao; Ou, Chao; Rong, Liang; Drummen, Gregor P C

2014-09-01

All-trans retinoic acid (ATRA) exerts various effects on physiological processes such as cell growth, differentiation, apoptosis and inflammation. Prohibitins (PHB), including prohibitin 1 (PHB1) and prohibitin 2 (PHB2), are evolutionary conserved and pleiotropic proteins implicated in various cellular functions, including proliferation, tumor suppression, apoptosis, transcription, and mitochondrial protein folding. The renin-angiotensin-aldosterone system plays a pivotal role in the regulation of blood pressure and volume homeostasis. All these factors and systems have been implicated in renal interstitial fibrosis. Therefore, the objective of this study was to investigate the effect of ATRA treatment on the renin-angiotensin-aldosterone system and expression of prohibitins to further understand its role in the processes leading to renal interstitial fibrosis. The hypoxic and oxidative stress conditions in obstructive renal disease were simulated in a hypoxia/reoxygenation model with renal tubular epithelial cells (RTEC) as a model system. Subsequently, the effect of ATRA on mRNA and protein expression levels was determined and correlations were established between factors involved in the renin-angiotensin-aldosterone system, the prohibitins, cellular redox status, renal interstitial fibrosis and ATRA treatment. Correlation analysis showed that both PHB1 and PHB2 protein levels were negatively correlated with angiotensin I, ACE1, angiotensin II, TGF-β1, Col-IV, FN, ROS, and MDA (PHB1: r = -0.792, -0.834, -0.805, -0.795, -0.778, -0.798, -0.751, -0.682; PHB2: r = -0.872, -0.799, -0.838, -0.773, -0.769, -0.841, -0.794, -0.826; each p < 0.05), but positively correlated with ACE2, SOD, and GSH (PHB1: r = 0.796, 0.879, 0.824; PHB2: r = 0.785, 0.914, 0.849; each p < 0.05). ACE1 was positively correlated with angiotensin I, angiotensin II, TGF-β1, Col-IV, FN, ROS, and MDA, and negatively correlated with ACE2, SOD, and GSH (each p < 0.05). ACE2 was negatively correlated with ACE1, angiotensin I, angiotensin II, TGF-β1, Col-IV, FN, ROS, and MDA, and positively correlated with SOD and GSH (each p < 0.05). The results suggest that ATRA acts as a positive regulator of PHB1, PHB2 and ACE2, and as a negative regulator of ACE1, angiotensin I, and angiotensin II in a RTEC model system under hypoxia/reoxygenation conditions. © The Author(s) 2014.

METABOLISM AND DISPOSITION OF ACETAMINOPHEN: RECENT ADVANCES IN RELATION TO HEPATOTOXICITY AND DIAGNOSIS

PubMed Central

McGill, Mitchell R.; Jaeschke, Hartmut

2013-01-01

Acetaminophen (APAP) is one of the most widely used drugs. Though safe at therapeutic doses, overdose causes mitochondrial dysfunction and centrilobular necrosis in the liver. The first studies of APAP metabolism and activation were published more than forty years ago. Most of the drug is eliminated by glucuronidation and sulfation. These reactions are catalyzed by UDP-glucuronosyltransferases (UGT1A1 and 1A6) and sulfotransferases (SULT1A1, 1A3/4, and 1E1), respectively. However, some is converted by CYP2E1 and other cytochrome P450 enzymes to a reactive intermediate that can bind to sulfhydryl groups. The metabolite can deplete liver glutathione (GSH) and modify cellular proteins. GSH binding occurs spontaneously, but may also involve GSH-S-transferases. Protein binding leads to oxidative stress and mitochondrial damage. The glucuronide, sulfate, and GSH conjugates are excreted by transporters in the canalicular (Mrp2 and Bcrp) and basolateral (Mrp3 and Mrp4) hepatocyte membranes. Conditions that interfere with metabolism and metabolic activation can alter the hepatotoxicity of the drug. Recent data providing novel insights into these processes, particularly in humans, are reviewed in the context of earlier work, and the effects of altered metabolism and reactive metabolite formation are discussed. Recent advances in the diagnostic use of serum adducts are covered. PMID:23462933

[Effect of Coriolus versicolor polysaccharide B on membrane glycosaminoglycans and cellular glutathione changes in RAW264.7 macrophages exposed to angiotensin II].

PubMed

Lou, Ning; Ma, Gang; Wang, Dao-feng; Zhu, Zhi-wei; Su, Quan-guan; Fang, Yi

2007-12-01

To investigate the effect of Coriolus versicolor polysaccharide B (CVP-B) on increased membrane glycosaminoglycans (GAG) expression and intracellular glutathione (GSH) of RAW264.7 macrophages exposed to angiotensin II (Ang II). The plasma membrane of RAW264.7 macrophages exposed to Ang II treatment was isolated by ultracentrifugation, and the membrane GAG expression was analyzed using 1, 9-dimethylmethylene blue (DMMB) spectrophotometric assay for sulfated GAG. The intracellular reduced GSH was determined using fluorophotometry. The GAG content in the macrophage membranes increased by up to 54% following cell exposure to 1.0 micromol/L Ang II, whereas in presence of 1.0 micromol;/L Ang II, CVP-B at 1, 10, and 50 microg/ml decreased the GAG content by 13%, 43% (P<0.01), and 52% (P<0.01), respectively. The macrophage GSH activity decreased by 69% following incubation with 1.0 micromol;/L Ang II for 24 h, and CVP-B treatment at 1, 10, and 50 microg/ml in presence of 1.0 micromol;/L Ang II resulted in significant increment of GSH activity by 31%(P<0.05), 104% (P<0.01), and 168% (P<0.01), respectively. These data provide the first evidence that CVP-B inhibits elevated GAG expression in RAW264.7 macrophage membrane induced by Ang II.

A Novel Polyphenol Conjugate Sensitizes Cisplatin-Resistant Head and Neck Cancer Cells to Cisplatin via Nrf2 Inhibition.

PubMed

Kim, Eun Hye; Jang, Hyejin; Roh, Jong-Lyel

2016-11-01

Many cancer cells show acquired resistance to chemotherapeutic agents, such as cisplatin. This is a major cause of cancer treatment failure, and novel agents to overcome resistance are thus urgently required. A novel synthetic polyphenol conjugate, (E)-3-(3,5-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (DPP-23), selectively kills tumor cells via the reactive oxygen species (ROS)-mediated unfolded protein response. We investigated the ability of DPP-23 to overcome cisplatin resistance in head and neck cancer (HNC) cells and further clarified its molecular mechanisms of action. Cisplatin-resistant HNC cell lines and their parental and other HNC cell lines were used. The effects of cisplatin and DPP-23 were assessed alone and in combination in HNC and normal cells using cell viability, cell cycle, and cell death assays, by measuring glutathione (GSH), ROS, and protein levels, and via preclinical mouse studies. DPP-23 induced selective cell death in HNC cells, including cisplatin-resistant HNC cells, but spared normal cells, via cellular GSH depletion and ROS accumulation. The effect was blocked by the antioxidant N-acetyl-L-cysteine. DPP-23 activated p53 and its related cell death pathways via a robust accumulation of cellular ROS that involved inhibition of nuclear factor erythroid 2-related factor 2 antioxidant defense mechanisms. Thus, DPP-23 significantly overcame cisplatin resistance in HNC cells in vitro and in vivo As a promising anticancer strategy, ROS generation and subsequent selective cancer cell killing by DPP-23 might help to overcome cisplatin resistance in HNC. Mol Cancer Ther; 15(11); 2620-9. ©2016 AACR. ©2016 American Association for Cancer Research.

Effects of dietary selenium of organic form against lead toxicity on the antioxidant system in Cyprinus carpio.

PubMed

Özkan-Yilmaz, Ferbal; Özlüer-Hunt, Arzu; Gündüz, Suna Gül; Berköz, Mehmet; Yalin, Serap

2014-04-01

In this study was evaluated potential protective effect of organic selenium (Se) on heavy metal stress induced by lead (Pb) in Cyprinus carpio. For this reason, C. carpio was exposed to sublethal concentration of Pb (1.5 mg/L Pb(NO3)2) for 14 days. The fish were fed a basal (control; measured 0.55 mg/kg Se) diet or a basal diet supplemented with 2.50 mg/kg (measured 2.92 mg/kg Se) organic Se (Sel-Plex(®)) during the experiment period. The variations in glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) activities, and levels of reduced glutathione (GSH) with malondialdehyde (MDA) in liver and brain tissues of C. carpio were investigated in experimental groups. GSH levels in liver and brain tissues were significantly decreased by exposure to Pb. GST activity was significantly increased (p < 0.05) in liver tissue, but decreased in brain of treated fish by exposure to Pb. Also, GSH-Px activity was significantly increased in liver tissue, but decreased in brain of Pb-treated fish. Levels of MDA were increased in liver and brain of Pb-treated fish. The organic Se treatment for Pb-intoxicated animals improved activities of GSH-Px, GST and levels of MDA within normal limits. Supplemented Se could be able to improve Pb-induced oxidative stress by decreasing lipid peroxidation and regulating antioxidant defense system in tissues.

Reduction potentials of protein disulfides and catalysis of glutathionylation and deglutathionylation by glutaredoxin enzymes.

PubMed

Ukuwela, Ashwinie A; Bush, Ashley I; Wedd, Anthony G; Xiao, Zhiguang

2017-11-09

Glutaredoxins (Grxs) are a class of GSH (glutathione)-dependent thiol-disulfide oxidoreductase enzymes. They use the cellular redox buffer GSSG (glutathione disulfide)/GSH directly to catalyze these exchange reactions. Grxs feature dithiol active sites and can shuttle rapidly between three oxidation states, namely dithiol Grx(SH) 2 , mixed disulfide Grx(SH)(SSG) and oxidized disulfide Grx(SS). Each is characterized by a distinct standard reduction potential [Formula: see text] The [Formula: see text] values for the redox couple Grx(SS)/Grx(SH) 2 are available, but a recent estimate differs by over 100 mV from the literature values. No estimates are available for [Formula: see text] for the mixed disulfide couple Grx(SH)(SSG)/(Grx(SH) 2  + GSH). This work determined both [Formula: see text] and [Formula: see text] for two representative Grx enzymes, Homo sapiens HsGrx1 and Escherichia coli EcGrx1. The empirical approaches were verified rigorously to overcome the sensitivity of these redox-labile enzymes to experimental conditions. The classic method of acid 'quenching' was demonstrated to shift the thiol-disulfide redox equilibria. Both enzymes exhibit an [Formula: see text] (vs. SHE) at a pH of 7.0. Their [Formula: see text] values (-213 and -230 mV for EcGrx1 and HsGrx1, respectively) are slightly less negative than that ([Formula: see text]) of the redox buffer GSSG/2GSH. Both [Formula: see text] and [Formula: see text] vary with log [GSH], but the former more sensitively by a factor of 2. This confers dual catalytic functions to a Grx enzyme as either an oxidase at low [GSH] or as a reductase at high [GSH]. Consequently, these enzymes can participate efficiently in either glutathionylation or deglutathionylation. The catalysis is demonstrated to proceed via a monothiol ping-pong mechanism relying on a single Cys residue only in the dithiol active site. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

p-Aminophenol-induced hepatotoxicity in hamsters: role of glutathione.

PubMed

Fu, Xin; Chen, Theresa S; Ray, Mukunda B; Nagasawa, Herbert T; Williams, Walter M

2004-01-01

p-Aminophenol (PAP) is a widely used industrial chemical and a known nephrotoxin. Recently, it was found to also cause hepatotoxicity and glutathione (GSH) depletion in mice. The exact mechanism of liver toxicity is not known. The aims of this study were to determine whether PAP can cause acute hepatotoxicity in hamsters and to further investigate the role of GSH in PAP-induced toxicity. PAP was administered ip to hamsters in doses of 200-800 mg/kg. Liver damage at 24 h after PAP administration was assessed by elevations in plasma enzyme activities and histopathologic examination. GSH and cysteine (Cys) levels in liver at 4 h were determined by HPLC. PAP decreased hepatic GSH concentration to 8% and Cys to 30% of vehicle control values. It increased plasma glutamic pyruvic transaminase (GPT) activity by 47-fold and sorbitol dehydrogenase (SDH) activity by 113-fold. PAP also caused severe centrilobular hepatocellular necrosis. 2(RS)-n-Propylthiazolidine-4(R)-carboxylic acid (PTCA), a Cys precursor, attenuated the PAP-induced decreases in hepatic sulfhydryl levels; GSH and Cys were 39% and 78% of vehicle controls, respectively. PTCA also attenuated the PAP-induced elevations in plasma enzyme activities and hepatic necrosis. It was concluded that PAP hepatotoxicity is associated with depletion of hepatic GSH and can be prevented by PTCA. Copyright 2004 Wiley Periodicals, Inc.

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones

PubMed Central

Clemente, Maria R.; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K.; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

2012-01-01

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1–48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24–48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses. PMID:22442424

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones.

PubMed

Clemente, Maria R; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

2012-06-01

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1-48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24-48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.

Redox-sensitive shell-crosslinked polypeptide-block-polysaccharide micelles for efficient intracellular anticancer drug delivery.

PubMed

Zhang, Aiping; Zhang, Zhe; Shi, Fenghua; Xiao, Chunsheng; Ding, Jianxun; Zhuang, Xiuli; He, Chaoliang; Chen, Li; Chen, Xuesi

2013-09-01

Redox-responsive SCMs based on amphiphilic PBLG-b-dextran with good biocompatibility are synthesized and used for efficient intracellular drug delivery. The molecular structures and SCMs characteristics are characterized by (1) H NMR, FT-IR, TEM, and DLS. The hydrodynamic radius of SCMs increases gradually in PBS due to the cleavage of disulfide bond in micellar shell caused by the presence of GSH. The encapsulation efficiency and release kinetics of DOX are investigated. The fastest DOX release is observed under intracellular-mimicking reductive environments. An MTT assay demonstrates that DOX-loaded SCMs show higher cellular proliferation inhibition against GSH-OEt pretreated HeLa and HepG2 than that of the non-pretreated and BSO-pretreated ones. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The mycotoxin patulin reacts with DNA bases with and without previous conjugation to GSH: implication for related α,β-unsaturated carbonyl compounds?

PubMed

Pfenning, Carolin; Esch, Harald L; Fliege, Ralph; Lehmann, Leane

2016-02-01

The α,β-unsaturated carbonyl group is recognized as alert for mutagenicity, attributed to (1) its direct reaction with DNA, counteractable by glutathione (GSH), and (2) oxidative stress caused indirectly by GSH depletion. Accordingly, the α,β,γ,δ-unsaturated lactone patulin (PAT), a mycotoxin detected in fruits and products derived thereof, is known to induce gene, chromosome, and genome mutations in vitro, its mutagenicity correlating inversely with intracellular GSH levels. Thus, the reactivity of PAT against DNA bases and nucleosides in the absence and presence of GSH and glutathione S-transferases (GSTs) was investigated under cell-free conditions using HPLC mass spectrometry techniques for identification of reaction products. Adduct formation with all four nucleobases as well as with purine base nucleosides occurred even in the presence of GSH, revealing several adducts of PAT, mono- and disubstituted with nucleobases/nucleosides as well as novel GSH-PAT adducts. In addition, novel mixed GSH-PAT-nucleobase adducts were observed. These adducts exhibited a ketohexanoic acid-type structure of the PAT molecule, C6 substituted with GSH and linking C1 of PAT with nitrogens of nucleobases/nucleosides via an amide bond. Formation of GSH-PAT-adenine adducts was not prevented by GSTs, and excess of GSH needed to reduce their formation was higher than for PAT-adenine adducts. The formation of mixed GSH-DNA base adducts has not been described for PAT or any other α,β-unsaturated carbonyl before, although the reaction mechanism seems to be applicable to a variety of α,β-unsaturated carbonyls occurring in food and in the environment.

Do glutathione levels decline in aging human brain?

PubMed

Tong, Junchao; Fitzmaurice, Paul S; Moszczynska, Anna; Mattina, Katie; Ang, Lee-Cyn; Boileau, Isabelle; Furukawa, Yoshiaki; Sailasuta, Napapon; Kish, Stephen J

2016-04-01

For the past 60 years a major theory of "aging" is that age-related damage is largely caused by excessive uncompensated oxidative stress. The ubiquitous tripeptide glutathione is a major antioxidant defense mechanism against reactive free radicals and has also served as a marker of changes in oxidative stress. Some (albeit conflicting) animal data suggest a loss of glutathione in brain senescence, which might compromise the ability of the aging brain to meet the demands of oxidative stress. Our objective was to establish whether advancing age is associated with glutathione deficiency in human brain. We measured reduced glutathione (GSH) levels in multiple regions of autopsied brain of normal subjects (n=74) aged one day to 99 years. Brain GSH levels during the infancy/teenage years were generally similar to those in the oldest examined adult group (76-99 years). During adulthood (23-99 years) GSH levels remained either stable (occipital cortex) or increased (caudate nucleus, frontal and cerebellar cortices). To the extent that GSH levels represent glutathione antioxidant capacity, our postmortem data suggest that human brain aging is not associated with declining glutathione status. We suggest that aged healthy human brains can maintain antioxidant capacity related to glutathione and that an age-related increase in GSH levels in some brain regions might possibly be a compensatory response to increased oxidative stress. Since our findings, although suggestive, suffer from the generic limitations of all postmortem brain studies, we also suggest the need for "replication" investigations employing the new (1)H MRS imaging procedures in living human brain. Copyright © 2016 Elsevier Inc. All rights reserved.

Pivotal role of glutathione depletion in plasma-induced endothelial oxidative stress during sepsis.

PubMed

Huet, Olivier; Cherreau, Christaine; Nicco, Carole; Dupic, Laurent; Conti, Marc; Borderie, Didier; Pene, Frédéric; Vicaut, Eric; Benhamou, Dan; Mira, Jean-Paul; Duranteau, Jacques; Batteux, Frédéric

2008-08-01

Plasma from septic shock patients can induce production of reactive oxygen species (ROS) by human umbilical vein endothelial cells (HUVEC) in vitro. How endothelial cells defend themselves against ROS under increased oxidative stress has not yet been examined. This study investigates the antioxidant defenses of HUVEC exposed to plasma obtained from either septic shock patients or healthy volunteers. Prospective, observational study. Medical intensive care unit in a university hospital. Twenty-five patients with septic shock and 10 healthy volunteers. Blood samples were collected within the first 24 hrs of septic shock. In vitro HUVEC production of ROS was studied by spectrofluorimetry using 2',7'-dichlorodihydrofluorescein diacetate fluorescent dye. Reactive nitrogen species were also assessed. Intracellular reduced glutathione (GSH) levels were measured using monochlorobimane fluorescent dye. Activity of catalase and superoxide dismutase in HUVEC were also measured. Cell death was assessed using YOPRO fluorescent dye and the MTT assay. On admission, the septic shock population's mean age was 55 yrs old, the mean Sequential Organ Failure Assessment score was 12, mean simplified acute physiology score was 50, and intensive care unit mortality rate was 45%. Evaluation of HUVEC antioxidant defenses showed a significantly decreased GSH level, increased catalase activity, and unchanged superoxide dismutase activity. ROS levels and cell death were significantly reduced when cells were pretreated with N-acetylcysteine or GSH, but no changes in reactive nitrogen species were observed. This study demonstrates that plasma-induced ROS production by HUVEC is associated with an intracellular decrease in reduced GSH. Both ROS levels and cell death decreased when N-acetylcysteine or GSH were added before exposing the cells to plasma. These data suggest a pivotal role of alterations in GSH in damage caused by sepsis-generated ROS in endothelial cell.

N-acetylcysteine in a Double-Blind Randomized Placebo-Controlled Trial: Toward Biomarker-Guided Treatment in Early Psychosis

PubMed Central

Conus, Philippe; Seidman, Larry J; Fournier, Margot; Xin, Lijing; Cleusix, Martine; Baumann, Philipp S; Ferrari, Carina; Cousins, Ann; Alameda, Luis; Gholam-Rezaee, Mehdi; Golay, Philippe; Jenni, Raoul; Woo, T -U Wilson; Keshavan, Matcheri S; Eap, Chin B; Wojcik, Joanne; Cuenod, Michel; Buclin, Thierry; Gruetter, Rolf

2018-01-01

Abstract Biomarker-guided treatments are needed in psychiatry, and previous data suggest oxidative stress may be a target in schizophrenia. A previous add-on trial with the antioxidant N-acetylcysteine (NAC) led to negative symptom reductions in chronic patients. We aim to study NAC’s impact on symptoms and neurocognition in early psychosis (EP) and to explore whether glutathione (GSH)/redox markers could represent valid biomarkers to guide treatment. In a double-blind, randomized, placebo-controlled trial in 63 EP patients, we assessed the effect of NAC supplementation (2700 mg/day, 6 months) on PANSS, neurocognition, and redox markers (brain GSH [GSHmPFC], blood cells GSH levels [GSHBC], GSH peroxidase activity [GPxBC]). No changes in negative or positive symptoms or functional outcome were observed with NAC, but significant improvements were found in favor of NAC on neurocognition (processing speed). NAC also led to increases of GSHmPFC by 23% (P = .005) and GSHBC by 19% (P = .05). In patients with high-baseline GPxBC compared to low-baseline GPxBC, subgroup explorations revealed a link between changes of positive symptoms and changes of redox status with NAC. In conclusion, NAC supplementation in a limited sample of EP patients did not improve negative symptoms, which were at modest baseline levels. However, NAC led to some neurocognitive improvements and an increase in brain GSH levels, indicating good target engagement. Blood GPx activity, a redox peripheral index associated with brain GSH levels, could help identify a subgroup of patients who improve their positive symptoms with NAC. Thus, future trials with antioxidants in EP should consider biomarker-guided treatment. PMID:29462456

Cytotoxicity and the induction of the stress protein Hsp 70 in Chang liver cells in response to zearalenone-induced oxidative stress.

PubMed

Lee, Hyungkyoung; Kang, Changgeun; Yoo, Yong-San; Hah, Do-Yun; Kim, Chung Hui; Kim, Euikyung; Kim, Jong Shu

2013-09-01

Zearalenone (ZEN) has been implicated in several cases of mycotoxicosis in farm animals and humans. The toxic effects of ZEN have been well characterized, but little is known regarding the mechanisms of ZEN toxicity, including the involvement of the oxidative stress pathway. Using Chang liver cells as a model, the aim of this study was to determine if ZEN could elevate the expression of the heat shock protein Hsp 70, induce cytotoxicity and modulate the levels of glutathione (GSH) and thiobarbituric acid reactive substance (TBARS). In addition, the cytoprotective effects of N-acetylcysteine amide (NACA) pre-treatment were assessed. Finally, the involvement of oxidative stress in ZEN-induced toxicity was confirmed. The results of this study demonstrated that ZEN-induced Hsp 70 expression in a dose- and time-dependent manners. This effect occurred at low-ZEN concentrations, and could therefore be considered a biomarker of ZEN-induced toxicity. The cytotoxicity was reduced when Chang liver cells were exposed to sub-lethal heat shock prior to ZEN treatment, demonstrating a cytoprotective effect of Hsp 70. This cytoprotective effect suggested that Hsp 70 might play a key role in the cellular defense mechanism. When cells were pre-treated with NACA prior to ZEN treatment, the cells were also protected from toxicity. This NACA cytoprotective effect suggested the involvement of oxidative stress in ZEN-induced toxicity, and this mechanism was supported by reduced Hsp 70 expression, inhibited cytolethality, increased GSH levels and decreased TBARS formation when cells were pre-treated with NACA prior to ZEN exposure. Our data clearly demonstrated that ZEN induced cytotoxicity in Chang liver cells by inhibiting cell proliferation, decreasing GSH levels and increasing TBARS formation in a dose-dependent manner. ZEN also, induced Hsp 70 expression, and the side effects of ZEN were significantly alleviated by pre-treatment with NACA. Oxidative stress is likely to be one of the primary pathways of ZEN toxicity. This oxidative stress may contribute, at least in part, to the mechanism of ZEN-induced cytotoxicity. Copyright © 2013 Elsevier B.V. All rights reserved.

Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its D,L-polylactide microparticle formulation.

PubMed

Bartolini, D; Piroddi, M; Tidei, C; Giovagnoli, S; Pietrella, D; Manevich, Y; Tew, K D; Giustarini, D; Rossi, R; Townsend, D M; Santi, C; Galli, F

2015-01-01

Catalytic properties and cellular effects of the glutathione peroxidase (GPx)-mimetic compound PhSeZnCl or its d,l-lactide polymer microencapsulation form (M-PhSeZnCl) were investigated and compared with the prototypical Se-organic compounds ebselen and diselenide (PhSe)2. PhSeZnCl was confirmed to catalyze the ping-pong reaction of GPx with higher Vmax than ebselen and (PhSe)2, but the catalytic efficiency calculated for the cosubstrates glutathione (GSH) and H2O2, and particularly the high reactivity against thiols (lowest KM for GSH in the series of test molecules), suggested poor biological applicability of PhSeZnCl as a GPx mimetic. Cytotoxicity of PhSeZnCl was demonstrated in various cancer cell lines via increased reactive oxygen species (ROS) generation, depletion of intracellular thiols, and induction of apoptosis. Experiments carried out in GSH S-transferase P (GSTP)-overexpressing K562 human erythroleukemia cells and in GSTP1-1-knockout murine embryonic fibroblasts (MEFs) demonstrated that this cytosolic enzyme represents a preferential target of the redox disturbances produced by this Se-compound with a key role in controlling H2O2 generation and the perturbation of stress/survival kinase signaling. Microencapsulation was adopted as a strategy to control the thiol reactivity and oxidative stress effects of PhSeZnCl, then assessing applications alternative to anticancer. The uptake of this "depowered" GPx-mimetic formulation, which occurred through an endocytosis-like mechanism, resulted in a marked reduction of cytotoxicity. In MCF-7 cells transfected with different allelic variants of GSTP, M-PhSeZnCl lowered the burst of cellular ROS induced by the exposure to extracellular H2O2, and the extent of this effect changed between the GSTP variants. Microencapsulation is a straightforward strategy to mitigate the toxicity of thiol-reactive Se-organic drugs that enhanced the antioxidant and cellular protective effects of PhSeZnCl. A mechanistic linkage of these effects with the expression pattern and signaling properties of GSTP . This has overcome the GPx-mimetic paradigm proposed for Se-organic drugs with a more pragmatic concept of GSTP signaling modulators. Copyright © 2014 Elsevier Inc. All rights reserved.

Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its d,l-polylactide microparticle formulation

PubMed Central

Bartolini, D.; Piroddi, M.; Tidei, C.; Giovagnoli, S.; Pietrella, D.; Manevich, Y.; Tew, K.D.; Giustarini, D.; Rossi, R.; Townsend, D.M.; Santi, C.; Galli, F.

2015-01-01

Catalytic properties and cellular effects of the glutathione peroxidase (GPx)-mimetic compound PhSeZnCl or its d,l-lactide polymer microencapsulation form (M-PhSeZnCl) were investigated and compared with the prototypical Se-organic compounds ebselen and diselenide (PhSe)2. PhSeZnCl was confirmed to catalyze the ping-pong reaction of GPx with higher Vmax than ebselen and (PhSe)2, but the catalytic efficiency calculated for the cosubstrates glutathione (GSH) and H2O2, and particularly the high reactivity against thiols (lowest KM for GSH in the series of test molecules), suggested poor biological applicability of PhSeZnCl as a GPx mimetic. Cytotoxicity of PhSeZnCl was demonstrated in various cancer cell lines via increased reactive oxygen species (ROS) generation, depletion of intracellular thiols, and induction of apoptosis. Experiments carried out in GSH S-transferase P (GSTP)-overexpressing K562 human erythroleukemia cells and in GSTP1-1-knockout murine embryonic fibroblasts (MEFs) demonstrated that this cytosolic enzyme represents a preferential target of the redox disturbances produced by this Se-compound with a key role in controlling H2O2 generation and the perturbation of stress/survival kinase signaling. Microencapsulation was adopted as a strategy to control the thiol reactivity and oxidative stress effects of PhSeZnCl, then assessing applications alternative to anticancer. The uptake of this “depowered” GPx-mimetic formulation, which occurred through an endocytosis-like mechanism, resulted in a marked reduction of cytotoxicity. In MCF-7 cells transfected with different allelic variants of GSTP, M-PhSeZnCl lowered the burst of cellular ROS induced by the exposure to extracellular H2O2, and the extent of this effect changed between the GSTP variants. Microencapsulation is a straightforward strategy to mitigate the toxicity of thiol-reactive Se-organic drugs that enhanced the antioxidant and cellular protective effects of PhSeZnCl. A mechanistic linkage of these effects with the expression pattern and signaling properties of GSTP. This has overcome the GPx-mimetic paradigm proposed for Se-organic drugs with a more pragmatic concept of GSTP signaling modulators. PMID:25452145

Wound Healing Potential of Intermittent Negative Pressure under Limited Access Dressing in Burn Patients: Biochemical and Histopathological Study

PubMed Central

Muguregowda, Honnegowda Thittamaranahalli; Kumar, Pramod; Govindarama, Padmanabha Udupa Echalasara

2018-01-01

BACKGROUND Malondialdehyde (MDA) is an oxidant that causes damage to membranes, DNA, proteins, and lipids at the cellular level. Antioxidants minimize the effects of oxidants and thus help in formation of healthy granulation tissues with higher level of hydroxyproline and total protein. This study compared the effect of limited access dressing (LAD) with conventional closed dressing biochemically and histopathologically. METHODS Seventy-two 12-65 years old burn patients with mean wound size of 14 cm2 were divided to two groups of LAD (n=37), and conventional dressing groups (n=35). Various biochemical parameters were measured in granulation tissue. Histopathological analysis of the granulation tissue was studied too. RESULTS LAD group showed significant increase in hydroxyproline, total protein, GSH, and GPx and decrease in MDA levels compared to conventional dressing group. A significant negative correlation between GSH and MDA was noted in LAD group, but in conventional dressing group there was no significant correlation. A significant negative correlation between GPx and MDA was noticed in LAD group, but in conventional dressing group was not significant. There was a histologically fewer inflammatory cells, increased and well organized extracellular matrix deposit, more angiogenesis in LAD group after 10 days while the difference was significant between the groups. CONCLUSION Our study showed a significant reduction in oxidative stress biomarker of MDA, increase in hydroxyproline, total protein, antioxidants and amount of ECM deposition, number of blood vessels and a decrease in the amount of inflammatory cells and necrotic tissues in LAD group indicating the better healing effect of burn wounds. PMID:29651393

Protective effect of polysaccharides from Sargassum horneri against oxidative stress in RAW264.7 cells.

PubMed

Wen, Zheng-Shun; Liu, Li-Jia; OuYang, Xiao-Kun; Qu, You-Le; Chen, Yin; Ding, Guo-Fang

2014-07-01

This study was designed to investigate chemical composition and the protective effects of polysaccharides isolated from Sargassum horneri against hydrogen peroxide (H2O2)-induced oxidative injury in RAW264.7 cells. Results showed that isolated polysaccharides (SHSc) and the major fractions (SHS1, SHS0.5) contained sulfate ester, and SHS1 was high fucose-containing sulfated polysaccharide. After preincubation with three isolated polysaccharides, RAW264.7 cells viability were significantly restored and decreased in cellular LDH release (P<0.05). SHS1 and SHS0.5 decreased intracellular ROS level, intracellular NO and malonic dialdehyde (MDA) level (P<0.05), restoring activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) (P<0.05), decreasing inducible nitric oxide synthase (iNOS) (P<0.05). Moreover, preincubation of SHS1 with RAW264.7 cells resulted in the increase of the gene expression level of endogenous antioxidant enzymes such as MnSOD and GSH-Px (P<0.05). These results clearly showed that SHSc and its fractions could attenuate H2O2-induced stress injury in RAW264.7 cells, and a similar efficiency in protecting RAW264.7 cells against H2O2-induced oxidative injury between SHS1 and Vitamin C. Taken together, our findings suggested that SHS1 can effectively protect RAW264.7 cells against oxidative stress by H2O2, which might be used as a potential natural antioxidant in the functional food and pharmaceutical industries. Copyright © 2014 Elsevier B.V. All rights reserved.

Dose-dependent protective effect of sildenafil citrate on testicular injury after torsion/detorsion in rats.

PubMed

Yıldız, H; Durmus, A S; Şimşek, H; Yaman, M

2012-05-01

This experiment was designed to investigate the effect of sildenafil citrate on testicular injury after unilateral testicular torsion/detorsion (T/D). Thirty-seven adult male Wistar albino rats were divided into four groups: sham operated group (group 1), T/D+ saline (group 2), T/D+ 0.7 mg sildenafil citrate (group 3) and T/D+ 1.4 mg sildenafil citrate (group 4). Testicular torsion was created by rotating the right testis 720° in a clockwise direction for 2 h in other groups, except for group 1, which was served as sham group. The level of GSH (P < 0.05) in the testis in the group 2 were significantly lower (P < 0.05) and the levels of MDA and NO (P < 0.01 for both) in the testis were significantly higher when compared with those of the group 1. Administration of low dose sildenafil citrate prevented the increases in MDA and NO levels and decreases in GSH values induced by testicular torsion. However, administration of high dose sildenafil citrate did not have any effect on these testicular tissue parameters (P > 0.05). Also, mean values of seminiferous tubules diameters, germinal cell layer thicknesses and mean testicular biopsy score were significantly better in group 3 than groups 2 and 4. These results suggest that T/D injury occurred in testis after unilateral testicular T/D and that administration of low dose sildenafil citrate before detorsion prevents ischemia/reperfusion cellular damage in testicular torsion. Sildenafil citrate probably acts through reduction of reactive oxygen species and support antioxidant enzyme systems. © 2011 Blackwell Verlag GmbH.

Protective effect of sildenafil citrate on contralateral testis injury after unilateral testicular torsion/detorsion.

PubMed

Yíldíz, Hamit; Durmus, Ali Said; Simşek, Halil; Yaman, Mine

2011-01-01

This study was designed to investigate prevention of contralateral testicular injury with sildenafil citrate after unilateral testicular torsion/detorsion. Thirty-seven adult male rats were divided into four groups: sham operated (group 1, n = 7), torsion/detorsion + saline (group 2, n = 10), torsion/detorsion + 0.7 mg of sildenafil citrate (group 3, n = 10) and torsion/detorsion + 1.4 mg of sildenafil citrate (group 4, n = 10). Unilateral testicular torsion was created by rotating the right testis 720º in a clockwise direction for 2 h in other groups, except for group 1, which was served as sham group. After torsion (2 h) and detorsion (2 h) periods, rats were killed. The level of reduced glutathion (GSH) (p < 0.05) and the activities of catalase (p < 0.01) and glutathione peroxidase (p < 0.05) in the contralateral testis from group 2 were significantly lower and nitric oxide (NO) (p < 0.05) level in the contralateral testis were significantly higher than those of group 1. Administration of low-dose sildenafil citrate (group 3) prevented the increases in malondialdehyde and NO levels and decreases in glutathione peroxidase activities and GSH values induced by testicular torsion. However, administration of high-dose sildenafil citrate (group 4) had no effect on these testicular parameters (p > 0.05). Histopathological changes were detected in groups 2, 3 and 4. These results suggest that biochemically and histologically torsion/detorsion injury occurs in the contralateral testis following 2-h torsion and 2-h detorsion and that administration of low-dose sildenafil citrate before detorsion prevents ischemia/reperfusion cellular damage in testicular tissue.

The Mixture of Salvianolic Acids from Salvia miltiorrhiza and Total Flavonoids from Anemarrhena asphodeloides Attenuate Sulfur Mustard-Induced Injury

PubMed Central

Li, Jianzhong; Chen, Linlin; Wu, Hongyuan; Lu, Yiming; Hu, Zhenlin; Lu, Bin; Zhang, Liming; Chai, Yifeng; Zhang, Junping

2015-01-01

Sulfur mustard (SM) is a vesicating chemical warfare agent used in numerous military conflicts and remains a potential chemical threat to the present day. Exposure to SM causes the depletion of cellular antioxidant thiols, mainly glutathione (GSH), which may lead to a series of SM-associated toxic responses. MSTF is the mixture of salvianolic acids (SA) of Salvia miltiorrhiza and total flavonoids (TFA) of Anemarrhena asphodeloides. SA is the main water-soluble phenolic compound in Salvia miltiorrhiza. TFA mainly includes mangiferin, isomangiferin and neomangiferin. SA and TFA possess diverse activities, including antioxidant and anti-inflammation activities. In this study, we mainly investigated the therapeutic effects of MSTF on SM toxicity in Sprague Dawley rats. Treatment with MSTF 1 h after subcutaneous injection with 3.5 mg/kg (equivalent to 0.7 LD50) SM significantly increased the survival levels of rats and attenuated the SM-induced morphological changes in the testis, small intestine and liver tissues. Treatment with MSTF at doses of 60 and 120 mg/kg caused a significant (p < 0.05) reversal in SM-induced GSH depletion. Gene expression profiles revealed that treatment with MSTF had a dramatic effect on gene expression changes caused by SM. Treatment with MSTF prevented SM-induced differential expression of 93.8% (973 genes) of 1037 genes. Pathway enrichment analysis indicated that these genes were mainly involved in a total of 36 pathways, such as the MAPK signaling pathway, pathways in cancer, antigen processing and presentation. These data suggest that MSTF attenuates SM-induced injury by increasing GSH and targeting multiple pathways, including the MAPK signaling pathway, as well as antigen processing and presentation. These results suggest that MSTF has the potential to be used as a potential therapeutic agent against SM injuries. PMID:26501264

Glutathione maintenance mitigates age-related susceptibility to redox cycling agents.

PubMed

Thomas, Nicholas O; Shay, Kate P; Kelley, Amanda R; Butler, Judy A; Hagen, Tory M

2016-12-01

Isolated hepatocytes from young (4-6mo) and old (24-26mo) F344 rats were exposed to increasing concentrations of menadione, a vitamin K derivative and redox cycling agent, to determine whether the age-related decline in Nrf2-mediated detoxification defenses resulted in heightened susceptibility to xenobiotic insult. An LC 50 for each age group was established, which showed that aging resulted in a nearly 2-fold increase in susceptibility to menadione (LC 50 for young: 405μM; LC 50 for old: 275μM). Examination of the known Nrf2-regulated pathways associated with menadione detoxification revealed, surprisingly, that NAD(P)H: quinone oxido-reductase 1 (NQO1) protein levels and activity were induced 9-fold and 4-fold with age, respectively (p=0.0019 and p=0.018; N=3), but glutathione peroxidase 4 (GPX4) declined by 70% (p=0.0043; N=3). These results indicate toxicity may stem from vulnerability to lipid peroxidation instead of inadequate reduction of menadione semi-quinone. Lipid peroxidation was 2-fold higher, and GSH declined by a 3-fold greater margin in old versus young rat cells given 300µM menadione (p<0.05 and p≤0.01 respectively; N=3). We therefore provided 400µMN-acetyl-cysteine (NAC) to hepatocytes from old rats before menadione exposure to alleviate limits in cysteine substrate availability for GSH synthesis during challenge. NAC pretreatment resulted in a >2-fold reduction in cell death, suggesting that the age-related increase in menadione susceptibility likely stems from attenuated GSH-dependent defenses. This data identifies cellular targets for intervention in order to limit age-related toxicological insults to menadione and potentially other redox cycling compounds. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Assessment of phosphamidon-induced apoptosis in human peripheral blood mononuclear cells: protective effects of N-acetylcysteine and curcumin.

PubMed

Ahmed, Tanzeel; Tripathi, Ashok K; Ahmed, Rafat S; Banerjee, Basu Dev

2010-01-01

The molecular mechanism for noncholinergic toxicity of phosphamidon, an extensively used organophosphate pesticide, is still not clear. The aim of the present study is to find the possible molecular mechanism of this pesticide to induce apoptosis and the role of different drugs for attenuation of such effects. Human peripheral blood mononuclear cells (PBMC) were incubated with increasing concentrations of phosphamidon (0-20 μM) for 6-24 h. The MTT assay reveals that phosphamidon induces cytotoxicity in a dose-dependent manner. Cellular glutathione (GSH) is depleted in a dose-dependent manner from 55% to 70% at concentrations between 10 and 20 μM. The percentage of cells that bind to Annexin-V, which is a representative of cells either undergoing apoptosis or necrosis during 24 h incubation, increases in a dose-dependent manner. Above 5 μM, significant necrosis of cells was observed. DNA fragmentation assay revealed that at low concentration of phosphamidon (1 μM), no appreciable change in DNA fragmentation was seen; however, distinct fragmentation was observed beyond 2.5 μM. Phosphamidon was found to cause significant depletion of GSH, which correlates well with the percentage of cells undergoing apoptosis. An increasing trend in levels of cytochrome c was observed with increasing concentration of phosphamidon, indicating that the apoptotic effect of phosphamidon is mediated through cytochrome c release. Coadministration of the antioxidants N-acetylcysteine and curcumin attenuated phosphamidon-induced apoptosis. This further supports our hypothesis that oxidative stress, as indicated by GSH depletion, results in the induction of apoptosis by release of cytochrome c. Copyright 2010 Wiley Periodicals, Inc.

Anti-inflammatory polymersomes of redox-responsive polyprodrug amphiphiles with inflammation-triggered indomethacin release characteristics.

PubMed

Tan, Jiajia; Deng, Zhengyu; Liu, Guhuan; Hu, Jinming; Liu, Shiyong

2018-03-21

Inflammation serves as a natural defense mechanism to protect living organisms from infectious diseases. Nonsteroidal anti-inflammatory drugs (NSAIDs) can help relieve inflammatory reactions and are clinically used to treat pain, fever, and inflammation, whereas long-term use of NSAIDs may lead to severe side effects including gastrointestinal damage and cardiovascular toxicity. Therefore, it is of increasing importance to configure new dosing strategies and alleviate the side effects of NSAIDs. Towards this goal, glutathione (GSH)-responsive disulfide bonds and hydrogen peroxide (H 2 O 2 )-reactive phenylboronic ester linkages were utilized as triggering moieties in this work to design redox-responsive prodrug monomers and polyprodrug amphiphiles based on indomethacin (IND) drug. Note that IND is a widely prescribed NSAID in the clinic. Starting from three types of redox-reactive IND prodrug monomers, redox-responsive polyprodrug amphiphiles were synthesized through reversible addition-fragmentation chain transfer (RAFT) polymerizations of prodrug monomers using poly(ethylene oxide) (PEO)-based macroRAFT agent. The resultant polyprodrug amphiphiles with high IND loading contents (>33 wt%) could self-assemble into polymersomes with PEO shielding coronas and redox-responsive bilayer membranes composed of IND prodrugs. Upon incubation with GSH or H 2 O 2 , controlled release of intact IND in the active form from polyprodrug polymersomes was actuated by GSH-mediated disulfide cleavage reaction and H 2 O 2 -mediated oxidation of phenylboronic ester moieties, respectively, followed by self-immolative degradation events. Furthermore, in vitro studies at the cellular level revealed that redox-responsive polymersomes could efficiently relieve inflammatory responses induced by lipopolysaccharide (LPS) in RAW264.7 macrophage cells. Copyright © 2018. Published by Elsevier Ltd.

Isocitrate dehydrogenase 1 mutant R132H sensitizes glioma cells to BCNU-induced oxidative stress and cell death.

PubMed

Mohrenz, Isabelle Vanessa; Antonietti, Patrick; Pusch, Stefan; Capper, David; Balss, Jörg; Voigt, Sophia; Weissert, Susanne; Mukrowsky, Alicia; Frank, Jan; Senft, Christian; Seifert, Volker; von Deimling, Andreas; Kögel, Donat

2013-11-01

Isocitrate dehydrogenase 1 (IDH1) decarboxylates isocitrate to α-ketoglutarate (α-KG) leading to generation of NADPH, which is required to regenerate reduced glutathione (GSH), the major cellular ROS scavenger. Mutation of R132 of IDH1 abrogates generation of α-KG and leads to conversion of α-KG to 2-hydroxyglutarate. We hypothesized that glioma cells expressing mutant IDH1 have a diminished antioxidative capacity and therefore may encounter an ensuing loss of cytoprotection under conditions of oxidative stress. Our study was performed with LN229 cells stably overexpressing IDH1 R132H and wild type IDH1 or with a lentiviral IDH1 knockdown. Quantification of GSH under basal conditions and following treatment with the glutathione reductase inhibitor BCNU revealed significantly lower GSH levels in IDH1 R132H expressing cells and IDH1 KD cells compared to their respective controls. FACS analysis of cell death and ROS production also demonstrated an increased sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to BCNU, but not to temozolomide. The sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to ROS induction and cell death was further enhanced with the transaminase inhibitor aminooxyacetic acid and under glutamine free conditions, indicating that these cells were more addicted to glutaminolysis. Increased sensitivity to BCNU-induced ROS production and cell death was confirmed in HEK293 cells inducibly expressing the IDH1 mutants R132H, R132C and R132L. Based on these findings we propose that in addition to its established pro-tumorigenic effects, mutant IDH1 may also limit the resistance of gliomas to specific death stimuli, therefore opening new perspectives for therapy.

A Neuroprotective Sericin Hydrogel As an Effective Neuronal Cell Carrier for the Repair of Ischemic Stroke.

PubMed

Wang, Zheng; Wang, Jian; Jin, Yang; Luo, Zhen; Yang, Wen; Xie, Hongjian; Huang, Kai; Wang, Lin

2015-11-11

Ischemic stroke causes extensive cellular loss that impairs brain functions, resulting in severe disabilities. No effective treatments are currently available for brain tissue regeneration. The need to develop effective therapeutic approaches for treating stroke is compelling. A tissue engineering approach employing a hydrogel carrying both cells and neurotrophic cytokines to damaged regions is an encouraging alternative for neuronal repair. However, this approach is often challenged by low in vivo cell survival rate, and low encapsulation efficiency and loss of cytokines. To address these limitations, we propose to develop a biomaterial that can form a matrix capable of improving in vivo survival of transplanted cells and reducing in vivo loss of cytokines. Here, we report that using sericin, a natural protein from silk, we have fabricated a genipin-cross-linked sericin hydrogel (GSH) with porous structure and mild swelling ratio. The GSH supports the effective attachment and growth of neurons in vitro. Strikingly, our data reveal that sericin protein is intrinsically neurotrophic and neuroprotective, promoting axon extension and branching as well as preventing primary neurons from hypoxia-induced cell death. Notably, these functions are inherited by the GSH's degradation products, which might spare a need of incorporating costly cytokines. We further demonstrate that this neurotrophic effect is dependent on the Lkb1-Nuak1 pathway, while the neuroprotective effect is realized through regulating the Bcl-2/Bax protein ratio. Importantly, when transplanted in vivo, the GSH gives a high cell survival rate and allows the cells to continuously proliferate. Together, this work unmasks the neurotrophic and neuroprotective functions for sericin and provides strong evidence justifying the GSH's suitability as a potential neuronal cell delivery vehicle for ischemic stroke repair.

Synthesis and biological evaluation of 2-substituted-5-(4-nitrophenylsulfonamido)benzoxazoles as human GST P1-1 inhibitors, and description of the binding site features.

PubMed

Ertan-Bolelli, Tuğba; Musdal, Yaman; Bolelli, Kayhan; Yilmaz, Serap; Aksoy, Yasemin; Yildiz, Ilkay; Aki-Yalcin, Esin; Yalcin, Ismail

2014-05-01

Glutathione-S-transferases (GSTs) are enzymes involved in cellular detoxification by catalyzing the nucleophilic attack of glutathione (GSH) on the electrophilic center of numerous of toxic compounds and xenobiotics, including chemotherapeutic drugs. Human GST P1-1, which is known as the most prevalent isoform of the mammalian cytosolic GSTs, is overexpressed in many cancers and contributes to multidrug resistance by directly conjugating to chemotherapeutics. It is suggested that this resistance is related to the high expression of GST P1-1 in cancers, thereby contributing to resistance to chemotherapy. In addition, GSTs exhibit sulfonamidase activity, thereby catalyzing the GSH-mediated hydrolysis of sulfonamide bonds. Such reactions are of interest as potential tumor-directed prodrug activation strategies. Herein we report the design and synthesis of some novel sulfonamide-containing benzoxazoles, which are able to inhibit human GST P1-1. Among the tested compounds, 2-(4-chlorobenzyl)-5-(4-nitrophenylsulfonamido)benzoxazole (5 f) was found as the most active hGST P1-1 inhibitor, with an IC50 value of 10.2 μM, showing potency similar to that of the reference drug ethacrynic acid. Molecular docking studies performed with CDocker revealed that the newly synthesized 2-substituted-5-(4-nitrophenylsulfonamido)benzoxazoles act as catalytic inhibitors of hGST P1-1 by binding to the H-site and generating conjugates with GSH to form S-(4-nitrophenyl)GSH (GS-BN complex) via nucleophilic aromatic substitution reaction. The 4-nitrobenzenesulfonamido moiety at position 5 of the benzoxazole ring is essential for binding to the H-site and for the formation of the GST-mediated GSH conjugate. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Leptin Level and Oxidative Stress Contribute to Obesity-Induced Low Testosterone in Murine Testicular Tissue

PubMed Central

Zhao, Jian; Zhai, Lingling; Liu, Zheng; Wu, Shuang; Xu, Liping

2014-01-01

Objective. This study evaluated the effects of obesity on the function of reproductive organs in male mice and the possible mechanism of male secondary hypogonadism (SH) in obesity. Methods. Ninety-six mice were randomly assigned to three groups: the control group, diet-induced obesity group, and diet-induced obesity resistant group for 8 weeks and 19 weeks. The effects of short- and long-term high-fat diet on the reproductive organs were determined by measuring sperm count and motility, relative testis weight, testosterone level, pathological changes and apoptosis of Leydig cells. Oxidative stress was evaluated by determining malondialdehyde, H2O2, NO levels, and GSH in testis tissues. CAT, SOD, GSH-Px and Nrf2 mRNA were measured by real-time PCR. Results. Short- and long-term high-fat diet decreased sperm count and motility, relative testis weight, testosterone level; decreased CAT, SOD, GSH-Px and Nrf2 mRNA expression; increased MDA, H2O2, NO and leptin levels; inhibited the activity of CAT and GSH-Px enzymes. Pathological injury and apoptosis of Leydig cells were found in testis tissue. Conclusions. Pathological damage of Leydig cells, oxidative stress in testis tissue, and high level of leptin may provide some evidence to clarify the mechanisms of male SH in obesity. PMID:24829619

New Approach in Translational Medicine: Effects of Electrolyzed Reduced Water (ERW) on NF-κB/iNOS Pathway in U937 Cell Line under Altered Redox State

PubMed Central

Franceschelli, Sara; Gatta, Daniela Maria Pia; Pesce, Mirko; Ferrone, Alessio; Patruno, Antonia; de Lutiis, Maria Anna; Grilli, Alfredo; Felaco, Mario; Croce, Fausto; Speranza, Lorenza

2016-01-01

It is known that increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) can exert harmful effects, altering the cellular redox state. Electrolyzed Reduced Water (ERW) produced near the cathode during water electrolysis exhibits high pH, high concentration of dissolved hydrogen and an extremely negative redox potential. Several findings indicate that ERW had the ability of a scavenger free radical, which results from hydrogen molecules with a high reducing ability and may participate in the redox regulation of cellular function. We investigated the effect of ERW on H2O2-induced U937 damage by evaluating the modulation of redox cellular state. Western blotting and spectrophotometrical analysis showed that ERW inhibited oxidative stress by restoring the antioxidant capacity of superoxide dismutase, catalase and glutathione peroxidase. Consequently, ERW restores the ability of the glutathione reductase to supply the cell of an important endogenous antioxidant, such as GSH, reversing the inhibitory effect of H2O2 on redox balance of U937 cells. Therefore, this means a reduction of cytotoxicity induced by peroxynitrite via a downregulation of the NF-κB/iNOS pathway and could be used as an antioxidant for preventive and therapeutic application. In conclusion, ERW can protect the cellular redox balance, reducing the risk of several diseases with altered cellular homeostasis such as inflammation. PMID:27598129

UV-B radiation-induced oxidative stress and p38 signaling pathway involvement in the benthic copepod Tigriopus japonicus.

PubMed

Kim, Bo-Mi; Rhee, Jae-Sung; Lee, Kyun-Woo; Kim, Min-Jung; Shin, Kyung-Hoon; Lee, Su-Jae; Lee, Young-Mi; Lee, Jae-Seong

2015-01-01

Ultraviolet B (UV-B) radiation presents an environmental hazard to aquatic organisms. To understand the molecular responses of the intertidal copepod Tigriopus japonicus to UV-B radiation, we measured the acute toxicity response to 96 h of UV-B radiation, and we also assessed the intracellular reactive oxygen species (ROS) levels, glutathione (GSH) content, and antioxidant enzyme (GST, GR, GPx, and SOD) activities after 24 h of exposure to UV-B with LD50 and half LD50 values. Also, expression patterns of p53 and hsp gene families with phosphorylation of p38 MAPK were investigated in UV-B-exposed copepods. We found that the ROS level, GSH content, and antioxidant enzyme activity levels were increased with the transcriptional upregulation of antioxidant-related genes, indicating that UV-B induces oxidative stress by generating ROS and stimulating antioxidant enzymatic activity as a defense mechanism. Additionally, we found that p53 expression was significantly increased after UV-B irradiation due to increases in the phosphorylation of the stress-responsive p38 MAPK, indicating that UV-B may be responsible for inducing DNA damage in T. japonicus. Of the hsp family genes, transcriptional levels of hsp20, hsp20.7, hsp70, and hsp90 were elevated in response to a low dose of UV-B radiation (9 kJ m(-2)), suggesting that these hsp genes may be involved in cellular protection against UV-B radiation. In this paper, we performed a pathway-oriented mechanistic analysis in response to UV-B radiation, and this analysis provides a better understanding of the effects of UV-B in the intertidal benthic copepod T. japonicus. Copyright © 2014 Elsevier Inc. All rights reserved.

Mangiferin, a Dietary Xanthone Protects Against Mercury-Induced Toxicity in HepG2 Cells

PubMed Central

Agarwala, Sobhika; Rao, B. Nageshwar; Mudholkar, Kaivalya; Bhuwania, Ridhirama; Rao, B. S. Satish

2012-01-01

Mercury is one of the noxious heavy metal environmental toxicants and is a cause of concern for human exposure. Mangiferin (MGN), a glucosylxanthone found in Mangifera indica, reported to have a wide range of pharmacological properties. The objective of this study was to evaluate the cytoprotective potential of MGN, against mercury chloride (HgCl2) induced toxicity in HepG2 cell line. The cytoprotective effect of MGN on HgCl2 induced toxicity was assessed by colony formation assay, while antiapoptotic effect by fluorescence microscopy, flow cytometric DNA analysis, and DNA fragmentation pattern assays. Further, the cytoprotective effect of MGN against HgCl2 toxicity was assessed by using biochemical parameters like reduced glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) by spectrophotometrically, mitochondrial membrane potential by flowcytometry and the changes in reactive oxygen species levels by DCFH-DA spectrofluoremetric analysis. A significant increase in the surviving fraction was observed with 50 µM of MGN administered two hours prior to various concentrations of HgCl2. Further, pretreatment of MGN significantly decreased the percentage of HgCl2 induced apoptotic cells. Similarly, the levels of ROS generated by the HgCl2 treatment were inhibited significantly (P < 0.01) by MGN. MGN also significantly (P < 0.01) inhibited the HgCl2 induced decrease in GSH, GST, SOD, and CAT levels at all the post incubation intervals. Our study demonstrated the cytoprotective potential of MGN, which may be attributed to quenching of the ROS generated in the cells due to oxidative stress induced by HgCl2, restoration of mitochondrial membrane potential and normalization of cellular antioxidant levels. PMID:20629087

Lead tolerance in plants: strategies for phytoremediation.

PubMed

Gupta, D K; Huang, H G; Corpas, F J

2013-04-01

Lead (Pb) is naturally occurring element whose distribution in the environment occurs because of its extensive use in paints, petrol, explosives, sludge, and industrial wastes. In plants, Pb uptake and translocation occurs, causing toxic effects resulting in decrease of biomass production. Commonly plants may prevent the toxic effect of heavy metals by induction of various celular mechanisms such as adsorption to the cell wall, compartmentation in vacuoles, enhancement of the active efflux, or induction of higher levels of metal chelates like a protein complex (metallothioneins and phytochelatins), organic (citrates), and inorganic (sulphides) complexes. Phyotochelains (PC) are synthesized from glutathione (GSH) and such synthesis is due to transpeptidation of γ-glutamyl cysteinyl dipeptides from GSH by the action of a constitutively present enzyme, PC synthase. Phytochelatin binds to Pb ions leading to sequestration of Pb ions in plants and thus serves as an important component of the detoxification mechanism in plants. At cellular level, Pb induces accumulation of reactive oxygen species (ROS), as a result of imbalanced ROS production and ROS scavenging processes by imposing oxidative stress. ROS include superoxide radical (O2(.-)), hydrogen peroxide (H2O2) and hydroxyl radical ((·)OH), which are necessary for the correct functioning of plants; however, in excess they caused damage to biomolecules, such as membrane lipids, proteins, and nucleic acids among others. To limit the detrimental impact of Pb, efficient strategies like phytoremediation are required. In this review, it will discuss recent advancement and potential application of plants for lead removal from the environment.

Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress

PubMed Central

Cavalheiro, Renan P.; Machado, Daisy; Cruz, Bread L. G.; Paredes-Gamero, Edgar J.; Gomes-Marcondes, Maria C. C.; Zambuzzi, Willian F.; Vasques, Luciana; Nader, Helena B.; Souza, Ana Carolina S.; Justo, Giselle Z.

2015-01-01

Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death. PMID:25781955

Tributyltin bioaccumulation and toxic effects in freshwater gastropods Pomacea canaliculata after a chronic exposure: field and laboratory studies.

PubMed

Martínez, María L; Piol, María N; Sbarbati Nudelman, Norma; Verrengia Guerrero, Noemí R

2017-07-01

Freshwater samples and gastropod mollusks (Pomacea canaliculata) were collected at 5 sampling stations located along the lower Río de la Plata basin, Argentina, to assess the extent of tributyltin (TBT) contamination. Determined data revealed the presence of TBT and some of its breakdown products (dibutyltin: DBT, and monobutyltin: MBT) in all freshwater samples and also in soft tissues of P. canaliculata gastropods. Chronic bioassays (6 months) were performed using female gastropods that had been reared under laboratory conditions and exposed to a similar TBT concentration than the value determined in freshwater samples (1 µg L -1 ). The aims of this study were to evaluate the extent of TBT accumulation, the tissue distribution, and the effects on selected biomarkers (activity of superoxide dismutasa: SOD, activity of catalase: CAT, levels of total glutathione: t-GSH, lipid peroxidation, and activity of acetylcholinesterase: AChE). Gonads presented the highest accumulation, followed by the cephalopedal region, albumin gland, and finally hepatopancreas. Both metabolites, DBT and MBT, were also found. All exposed female animals presented development of a penis reflecting the potential of TBT as an endocrine disrupting chemical for this gastropod species. Results on the selected biomarkers confirmed additional adverse effects induced by TBT. An increase in CAT activity and changes in t-GSH levels are indicative of alterations on the cellular redox status. The inhibition of AChE could reflect signs of neurotoxicity. Altogether, these results reveal a negative impact on the health of this gastropod population.

Distribution and abundance of organic thiols

NASA Technical Reports Server (NTRS)

Fahey, R.

1985-01-01

The role of glutathione (GSH) in protecting against the toxicity of oxygen and oxygen by products is well established for all eukaryotes studied except Entamoeba histolytica which lacks mitochrondria, chloroplasts, and microtubules. The GSH is not universal among prokaryotes. Entamoeba histolytica does not produce GSH or key enzymes of GSH metabolism. A general method of thiol analysis based upon fluorescent labeling with monobromobimane and HPLC separation of the resulting thiol derivatives was developed to determine the occurrence of GSH and other low molecular weight thiols in bacteria. Glutathione is the major thiol in cyanobacteria and in most bacteria closely related to the purple photosynthetic bacteria, but GSH was not found in archaebacteria, green bacteria, or GRAM positive bacteria. It suggested that glutathione metabolism was incorporated into eukaryotes at the time that mitochondria and chloroplasts were acquired by endosymbiosis. In Gram positive aerobes, coenzyme A occurs at millimolar levels and CoA disulfide reductases are identified. The CoA, rather than glutathione, may function in the oxygen detoxification processes of these organisms.

GSM base station electromagnetic radiation and oxidative stress in rats.

PubMed

Yurekli, Ali Ihsan; Ozkan, Mehmed; Kalkan, Tunaya; Saybasili, Hale; Tuncel, Handan; Atukeren, Pinar; Gumustas, Koray; Seker, Selim

2006-01-01

The ever increasing use of cellular phones and the increasing number of associated base stations are becoming a widespread source of nonionizing electromagnetic radiation. Some biological effects are likely to occur even at low-level EM fields. In this study, a gigahertz transverse electromagnetic (GTEM) cell was used as an exposure environment for plane wave conditions of far-field free space EM field propagation at the GSM base transceiver station (BTS) frequency of 945 MHz, and effects on oxidative stress in rats were investigated. When EM fields at a power density of 3.67 W/m2 (specific absorption rate = 11.3 mW/kg), which is well below current exposure limits, were applied, MDA (malondialdehyde) level was found to increase and GSH (reduced glutathione) concentration was found to decrease significantly (p < 0.0001). Additionally, there was a less significant (p = 0.0190) increase in SOD (superoxide dismutase) activity under EM exposure.

Intravenous Administration of Stable-Labeled N-Acetylcysteine Demonstrates an Indirect Mechanism for Boosting Glutathione and Improving Redox Status.

PubMed

Zhou, Jie; Coles, Lisa D; Kartha, Reena V; Nash, Nardina; Mishra, Usha; Lund, Troy C; Cloyd, James C

2015-08-01

There is an increasing interest in using N-acetylcysteine (NAC) as a treatment for neurodegenerative disorders to increase glutathione (GSH) levels and its redox status. The purpose of this study was to characterize the biosynthesis of NAC to GSH using a novel stable isotope-labeled technique, and investigate the pharmacodynamics of NAC in vivo. Female wild-type mice were given a single intravenous bolus dose of 150 mg kg(-1) stable-labeled NAC. Plasma, red blood cells (RBC), and brain tissues were collected at predesignated time points. Stable-labeled NAC and its metabolite GSH (both labeled and unlabeled forms) were quantified in blood and brain samples. Molar ratios of the reduced and oxidized forms of GSH (GSH divided by glutathione disulfide, redox ratio) were also determined. The elimination phase half-life of NAC was approximately 34 min. Both labeled and unlabeled GSH in RBC were found to increase; however, the area under the curve above baseline (AUCb0-280 ) of labeled GSH was only 1% of the unlabeled form. These data indicate that NAC is not a direct precursor of GSH. In addition, NAC has prolonged effects in brain even when the drug has been eliminated from systemic circulation. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Short-term exercise worsens cardiac oxidative stress and fibrosis in 8-month-old db/db mice by depleting cardiac glutathione.

PubMed

Laher, Ismail; Beam, Julianne; Botta, Amy; Barendregt, Rebekah; Sulistyoningrum, Dian; Devlin, Angela; Rheault, Mark; Ghosh, Sanjoy

2013-01-01

Moderate exercise improves cardiac antioxidant status in young humans and animals with Type-2 diabetes (T2D). Given that both diabetes and advancing age synergistically decrease antioxidant expression in most tissues, it is unclear whether exercise can upregulate cardiac antioxidants in chronic animal models of T2D. To this end, 8-month-old T2D and normoglycemic mice were exercised for 3 weeks, and cardiac redox status was evaluated. As expected, moderate exercise increased cardiac antioxidants and attenuated oxidative damage in normoglycemic mice. In contrast, similar exercise protocol in 8-month-old db/db mice worsened cardiac oxidative damage, which was associated with a specific dysregulation of glutathione (GSH) homeostasis. Expression of enzymes for GSH biosynthesis [γ-glutamylcysteine synthase, glutathione reductase] as well as for GSH-mediated detoxification (glutathione peroxidase, glutathione-S-transferase) was lower, while toxic metabolites dependent on GSH for clearance (4-hydroxynonenal) were increased in exercised diabetic mice hearts. To validate GSH loss as an important factor for such aggravated damage, daily administration of GSH restored cardiac GSH levels in exercised diabetic mice. Such supplementation attenuated both oxidative damage and fibrotic changes in the myocardium. Expression of transforming growth factor beta (TGF-β) and its regulated genes which are responsible for such profibrotic changes were also attenuated with GSH supplementation. These novel findings in a long-term T2D animal model demonstrate that short-term exercise by itself can deplete cardiac GSH and aggravate cardiac oxidative stress. As GSH administration conferred protection in 8-month-old diabetic mice undergoing exercise, supplementation with GSH-enhancing agents may be beneficial in elderly diabetic patients undergoing exercise.

Preclinical High-Dose Acetaminophen With N-Acetylcysteine Rescue Enhances the Efficacy of Cisplatin Chemotherapy in Atypical Teratoid Rhabdoid Tumors

PubMed Central

Neuwelt, Alexander J.; Nguyen, Tam; Wu, Y. Jeffrey; Donson, Andrew M.; Vibhakar, Rajeev; Venkatamaran, Sujatha; Amani, Vladimir; Neuwelt, Edward A.; Rapkin, Louis B.; Foreman, Nicholas K.

2016-01-01

Background Atypical teratoid rhabdoid tumors (AT-RT) are pediatric tumors of the central nervous system with limited treatment options and poor survival rate. We investigated whether enhancing chemotherapy toxicity by depleting intracellular glutathione (GSH; a key molecule in cisplatin resistance) with high dose acetaminophen (AAP), may improve therapeutic efficacy in AT-RT in vitro. Procedure BT16 (cisplatin-resistant) and BT12 (cisplatin-sensitive) AT-RT cell lines were treated with combinations of AAP, cisplatin, and the anti-oxidant N-acetylcysteine (NAC). Cell viability, GSH and peroxide concentrations, mitochondrial damage, and apoptosis were evaluated in vitro. Results AAP enhanced cisplatin cytotoxicity in cisplatin-resistant BT16 cells but not cisplatin-sensitive BT12 cells. Baseline GSH levels were elevated in BT16 cells compared to BT12 cells, and AAP decreased GSH to a greater magnitude in BT16 cells than BT12 cells. Unlike BT12 cells, BT16 cells did not have elevated peroxide levels upon treatment with cisplatin alone, but did have elevated levels when treated with AAP + cisplatin. Both cell lines had markedly increased mitochondrial injury when treated with AAP + cisplatin relative to either drug treatment alone. The enhanced toxic effects were partially reversed with concurrent administration of NAC. Conclusions Our results suggest that AAP could be used as a chemo-enhancement agent to potentiate cisplatin chemotherapeutic efficacy particularly in cisplatin-resistant AT-RT tumors with high GSH levels in clinical settings. PMID:23956023

Over-expression of bacterial gamma-glutamylcysteine synthetase (GSH1) in plastids affects photosynthesis, growth and sulphur metabolism in poplar (Populus tremula x Populus alba) dependent on the resulting gamma-glutamylcysteine and glutathione levels.

PubMed

Herschbach, Cornelia; Rizzini, Luca; Mult, Susanne; Hartmann, Tanja; Busch, Florian; Peuke, Andreas D; Kopriva, Stanislav; Ensminger, Ingo

2010-07-01

We compared three transgenic poplar lines over-expressing the bacterial gamma-glutamylcysteine synthetase (GSH1) targeted to plastids. Lines Lggs6 and Lggs12 have two copies, while line Lggs20 has three copies of the transgene. The three lines differ in their expression levels of the transgene and in the accumulation of gamma-glutamylcysteine (gamma-EC) and glutathione (GSH) in leaves, roots and phloem exudates. The lowest transgene expression level was observed in line Lggs6 which showed an increased growth, an enhanced rate of photosynthesis and a decreased excitation pressure (1-qP). The latter typically represents a lower reduction state of the plastoquinone pool, and thereby facilitates electron flow along the electron transport chain. Line Lggs12 showed the highest transgene expression level, highest gamma-EC accumulation in leaves and highest GSH enrichment in phloem exudates and roots. This line also exhibited a reduced growth, and after a prolonged growth of 4.5 months, symptoms of leaf injury. Decreased maximum quantum yield (F(v)/F(m)) indicated down-regulation of photosystem II reaction centre (PSII RC), which correlates with decreased PSII RC protein D1 (PsbA) and diminished light-harvesting complex (Lhcb1). Potential effects of changes in chloroplastic and cytosolic GSH contents on photosynthesis, growth and the whole-plant sulphur nutrition are discussed for each line.

Acute acetaminophen toxicity in transgenic mice with elevated hepatic glutathione.

PubMed

Rzucidlo, S J; Bounous, D I; Jones, D P; Brackett, B G

2000-06-01

Previous studies demonstrated that elevation of hepatic glutathione (GSH) concentrations protect against acetaminophen (APAP) hepatotoxicity in mice. Employing transgenic mice overexpressing glutathione synthetase, this study was conducted to determine if sustained elevation of hepatic GSH concentrations could ameliorate or prevent APAP toxicity. International Cancer Research transgenic mouse males and matched (ie same strain, sex, and age) control nontransgenic mice were pretreated ip with GSH synthetase substrate gamma-glutamylcysteinyl ethyl ester (gamma-GCE) or with saline. After a 16-h fast, mice received a single dose of 500 mg APAP/kg bw in saline ip and were sacrificed 4 h later. Other mice similarly pretreated were killed without APAP challenge. The elevated GSH concentrations in transgenic mice livers did not lessen APAP hepatotoxicity. Instead higher degrees of hepatotoxicity and nephrotoxicity were observed in transgenic mice than in controls as indicated by higher serum alanine aminotransferase activity and more severe histopathological lesions in transgenic mice livers and kidneys. Pretreatment with gamma-GCE did not affect either initial or post-APAP treatment tissue GSH concentrations or observed degrees of toxicity. Detection of a higher level of serum APAP in transgenic mice and the histopathological lesions found in transgenic mice kidneys together with no observable nephrotoxicity in control mice indicated early kidney damage in transgenic mice. Our findings suggest that high levels of GSH-APAP conjugates resulting from increased GSH concentrations in the livers of transgenic mice caused rapid kidney damage. Compromised excretory ability may have caused retention of APAP, which, in effect, elicited higher hepatotoxicity than that observed in nontransgenic mice.

A guanine-ethylthioethyl-glutathione adduct as a major DNA lesion in the skin and in organs of mice exposed to sulfur mustard.

PubMed

Batal, Mohamed; Rebelo-Moreira, Silvestre; Hamon, Nadège; Bayle, Pierre-Alain; Mouret, Stéphane; Cléry-Barraud, Cécile; Boudry, Isabelle; Douki, Thierry

2015-02-17

Sulfur mustard (SM) is an old chemical warfare but it remains a threat to both militaries and civilians. SM mainly targets skin, eyes and lungs and diffuses to internal organs. At the molecular level, SM is able to damage DNA through the formation of monoadducts and biadduct. Glutathione (GSH) is another critical target of SM in cells since it is part of the detoxification mechanism against alkylating agents. In the present work, we investigated whether SM could form covalent bonds simultaneously with a DNA base and the sulfhydryl group of GSH. The expected guanine adduct, S-[2-(N7-guanyl)-ethylthioethyl]-glutathione (N7Gua-ETE-GSH), was synthesized and detected in several tissues of SKH-1 mice exposed to 60mg/kg of SM in the dorsal-lumbar region. N7Gua-ETE-GSH was detected in all organs studied, except in the liver. The tissue exhibiting the highest levels of N7Gua-ETE-GSH was skin, followed by brain, lungs, kidneys and spleen. N7Gua-ETE-GSH was detected in skin, brain and lungs as long as two weeks after exposure. The persistence was less in other organs. The observation of the formation of N7Gua-ETE-GSH in vivo confirms the variety of damages induced by SM in DNA. It also provides another example of the formation of DNA adducts involving glutathione following in vivo exposure to bifunctional alkylating compounds. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Comparison of in vitro bioactivation of flutamide and its cyano analogue: evidence for reductive activation by human NADPH:cytochrome P450 reductase.

PubMed

Wen, Bo; Coe, Kevin J; Rademacher, Peter; Fitch, William L; Monshouwer, Mario; Nelson, Sidney D

2008-12-01

Flutamide (FLU), a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with idiosyncratic hepatotoxicity in patients. It is proposed that bioactivation of FLU and subsequent binding of reactive metabolite(s) to cellular proteins play a causative role. A toxicogenomic study comparing FLU and its nitro to cyano analogue (CYA) showed that the nitroaromatic group of FLU enhanced cytotoxicity to hepatocytes, indicating that reduction of the nitroaromatic group may represent a potential route of FLU-induced hepatotoxicity [Coe et al. (2007) Chem. Res. Toxicol. 20, 1277-1290]. In the current study, we compared in vitro bioactivation of FLU and CYA in human liver microsomes and cryopreserved human hepatocytes. A nitroreduction metabolite FLU-6 was formed in liver microsomal incubations of FLU under atmospheric oxygen levels and, to a greater extent, under anaerobic conditions. Seven glutathione (GSH) adducts of FLU, FLU-G1-7, were tentatively identified in human liver microsomal incubations using liquid chromatography-tandem mass spectrometry (LC/ MS/MS), while CYA formed only four corresponding GSH adducts, CYA-G1-4, under the same conditions. Of particular interest was the formation of FLU-G5-7 from FLU, where the nitroaromatic group of FLU was reduced to an amino group. A tentative pathway is that upon nitroreduction, the para-diamines undergo cytochrome P450 (P450)-catalyzed two-electron oxidations to form corresponding para-diimine intermediates that react with GSH to form GSH adducts FLU-G5-7, respectively. The identities of FLU-G5-7 were further confirmed by LC/MS/MS analyses of microsomal incubations of a synthesized standard FLU-6. In an attempt to identify enzymes involved in the nitroreduction of FLU, NADPH:cytochrome P450 reductase (CPR) was shown to reduce FLU to FLU-6 under both aerobic and anaerobic conditions. Furthermore, the formation of FLU-G5-7 was completely blocked by the addition of a reversible CPR inhibitor, alpha-lipoic acid, to the incubations of FLU under aerobic conditions. In summary, these results clearly demonstrate that nitroreduction of FLU by CPR contributes to bioactivation and potentially to hepatotoxicity of FLU.

Glutathione reductase mediates drug resistance in glioblastoma cells by regulating redox homeostasis.

PubMed

Zhu, Zhongling; Du, Shuangshuang; Du, Yibo; Ren, Jing; Ying, Guoguang; Yan, Zhao

2018-01-01

Glutathione (GSH) and GSH-related enzymes constitute the most important defense system that protects cells from free radical, radiotherapy, and chemotherapy attacks. In this study, we aim to explore the potential role and regulatory mechanism of the GSH redox cycle in drug resistance in glioblastoma multiforme (GBM) cells. We found that temozolomide (TMZ)-resistant glioma cells displayed lower levels of endogenous reactive oxygen species and higher levels of total antioxidant capacity and GSH than sensitive cells. Moreover, the expression of glutathione reductase (GSR), the key enzyme of the GSH redox cycle, was higher in TMZ-resistant cells than in sensitive cells. Furthermore, silencing GSR in drug-resistant cells improved the sensitivity of cells to TMZ or cisplatin. Conversely, the over-expression of GSR in sensitive cells resulted in resistance to chemotherapy. In addition, the GSR enzyme partially prevented the oxidative stress caused by pro-oxidant L-buthionine -sulfoximine. The modulation of redox state by GSH or L-buthionine -sulfoximine regulated GSR-mediated drug resistance, suggesting that the action of GSR in drug resistance is associated with the modulation of redox homeostasis. Intriguingly, a trend toward shorter progress-free survival was observed among GBM patients with high GSR expression. These results indicated that GSR is involved in mediating drug resistance and is a potential target for improving GBM treatment. © 2017 International Society for Neurochemistry.

Effects of dietary lipid, vitamins and minerals on total amounts and redox status of glutathione and ubiquinone in tissues of Atlantic salmon (Salmo salar): a multivariate approach.

PubMed

Hamre, Kristin; Torstensen, Bente E; Maage, Amund; Waagbø, Rune; Berge, Rolf K; Albrektsen, Sissel

2010-10-01

The hypothesis of the present study was that Atlantic salmon (Salmo salar) would respond to large variations in supplementation of dietary pro- and antioxidants, and marine lipid, with adjustment of the endogenously synthesised antioxidants, glutathione (GSH) and ubiquinone (UQ). An experiment with 2(7-3) reduced factorial design (the number of cases reduced systematically from 2(7) (full design) to 2(4) (reduced design)) was conducted, where vitamins, minerals and lipid were supplemented in the diet at high and low levels. For the vitamins and minerals the high levels were chosen to be just below anticipated toxic levels and the low levels were just above the requirement (vitamin C, 30 and 1000 mg/kg; vitamin E, 70 and 430 mg/kg; Fe, 70 and 1200 mg/kg; Cu, 8 and 110 mg/kg; Mn, 12 and 200 mg/kg). For astaxanthin, the dietary levels were 10 and 50 mg/kg and for lipid, 150 and 330 g/kg. The experiment was started with post-smolts (148 (sd 17 g)) and lasted for 5 months. The only effect on GSH was a minor increase ( < 10 %) in total concentration in the liver in response to high dietary lipid. GSH redox state was not affected. UQ responded to dietary lipid, astaxanthin and vitamin E, both with regard to total concentration and redox state. Except for an effect of Fe on plasma GSH, the trace elements and vitamin C had no effect on tissue levels and oxidation state of GSH and UQ. This shows that the endogenous redox state is quite robust with regard to variation of dietary pro- and antioxidants in Atlantic salmon.

Effects of third generation mobile phone-emitted electromagnetic radiation on oxidative stress parameters in eye tissue and blood of rats.

PubMed

Demirel, Soner; Doganay, Selim; Turkoz, Yusuf; Dogan, Zümrüt; Turan, Bahadir; Firat, Penpe Gul Bozgul

2012-06-01

To investigate the effects of electromagnetic radiation (EMR) emitted by a third generation (3G) mobile phone on the antioxidant and oxidative stress parameters in eye tissue and blood of rats. Eighteen Wistar albino rats were randomly assigned into two groups: Group I (n = 9) received a standardized a daily dose of 3G mobile phone EMR for 20 days, and Group II served as the control group (n = 9), receiving no exposure to EMR. Glutathione peroxidase (GSH-Px) and catalase (CAT) levels were measured in eye tissues; in addition, malondialdehyde (MDA) and reduced GSH levels were measured in blood. There was no significant difference between groups in GSH-Px (p = 0.99) and CAT (p = 0.18) activity in eye tissue. There was no significant difference between groups in MDA (p = 0.69) and GSH levels (p = 0.83) in blood. The results of this study suggest that under a short period of exposure, 3G mobile phone radiation does not lead to harmful effects on eye tissue and blood in rats.

A dual-channel fluorescent chemosensor for discriminative detection of glutathione based on functionalized carbon quantum dots.

PubMed

Huang, Yuanyuan; Zhou, Jin; Feng, Hui; Zheng, Jieyu; Ma, Hui-Min; Liu, Weidong; Tang, Cong; Ao, Hang; Zhao, Meizhi; Qian, Zhaosheng

2016-12-15

A convenient, fluorescent dual-channel chemosensor on the basis of bis(3-pyridylmethyl)amine-functionalized carbon quantum dots (BPMA-CQDs) nanoprobe was constructed, and it can discriminatively detect glutathione from its analogues cysteine and homocysteine based on two distinctive strategies. Two distinct fluorescence responses of BPMA-CQDs probe to Cu(II) and Ag(I) were identified and further employed to achieve selective detection of Cu(II) and Ag(I) respectively. Based on the BPMA-CQDs/Cu(II) conjugate, discriminative detection of GSH was achieved in terms of correlation between the amounts of GSH and fluorescence recovery. The addition of GSH into BPMA-CQDs/Cu(II) system induces the reduction of Cu(II) to Cu(I), which could efficiently block PET process resulting in the following fluorescence recovery. Based on the BPMA-CQDs/Ag(I) conjugate, GSH assay could also be established on the basis of fluorescence response to GSH. The introduction of GSH into the preceding system triggers the competitive coordination to Ag(I) between BPMA and GSH, and silver ions are finally taken away by GSH from the probe, where the fluorescence is restored to its original weak state. Both of the detection strategies can achieve discriminative detection of GSH from Cys and Hcy. The assays showed good stability and repeatability, and covered a broad linear range of up to 13.3μM with a lowest detection limit of 42.0nM. Moreover, both of them were utilized to monitor GSH level in live cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Influence of sildenafil and donepezil administration on the serum redox balance in experimentally induced lower limb critical ischemia.

PubMed

Constantinescu, Mihaela Ioana; Constantinescu, Dan Petru; Andercou, Aurel; Mironiuc, Ion Aurel

2013-01-01

Chronic lower limb ischemia (CLLI) leads to endothelial cell dysfunctions and endothelial lesions. The use of substances that release nitric oxide and activate endothelial nitric oxide synthase has proved to be useful in increasing angiogenesis and arteriogenesis under critical ischemia conditions. To investigate the therapeutic effect of Sildenafil and Donepezil with a vasodilating action in experimentally induced CLLI and on serum redox homeostasis. The research was performed in 3 groups of rats (n=10 animals/group) with experimentally induced CLLI: group I - control group; group II - animals treated postoperatively with a therapeutic dose of sildenafil, and group III - animals treated postoperatively with a therapeutic dose of donepezil. Oxidative stress (OS) indicators (malondialdehyde - MDA, protein carbonyls - PC), antioxidant (AO) defense indicators (reduced glutathione - GSH and oxidized glutathione - GSSH), and ceruloplasmin (CP) were determined on days 7, 14, 21 and 30. Statistical processing was performed using the Excel application (Microsoft Office 2007), with the StatsDirect v.2.7.2 software. Changes in OS were evidenced in all groups on account of a decrease in MDA and PC. The greatest OS decrease in all groups was on day 30. AO defence changes were represented by decreased levels of GSH and GSSG in all groups, at the studied moments. Intracellular AO defense in the cytosol, nucleus and mitochondria was similar in all groups, (decreased GSH, GSSG and GSH/GSSG ratio). We found increased extracellular levels of GSH, GSSG, and CP and increased extracellular GSH/GSSG ratio at level compared to values on day 7. 1) The administration of sildenafil (group II) and donepezil (group III) has favorable effects on reducing OS in experimentally induced CLLI. 2) Sildenafil and Donepezil administration stimulates extracellular AO defense on account of CP. 3) Sildenafil and Donepezil administration influences intracellular redox homeostasis on account of the GSH/GSSG couple, the major redox buffer in the body.

Redox Signaling Mediated by Thioredoxin and Glutathione Systems in the Central Nervous System.

PubMed

Ren, Xiaoyuan; Zou, Lili; Zhang, Xu; Branco, Vasco; Wang, Jun; Carvalho, Cristina; Holmgren, Arne; Lu, Jun

2017-11-01

The thioredoxin (Trx) and glutathione (GSH) systems play important roles in maintaining the redox balance in the brain, a tissue that is prone to oxidative stress due to its high-energy demand. These two disulfide reductase systems are active in various areas of the brain and are considered to be critical antioxidant systems in the central nervous system (CNS). Various neuronal disorders have been characterized to have imbalanced redox homeostasis. Recent Advances: In addition to their detrimental effects, recent studies have highlighted that reactive oxygen species/reactive nitrogen species (ROS/RNS) act as critical signaling molecules by modifying thiols in proteins. The Trx and GSH systems, which reversibly regulate thiol modifications, regulate redox signaling involved in various biological events in the CNS. In this review, we focus on the following: (i) how ROS/RNS are produced and mediate signaling in CNS; (ii) how Trx and GSH systems regulate redox signaling by catalyzing reversible thiol modifications; (iii) how dysfunction of the Trx and GSH systems causes alterations of cellular redox signaling in human neuronal diseases; and (iv) the effects of certain small molecules that target thiol-based signaling pathways in the CNS. Further study on the roles of thiol-dependent redox systems in the CNS will improve our understanding of the pathogenesis of many human neuronal disorders and also help to develop novel protective and therapeutic strategies against neuronal diseases. Antioxid. Redox Signal. 27, 989-1010.

The pulmonary inflammatory response to multiwalled carbon nanotubes is influenced by gender and glutathione synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cartwright, Megan M.; Schmuck, Stefanie C.; Corredor, Charlie

Here, inhalation of multiwalled carbon nanotubes (MWCNTs) during their manufacture or incorporation into various commercial products may cause lung inflammation, fibrosis, and oxidative stress in exposed workers. Some workers may be more susceptible to these effects because of differences in their ability to synthesize the major antioxidant and immune system modulator glutathione (GSH). Accordingly, in this study we examined the influence of GSH synthesis and gender on MWCNT-induced lung inflammation in C57BL/6 mice. GSH synthesis was impaired through genetic manipulation of Gclm, the modifier subunit of glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis. Twenty-four hours after aspirating 25more » µg of MWCNTs, all male mice developed neutrophilia in their lungs, regardless of Gclm genotype. However, female mice with moderate ( Gclm heterozygous) and severe ( Gclm null) GSH deficiencies developed significantly less neutrophilia. We found no indications of MWCNT-induced oxidative stress as reflected in the GSH content of lung tissue and epithelial lining fluid, 3-nitrotyrosine formation, or altered mRNA or protein expression of several redox-responsive enzymes. Our results indicate that GSH-deficient female mice are rendered uniquely susceptible to an attenuated neutrophil response. If the same effects occur in humans, GSH-deficient women manufacturing MWCNTs may be at greater risk for impaired neutrophil-dependent clearance of MWCNTs from the lung. In contrast, men may have effective neutrophil-dependent clearance, but may be at risk for lung neutrophilia regardless of their GSH levels.« less

The pulmonary inflammatory response to multiwalled carbon nanotubes is influenced by gender and glutathione synthesis

DOE PAGES

Cartwright, Megan M.; Schmuck, Stefanie C.; Corredor, Charlie; ...

2016-08-21

Here, inhalation of multiwalled carbon nanotubes (MWCNTs) during their manufacture or incorporation into various commercial products may cause lung inflammation, fibrosis, and oxidative stress in exposed workers. Some workers may be more susceptible to these effects because of differences in their ability to synthesize the major antioxidant and immune system modulator glutathione (GSH). Accordingly, in this study we examined the influence of GSH synthesis and gender on MWCNT-induced lung inflammation in C57BL/6 mice. GSH synthesis was impaired through genetic manipulation of Gclm, the modifier subunit of glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis. Twenty-four hours after aspirating 25more » µg of MWCNTs, all male mice developed neutrophilia in their lungs, regardless of Gclm genotype. However, female mice with moderate ( Gclm heterozygous) and severe ( Gclm null) GSH deficiencies developed significantly less neutrophilia. We found no indications of MWCNT-induced oxidative stress as reflected in the GSH content of lung tissue and epithelial lining fluid, 3-nitrotyrosine formation, or altered mRNA or protein expression of several redox-responsive enzymes. Our results indicate that GSH-deficient female mice are rendered uniquely susceptible to an attenuated neutrophil response. If the same effects occur in humans, GSH-deficient women manufacturing MWCNTs may be at greater risk for impaired neutrophil-dependent clearance of MWCNTs from the lung. In contrast, men may have effective neutrophil-dependent clearance, but may be at risk for lung neutrophilia regardless of their GSH levels.« less

The pulmonary inflammatory response to multiwalled carbon nanotubes is influenced by gender and glutathione synthesis.

PubMed

Cartwright, Megan M; Schmuck, Stefanie C; Corredor, Charlie; Wang, Bingbing; Scoville, David K; Chisholm, Claire R; Wilkerson, Hui-Wen; Afsharinejad, Zahra; Bammler, Theodor K; Posner, Jonathan D; Shutthanandan, Vaithiyalingam; Baer, Donald R; Mitra, Somenath; Altemeier, William A; Kavanagh, Terrance J

2016-10-01

Inhalation of multiwalled carbon nanotubes (MWCNTs) during their manufacture or incorporation into various commercial products may cause lung inflammation, fibrosis, and oxidative stress in exposed workers. Some workers may be more susceptible to these effects because of differences in their ability to synthesize the major antioxidant and immune system modulator glutathione (GSH). Accordingly, in this study we examined the influence of GSH synthesis and gender on MWCNT-induced lung inflammation in C57BL/6 mice. GSH synthesis was impaired through genetic manipulation of Gclm, the modifier subunit of glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis. Twenty-four hours after aspirating 25µg of MWCNTs, all male mice developed neutrophilia in their lungs, regardless of Gclm genotype. However, female mice with moderate (Gclm heterozygous) and severe (Gclm null) GSH deficiencies developed significantly less neutrophilia. We found no indications of MWCNT-induced oxidative stress as reflected in the GSH content of lung tissue and epithelial lining fluid, 3-nitrotyrosine formation, or altered mRNA or protein expression of several redox-responsive enzymes. Our results indicate that GSH-deficient female mice are rendered uniquely susceptible to an attenuated neutrophil response. If the same effects occur in humans, GSH-deficient women manufacturing MWCNTs may be at greater risk for impaired neutrophil-dependent clearance of MWCNTs from the lung. In contrast, men may have effective neutrophil-dependent clearance, but may be at risk for lung neutrophilia regardless of their GSH levels. Copyright © 2016. Published by Elsevier B.V.

Selenium requirement of shrimp Penaeus chinensis

NASA Astrophysics Data System (ADS)

Tian, Yuchuan; Liu, Fayi

1993-09-01

Penaeus chinensis were reared in fibreglass tanks for the study of their selenium requirements. The shrimp were fed semipurified diets containing graded levels of selenium, and weight gains, activities of glutatione peroxidase (GSH-Px) and selenium contents in muscle and hepatopancreas were determined. Weight gain and GSH-Px activity were the highest when the shrimp were fed diet containing 20 mg/kg selenium. Good linear correlation was found between GSH-Px activities and selenium contents in the diets, and the number of healthy shrimp. The experiment showed that 20 mg/kg selenium in the diet is optimal for the shrimp and that GSH-Px activity can be an important biochemical index of the selenium nutrition status of the animal.

Modulation of Oxidative Stress by Gamma-Glutamylcysteine (GGC) and Conjugated Linoleic Acid (CLA) Isomer Mixture in Human Umbilical Vein Endothelial Cells

DTIC Science & Technology

2012-04-02

during cutaneous wound healing . Mediators Inflamm. 2010, 342328. Ringseis, R., Muller, A., Herter, C., Gahler, S., Steinhart, H., Eder, K., 2006. CLA...glutamylcysteine (GGC), a dipeptide and precursor of glutathione (GSH), and conjugated linoleic acid (CLA), a trans-fatty acid, exhibit antioxidant properties...synthesis in human endothelial cells. Changes in levels of 8-epi-PGF2a, thiobarbituric acid reac- tive substances (TBARS), GSH, total antioxidants , GSH

l-glutamine and l-alanine supplementation increase glutamine-glutathione axis and muscle HSP-27 in rats trained using a progressive high-intensity resistance exercise.

PubMed

Leite, Jaqueline Santos Moreira; Raizel, Raquel; Hypólito, Thaís Menezes; Rosa, Thiago Dos Santos; Cruzat, Vinicius Fernandes; Tirapegui, Julio

2016-08-01

In this study we investigated the chronic effects of oral l-glutamine and l-alanine supplementation, either in their free or dipeptide form, on glutamine-glutathione (GLN-GSH) axis and cytoprotection mediated by HSP-27 in rats submitted to resistance exercise (RE). Forty Wistar rats were distributed into 5 groups: sedentary; trained (CTRL); and trained supplemented with l-alanyl-l-glutamine, l-glutamine and l-alanine in their free form (GLN+ALA), or free l-alanine (ALA). All trained animals were submitted to a 6-week ladder-climbing protocol. Supplementations were offered in a 4% drinking water solution for 21 days prior to euthanasia. Plasma glutamine, creatine kinase (CK), myoglobin (MYO), and erythrocyte concentration of reduced GSH and glutathione disulfide (GSSG) were measured. In tibialis anterior skeletal muscle, GLN-GSH axis, thiobarbituric acid reactive substances (TBARS), and the expression of heat shock factor 1 (HSF-1), 27-kDa heat shock protein (HSP-27), and glutamine synthetase were determined. In CRTL animals, high-intensity RE reduced muscle glutamine levels and increased GSSG/GSH rate and TBARS, as well as augmented plasma CK and MYO levels. Conversely, l-glutamine-supplemented animals showed an increase in plasma and muscle levels of glutamine, with a reduction in GSSG/GSH rate, TBARS, and CK. Free l-alanine administration increased plasma glutamine concentration and lowered muscle TBARS. HSF-1 and HSP-27 were high in all supplemented groups when compared with CTRL (p < 0.05). The results presented herein demonstrate that l-glutamine supplemented with l-alanine, in both a free or dipeptide form, improve the GLN-GSH axis and promote cytoprotective effects in rats submitted to high-intensity RE training.

Formation of the Thiol Conjugates and Active Metabolite of Clopidogrel by Human Liver Microsomes

PubMed Central

Lau, Wei C.; Hollenberg, Paul F.

2012-01-01

We reported previously the formation of a glutathionyl conjugate of the active metabolite (AM) of clopidogrel and the covalent modification of a cysteinyl residue of human cytochrome P450 2B6 in a reconstituted system (Mol Pharmacol 80:839–847, 2011). In this work, we extended our studies of the metabolism of clopidogrel to human liver microsomes in the presence of four reductants, namely, GSH, l-Cys, N-acetyl-l-cysteine (NAC), and ascorbic acid. Our results demonstrated that formation of the AM was greatly affected by the reductant used and the relative amounts of the AM formed were increased in the following order: NAC (17%) < l-Cys (53%) < ascorbic acid (61%) < GSH (100%). AM-thiol conjugates were observed in the presence of NAC, l-Cys, and GSH. In the case of GSH, the formation of both the AM and the glutathionyl conjugate was dependent on the GSH concentrations, with similar Km values of ∼0.5 mM, which indicates that formation of the thiol conjugates constitutes an integral part of the bioactivation processes for clopidogrel. It was observed that the AM was slowly converted to the thiol conjugate, with a half-life of ∼10 h. Addition of dithiothreitol to the reaction mixture reversed the conversion, which resulted in a decrease in AM-thiol conjugate levels and a concomitant increase in AM levels, whereas addition of NAC led to the formation of AM-NAC and a concomitant decrease in AM-GSH levels. These results not only confirm that the AM is formed through oxidative opening of the thiolactone ring but also suggest the existence of an equilibrium between the AM, the thiol conjugates, and the reductants. These factors may affect the effective concentrations of the AM in vivo. PMID:22584220

Modeling Exposures to the Oxidative Potential of PM10

PubMed Central

2012-01-01

Differences in the toxicity of ambient particulate matter (PM) due to varying particle composition across locations may contribute to variability in results from air pollution epidemiologic studies. Though most studies have used PM mass concentration as the exposure metric, an alternative which accounts for particle toxicity due to varying particle composition may better elucidate whether PM from specific sources is responsible for observed health effects. The oxidative potential (OP) of PM < 10 μm (PM10) was measured as the rate of depletion of the antioxidant reduced glutathione (GSH) in a model of human respiratory tract lining fluid. Using a database of GSH OP measures collected in greater London, U.K. from 2002 to 2006, we developed and validated a predictive spatiotemporal model of the weekly GSH OP of PM10 that included geographic predictors. Predicted levels of OP were then used in combination with those of weekly PM10 mass to estimate exposure to PM10 weighted by its OP. Using cross-validation (CV), brake and tire wear emissions of PM10 from traffic within 50 m and tailpipe emissions of nitrogen oxides from heavy-goods vehicles within 100 m were important predictors of GSH OP levels. Predictive accuracy of the models was high for PM10 (CV R2=0.83) but only moderate for GSH OP (CV R2 = 0.44) when comparing weekly levels; however, the GSH OP model predicted spatial trends well (spatial CV R2 = 0.73). Results suggest that PM10 emitted from traffic sources, specifically brake and tire wear, has a higher OP than that from other sources, and that this effect is very local, occurring within 50–100 m of roadways. PMID:22731499

The roles of ROS production-scavenging system in Lasiodiplodia theobromae (Pat.) Griff. & Maubl.-induced pericarp browning and disease development of harvested longan fruit.

PubMed

Sun, Junzheng; Lin, Hetong; Zhang, Shen; Lin, Yifen; Wang, Hui; Lin, Mengshi; Hung, Yen-Con; Chen, Yihui

2018-05-01

Effects of Lasiodiplodia theobromae on reactive oxygen species (ROS) production-scavenging system during L. theobromae-induced pericarp browning and disease development of harvested "Fuyan" longans were investigated. Compared with control longans, L. theobromae-inoculated longans exhibited higher pericarp browning index and fruit disease index, higher pericarp O 2 - generation rate and MDA content. Moreover, L. theobromae infection also resulted in lower contents of pericarp AsA and GSH, lower levels of pericarp DPPH radical scavenging ability and reducing power. Additionally, L. theobromae infection decreased the activities of pericarp SOD, CAT and APX from day 2 to day 5. These findings suggested that L. theobromae-induced pericarp browning and disease development of harvested longans might be due to reduction of ROS scavenging ability and increase in ROS production, which might stimulate membrane lipid peroxidation, disrupt cellular membrane structure, and cause the loss of cellular compartmentalization and disease resistance, in turn, resulting in pericarp browning and disease development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biosynthetic hydrogels--studies on chemical and physical characteristics on long-term cellular response for tissue engineering.

PubMed

Thankam, Finosh Gnanaprakasam; Muthu, Jayabalan

2014-07-01

Biosynthetic hydrogels can meet the drawbacks caused by natural and synthetic ones for biomedical applications. In the current article we present a novel biosynthetic alginate-poly(propylene fumarate) copolymer based chemically crosslinked hydrogel scaffolds for cardiac tissue engineering applications. Partially crosslinked PA hydrogel and fully cross linked PA-A hydrogel scaffolds were prepared. The influence of chemical and physical (morphology and architecture of hydrogel) characteristics on the long term cellular response was studied. Both these hydrogels were cytocompatible and showed no genotoxicity upon contact with fibroblast cells. Both PA and PA-A were able to resist deleterious effects of reactive oxygen species and sustain the viability of L929 cells. The hydrogel incubated oxidative stress induced cells were capable of maintaining the intra cellular reduced glutathione (GSH) expression to the normal level confirmed their protective effect. Relatively the PA hydrogel was found to be unstable in the cell culture medium. The PA-A hydrogel was able to withstand appreciable cyclic stretching. The cyclic stretching introduced complex macro and microarchitectural features with interconnected pores and more structured bound water which would provide long-term viability of around 250% after the 24th day of culture. All these qualities make PA-A hydrogel form a potent candidate for cardiac tissue engineering. © 2013 Wiley Periodicals, Inc.

Effect of vitamin A administration on free radicals and lactate levels in individuals exercised to exhaustion.

PubMed

Patlar, Suleyman; Baltaci, Abdulkerim Kasim; Mogulkoc, Rasim

2016-09-01

This study was performed to explore the effect of vitamin A administration on Free Radicals production and antioxidant system activity and lactate levels in individuals exercised to exhaustion The study registered 10 healthy sedentary males their mean age was 22,85±0,26 years. The subjects were orally administrated with 300 mg vitamin A (retinol) for 4 weeks and engaged in strenuous exercise (using the Bruce protocol) once a week. Blood samples were collected from the subjects at four different times, before and after the supplementation and before and after exercise to analyze Malondialdehyde (MDA), Nitric oxide (NO), Glutathione (GSH), Glutathione peroxidase (GSH-Px), Catalase (CAT), Superoxide dismutase (SOD) levels using colorimetric ELISA test kits and plasma lactate levels using an autoanalyzer. Exhaustion exercise leaded to an increase in both MDA, NO, and lactate, and GSH, GSH-Px, CAT and SOD levels compared to resting levels both before and after supplementation (p<0.05). Increased NO levels found in pre-supplementation exhaustion showed a significant decrease after the supplementation of vitamin A (p<0.05), but the other parameters were not changed after vitamin A administration. The results of our study demonstrate that the increase caused by 4-week strenuous exercise in the levels of the free radical NO was offset by vitamin A supplementation. It can be suggested that supplementation of vitamin A at physiological doses has a limited effect on lipid peroxidation caused by strenuous exercise.

Effect of selenium supplementation on the level of glutathione-peroxidase (GSH-Px) activity in the nursing rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barron, S.P.; Hittner, H.M.; Strength, D.R.

Prevention of retinopathy of prematurity using vitamin E as an antioxidant has been demonstrated. The purpose of this experiment was to study the antioxidant system, GSH-Px, (a selenoenzyme), in the retina. The effect of i.p. administration and dietary Se as selenite or selenomethionine (selmet) on tissue GSH-Px activity was determined in nursing pups. Dams were randomized into 3 dietary treatments (Basal, 0.15 ppm selenite, and 0.15 ppm selmet) and mated. Pups were sacrificed at 0, 7, and 14 days after delivery and GSH-Px was measured in pup eyes, hearts, livers, and kidneys, and dam livers. The pups of the damsmore » consuming the Basal diet were divided into 4 i.p. groups: none, saline, selenite, and selmet (3 ..mu..g Se/kg body wt). The i.p. Se had no effect on GSH-Px activity in eye or heart, but significantly increased GSH-Px activity in liver and kidney with no difference between selenite and selmet. The pups of the dams consuming selenite and selmet diets showed significantly higher GSH-Px activity in all tissues studied than those consuming the Basal diet. For all tissues GSH-Px activity was higher for pups and dams fed selmet than those fed selenite. This research demonstrates that there was a difference in selenium availability between diet and i.p. administration.« less

Upregulation of capacity for glutathione synthesis in response to amino acid deprivation: regulation of glutamate-cysteine ligase subunits

PubMed Central

Sikalidis, Angelos K.; Mazor, Kevin M.; Lee, Jeong-In; Roman, Heather B.; Hirschberger, Lawrence L.; Stipanuk, Martha H.

2014-01-01

Using HepG2/C3A cells and MEFs, we investigated whether induction of GSH synthesis in response to sulfur amino acid deficiency is mediated by the decrease in cysteine levels or whether it requires a decrease in GSH levels per se. Both the glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunit mRNA levels were upregulated in response to a lack of cysteine or other essential amino acids, independent of GSH levels. This upregulation did not occur in MEFs lacking GCN2 (general control non-derepressible 2, also known as eIF2α kinase 4) or in cells expressing mutant eIF2α lacking the eIF2α kinase Ser51 phosphorylation site, indicating that expression of both GCLC and GCLM was mediated by the GCN2/ATF4 stress response pathway. Only the increase in GCLM mRNA level, however, was accompanied by a parallel increase in protein expression, suggesting that the enhanced capacity for GSH synthesis depended largely on increased association of GCLC with its regulatory subunit. Upregulation of both GCLC and GLCM mRNA levels in response to cysteine deprivation was dependent on new protein synthesis, which is consistent with expression of GCLC and GCLM being mediated by proteins whose synthesis depends on activation of the GCN2/ATF4 pathway. Our data suggest that the regulation of GCLC expression may be mediated by changes in the abundance of transcriptional regulators, whereas the regulation of GCLM expression may be mediated by changes in the abundance of mRNA stabilizing or destabilizing proteins. Upregulation of GCLM levels in response to low cysteine levels may serve to protect the cell in the face of a future stress requiring GSH as an antioxidant or conjugating/detoxifying agent. PMID:24557597

Effect of Increasing Glutathione With Cysteine and Glycine Supplementation on Mitochondrial Fuel Oxidation, Insulin Sensitivity, and Body Composition in Older HIV-Infected Patients

PubMed Central

Nguyen, Dan; Hsu, Jean W.; Jahoor, Farook

2014-01-01

Background: HIV-infected patients are reported to have impaired oxidation of fatty acids despite increased availability, suggesting a mitochondrial defect. We investigated whether diminished levels of a key mitochondrial antioxidant, glutathione (GSH), was contributing to defective fatty acid oxidation in older HIV-infected patients, and if so, the metabolic mechanisms contributing to GSH deficiency in these patients. Methods: In an open-label design, 8 older GSH-deficient HIV-infected males were studied before and after 14 days of oral supplementation with the GSH precursors cysteine and glycine. A combination of stable-isotope tracers, calorimetry, hyperinsulinemic-euglycemic clamp, and dynamometry were used to measure GSH synthesis, fasted and insulin-stimulated (fed) mitochondrial fuel oxidation, insulin sensitivity, body composition, anthropometry, forearm-muscle strength, and lipid profiles. Results: Impaired synthesis contributed to GSH deficiency in the patients and was restored with cysteine plus glycine supplementation. GSH improvement was accompanied by marked improvements in fasted and fed mitochondrial fuel oxidation. Associated benefits included improvements in insulin sensitivity, body composition, anthropometry, muscle strength, and dyslipidemia. Conclusions: This work identifies 2 novel findings in older HIV-infected patients: 1) diminished synthesis due to decreased availability of cysteine and glycine contributes to GSH deficiency and can be rapidly corrected by dietary supplementation of these precursors and 2) correction of GSH deficiency is associated with improvement of mitochondrial fat and carbohydrate oxidation in both fasted and fed states and with improvements in insulin sensitivity, body composition, and muscle strength. The role of GSH on ameliorating metabolic complications in older HIV-infected patients warrants further investigation. PMID:24081740

Lower susceptibility of female mice to acetaminophen hepatotoxicity: Role of mitochondrial glutathione, oxidant stress and c-jun N-terminal kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Kuo; Williams, C. David; McGill, Mitchell R.

Acetaminophen (APAP) overdose causes severe hepatotoxicity in animals and humans. However, the mechanisms underlying the gender differences in susceptibility to APAP overdose in mice have not been clarified. In our study, APAP (300 mg/kg) caused severe liver injury in male mice but 69–77% lower injury in females. No gender difference in metabolic activation of APAP was found. Hepatic glutathione (GSH) was rapidly depleted in both genders, while GSH recovery in female mice was 2.6 fold higher in the mitochondria at 4 h, and 2.5 and 3.3 fold higher in the total liver at 4 h and 6 h, respectively. Thismore » faster recovery of GSH, which correlated with greater induction of glutamate-cysteine ligase, attenuated mitochondrial oxidative stress in female mice, as suggested by a lower GSSG/GSH ratio at 6 h (3.8% in males vs. 1.4% in females) and minimal centrilobular nitrotyrosine staining. While c-jun N-terminal kinase (JNK) activation was similar at 2 and 4 h post-APAP, it was 3.1 fold lower at 6 h in female mice. However, female mice were still protected by the JNK inhibitor SP600125. 17β-Estradiol pretreatment moderately decreased liver injury and oxidative stress in male mice without affecting GSH recovery. Conclusion: The lower susceptibility of female mice is achieved by the improved detoxification of reactive oxygen due to accelerated recovery of mitochondrial GSH levels, which attenuates late JNK activation and liver injury. However, even the reduced injury in female mice was still dependent on JNK. While 17β-estradiol partially protects male mice, it does not affect hepatic GSH recovery. - Highlights: • Female mice are less susceptible to acetaminophen overdose than males. • GSH depletion and protein adduct formation are similar in both genders. • Recovery of hepatic GSH levels is faster in females and correlates with Gclc. • Reduced oxidant stress in females leads to reduced JNK activation. • JNK activation and mitochondrial translocation are critical in females.« less

[Effect of flavonoids of ginkgo biloba on anti-oxidizing system of mice after acute alcohol administration].

PubMed

Yao, Ping; Liu, Lie-Gang; Jia, Wen-Bo; Song, Fang-Fang; Zhou, Shaoliang; Zhang, Xiping; Sun, Xiufa

2005-05-01

To investigate the protective effects of flavonoids of ginkgo biloba on anti-oxidizing system damaged by acute alcohol administration. Adult male Kunming mice were employed and divided into randomly flavonoid intervention group, normal control and ethanol control group according to body weight. After pretreated with flavonoids of ginkgo biloba (96mg/kg bw), the mice in flavonoid intervention group ingested alcohol (ethanol 4.8g/kg bw) via i.g. and were decapitated after 0.5, 1, 2, 4, 6, 9, and 15 h of ethanol administration. The same treatment was carried out on ethanol control group except that physiological saline was applied instead of flavonoid of ginkgo biloba. Meanwhile, the normal control group was established. The concentration of glutathione (GSH), malondialdehyde (MDA) and the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) in the serum and liver were determined. The experiment displays that the content of GSH and the activities of GSH-Px and SOD decreased rapidly after 1 h of treatment with alcohol and dropped to the lowest level at 4h of treatment. After 6h of treatment, these indexes came to the normal level rapidly. The level of MDA of serum and liver increased rapidly after 1 h of treatment and reached the climax at 4h and 6h respectively. It went back to the normal concentration until 15h and 9 h respectively. On a whole, there were similar curves between flavonoids intervention group and alcohol control group on the indexes. However, to some extent, the supplement of flavonoid of ginkgo biloba can prohibit the rise of MDA level and the decline of GSH-Px, SOD, GSH which were induced by acute alcohol intakes. Flavonoid of ginkgo biloba have some protective effects on the damage of anti-oxidizing system of mice induced by acute alcohol adminstration.

Effects of exogenous glutathione on arsenic burden and NO metabolism in brain of mice exposed to arsenite through drinking water.

PubMed

Wang, Yan; Zhao, Fenghong; Jin, Yaping; Zhong, Yuan; Yu, Xiaoyun; Li, Gexin; Lv, Xiuqiang; Sun, Guifan

2011-03-01

Chronic exposure to inorganic arsenic (iAs) is associated with neurotoxicity. Studies to date have disclosed that methylation of ingested iAs is the main metabolic pathway, and it is a process relying on reduced glutathione (GSH). The aim of this study was to explore the effects of exogenous GSH on arsenic burden and metabolism of nitric oxide (NO) in the brain of mice exposed to arsenite via drinking water. Mice were exposed to sodium arsenite through drinking water contaminated with 50 mg/L arsenic for 4 weeks and treated intraperitoneally with saline solution, 200 mg/kg body weight (b.w), 400 mg/kg b.w, or 800 mg/kg b.w GSH, respectively, at the 4th week. Levels of iAs, monomethylarsenic acid, and dimethylarsenic acid (DMAs) in the liver, blood, and brain were determined by method of hydride generation coupled with atomic absorption spectrophotometry. Activities of nitric oxide synthase (NOS) and contents of NO in the brain were determined by colorimetric method. Compared with mice exposed to arsenite alone, administration of GSH increased dose-dependently the primary and secondary methylation ratio in the liver, which caused the decrease in percent iAs and increase in percent DMAs in the liver, as a consequence, resulted in significant decrease in iAs levels in the blood and total arsenic levels in both blood and brain. NOS activities and NO levels in the brain of mice in iAs group were significantly lower than those in control; however, administration of GSH could increase significantly activities of NOS and contents of NO. Findings from this study suggested that exogenous GSH could promote both primary and secondary arsenic methylation capacity in the liver, which might facilitate excretion of arsenicals, and consequently reduce arsenic burden in both blood and brain and furthermore ameliorate the effects of arsenicals on NO metabolism in the brain.

Restoring Cytokine Balance in HIV-Positive Individuals with Low CD4 T Cell Counts

PubMed Central

Valdivia, Anddre; Ly, Judy; Gonzalez, Leslie; Hussain, Parveen; Saing, Tommy; Islamoglu, Hicret; Pearce, Daniel; Ochoa, Cesar

2017-01-01

Abstract HIV infects and destroys CD4+ T cells leading to a compromised immune system. In a double-blinded study, a group of HIV-infected individuals with CD4+ T cell counts below 350 cells/mm3 were given either an empty liposomal supplement or a liposomal glutathione (L-GSH) supplement to take over a 3-month period. Baseline measurements in HIV-positive subjects show a significant decrease in levels of interleukin (IL)-12, IL-2, and interferon (IFN)-γ, along with a substantial increase in the levels of IL-6, IL-10, transforming growth factor (TGF)-β, and free radicals, compared to healthy individuals. Supplementation of HIV-positive subjects with L-GSH for 3 months resulted in a notable increase in the levels of IL-12, IL-2, and IFN-γ, with a concomitant decrease in the levels of IL-6, IL-10, and free radicals, and stabilization in the levels of TGF-β, IL-1, and IL-17, compared to their placebo counterparts. Levels of free radicals in CD4+ T cells stabilized, while GSH levels increased in the treatment group. Those in the placebo group showed no significant difference throughout the study. In summary, supplementation with L-GSH in HIV-infected individuals with CD4+ T cell counts below 350 cells/mm3 can help restore redox homeostasis and cytokine balance, therefore aiding the immune system to control opportunistic infections. PMID:28398068

Glutathione Depletion Induced by c-Myc Downregulation Triggers Apoptosis on Treatment with Alkylating Agents1

PubMed Central

Biroccio, Annamaria; Benassi, Barbara; Fiorentino, Francesco; Zupi, Gabriella

2004-01-01

Abstract Here we investigate the mechanism(s) involved in the c-Myc-dependent drug response of melanoma cells. By using three M14-derived c-Myc low-expressing clones, we demonstrate that alkylating agents, cisplatin and melphalan, trigger apoptosis in the c-Myc antisense transfectants, but not in the parental line. On the contrary, topoisomerase inhibitors, adriamycin and camptothecin, induce apoptosis to the same extent regardless of c-Myc expression. Because we previously demonstrated that c-Myc downregulation decreases glutathione (GSH) content, we evaluated the role of GSH in the apoptosis induced by the different drugs. In control cells treated with one of the alkylating agents or the others, GSH depletion achieved by l-buthionine-sulfoximine preincubation opens the apoptotic pathway. The apoptosis proceeded through early Bax relocalization, cytochrome c release, and concomitant caspase-9 activation, whereas reactive oxygen species production and alteration of mitochondria membrane potential were late events. That GSH was determining in the c-Myc-dependent drug-induced apoptosis was demonstrated by altering the intracellular GSH content of the c-Myc low-expressing cells up to the level of controls. Indeed, GSH ethyl ester-mediated increase of GSH abrogated apoptosis induced by cisplatin and melphalan by inhibition of Bax/cytochrome c redistribution. The relationship among c-Myc, GSH content, and the response to alkylating agent has been also evaluated in the M14 Myc overexpressing clones as well as in the melanoma JR8 c-Myc antisense transfectants. All together, these results demonstrate that GSH plays a key role in governing c-Myc-dependent drug-induced apoptosis. PMID:15153331

Endophytic bacterium Sphingomonas SaMR12 promotes cadmium accumulation by increasing glutathione biosynthesis in Sedum alfredii Hance.

PubMed

Pan, Fengshan; Meng, Qian; Wang, Qiong; Luo, Sha; Chen, Bao; Khan, Kiran Yasmin; Yang, Xiaoe; Feng, Ying

2016-07-01

A hydroponic experiment was conducted to verify the effects of inoculation with endophytic bacteria Sphingomonas SaMR12 on root growth, cadmium (Cd) uptake, reactive oxygen species (ROS), antioxidases, glutathione (GSH) and the related gene expression of Sedum alfredii Hance under different levels of Cd such as 0, 10, 25, 100 and 400 μM. The results showed that inoculation of SaMR12 improved Cd accumulation and upregulated glutathione synthase (GS) expression, but slightly reduced malondialdehyde (MDA) concentration and alleviated Cd-induced damage in roots. However it didn't alter the activities of antioxidant enzymes. When Cd concentration exceeded 25 μM, SaMR12 increased the concentration of GSH and the expression level of GSH1. At high Cd treatment levels (100 and 400 μM), SaMR12 significantly reduced H2O2 concentration and enhanced expression level of 1-Cys peroxiredoxin PER1 and ATPS genes. These results indicate that although SaMR12 has no significant effects on antioxidases activities, it reduces H2O2 concentration by enhancing GSH concentration and relevant genes expression, and subsequently improves Cd tolerance and accumulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pseudomonas aeruginosa gshA Mutant Is Defective in Biofilm Formation, Swarming, and Pyocyanin Production

PubMed Central

Van Laar, Tricia A.; Esani, Saika; Birges, Tyler J.; Hazen, Bethany; Thomas, Jason M.

2018-01-01

ABSTRACT Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that can cause severe opportunistic infections. The principal redox buffer employed by this organism is glutathione (GSH). To assess the role of GSH in the virulence of P. aeruginosa, a number of analyses were performed using a mutant strain deficient in gshA, which does not produce GSH. The mutant strain exhibited a growth delay in minimal medium compared to the wild-type strain. Furthermore, the gshA mutant was defective in biofilm and persister cell formation and in swimming and swarming motility and produced reduced levels of pyocyanin, a key virulence factor. Finally, the gshA mutant strain demonstrated increased sensitivity to methyl viologen (a redox cycling agent) as well as the thiol-reactive antibiotics fosfomycin and rifampin. Taken together, these data suggest a key role for GSH in the virulence of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is a ubiquitous bacterium that can cause severe opportunistic infections, including many hospital-acquired infections. It is also a major cause of infections in patients with cystic fibrosis. P. aeruginosa is intrinsically resistant to a number of drugs and is capable of forming biofilms that are difficult to eradicate with antibiotics. The number of drug-resistant strains is also increasing, making treatment of P. aeruginosa infections very difficult. Thus, there is an urgent need to understand how P. aeruginosa causes disease in order to find novel ways to treat infections. We show that the principal redox buffer, glutathione (GSH), is involved in intrinsic resistance to the fosfomycin and rifampin antibiotics. We further demonstrate that GSH plays a role in P. aeruginosa disease and infection, since a mutant lacking GSH has less biofilm formation, is less able to swarm, and produces less pyocyanin, a pigment associated with infection. PMID:29669887

Development of Saccharomyces cerevisiae producing higher levels of sulfur dioxide and glutathione to improve beer flavor stability.

PubMed

Chen, Yefu; Yang, Xu; Zhang, Shijie; Wang, Xiaoqiong; Guo, Changhui; Guo, Xuewu; Xiao, Dongguang

2012-01-01

Sulfur compounds, such as sulfite (SO(2)), hydrogen sulfide (H(2)S), and glutathione (GSH), play different roles in beer flavor stability. SO(2) and GSH have antiaging effects which are helpful to improve the flavor stability of beer, whereas H(2)S is undesirable to beer flavor because of its unpleasant aroma. Here, we report the development of Saccharomyces cerevisiae which produces higher levels of SO(2) and GSH but lower level of H(2)S to improve beer flavor stability by nongenetic engineering approaches. After two rounds of UV mutagenesis coupled with specific plate screening methods, one promising mutant named MV16 was obtained. Compared with the original strain, the SO(2) and GSH production of MV16 in fermenting liquor increased by 31% and 30.2%, respectively, while H(2)S content decreased by 74.9%, and the DPPH radical clearance and the resistance staling value of beer fermented by MV16 increased by 24.6% and 33.0%, respectively. The antioxidizability of the mutant was improved significantly. The strategy adopted in our study could be used to obtain S. cerevisiae of improved antiaging properties, and the mutant would be safe for public use.

Cordyceps militaris extract attenuates D-galactose-induced memory impairment in mice.

PubMed

Li, Zaixin; Zhang, Zhi; Zhang, Jinshan; Jia, Jing; Ding, Jie; Luo, Rongzhen; Liu, Zhangqin

2012-12-01

Memory impairment is one of main clinical symptoms of brain senescence. To address the effects of Cordyceps militaris Link extract (CE) on memory impairment, a D-galactose (D-Gal)-induced aging mouse model was employed. Mice injected with D-Gal showed a significant learning and memory impairment that was rescued by CE treatment. The mechanism was further investigated by analyzing the protein level and activity of oxidant and antioxidant molecules, including malondialdehyde (MDA), monoamine oxidase (MAO), total super-oxide dismutase (T-SOD), total antioxidant capacity (T-AOC), glutathione (GSH), and glutathione peroxidase (GSH-px), which played critical roles in the development of brain senescence. The results showed that CE treatment resulted in a significant decrease in the oxidative activity of MAO and the level of MDA, and significantly increased the antioxidant activities of T-SOD and T-AOC in the cerebral cortices. Moreover, the level of GSH and the activity of antioxidant enzymes GSH-px in serum were significantly upregulated after CE treatment. Taken together, our results suggest that Cordyceps militaris extract could ameliorate experimental memory impairment in mice with D-Gal-induced aging through its potent antioxidant activities.

Bisphenol A, bisphenol S, bisphenol F and bisphenol AF induce different oxidative stress and damage in human red blood cells (in vitro study).

PubMed

Maćczak, Aneta; Cyrkler, Monika; Bukowska, Bożena; Michałowicz, Jaromir

2017-06-01

Bisphenol A (BPA) and its analogs are widely used in the production of various everyday use products, which leads to a common exposure of humans to these substances. The effect of bisphenols on oxidative stress parameters has not been described in detail in non-nucleated cells, therefore, we have decided to evaluate the impact of BPA and its analogs, i.e. bisphenol S (BPS), bisphenol F (BPF) and bisphenol AF (BPAF) on reactive oxygen species (ROS) formation, lipid peroxidation, glutathione (GSH) level and the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in human erythrocytes. The erythrocytes were incubated with the compounds studied in the concentrations ranging from 0.1 to 500μg/ml for 1, 4 or 24h. It has been found that bisphenols enhanced ROS (including • OH) formation, depleted GSH level, increased lipid peroxidation and changed the activities of SOD, CAT and GSH-Px. It has been noted that the strongest alterations in ROS formation, lipid peroxidation and the activity of antioxidant enzymes were induced by BPAF, which changed CAT and SOD activity even at 0.5μg/ml. It has also been shown that BPA caused the strongest changes in GSH level, while BPS, which is the main BPA substituent in the manufacture did not alter most parameters studied. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhanced Photodynamic Therapy by Reduced Levels of Intracellular Glutathione Obtained By Employing a Nano-MOF with CuII as the Active Center.

PubMed

Zhang, Wei; Lu, Jun; Gao, Xiaonan; Li, Ping; Zhang, Wen; Ma, Yu; Wang, Hui; Tang, Bo

2018-02-16

In photodynamic therapy (PDT), the level of reactive oxygen species (ROS) produced in the cell directly determines the therapeutic effect. Improvement in ROS concentration can be realized by reducing the glutathione (GSH) level or increasing the amount of photosensitizer. However, excessive amounts photosensitizer may cause side effects. Therefore, the development of photosensitizers that reduce GSH levels through synergistically improving ROS concentration in order to strengthen the efficacy of PDT for tumor is important. We report a nano-metal-organic framework (Cu II -metalated nano-MOF {CuL-[AlOH] 2 } n (MOF-2, H 6 L=mesotetrakis(4-carboxylphenyl)porphyrin)) based on Cu II as the active center for PDT. This MOF-2 is readily taken up by breast cancer cells, and high levels of ROS are generated under light irradiation. Meanwhile, intracellular GSH is considerably decreased owing to absorption on MOF-2; this synergistically increases ROS concentration and accelerates apoptosis, thereby enhancing the effect of PDT. Notably, based on the direct adsorption of GSH, MOF-2 showed a comparable effect with the commercial antitumor drug camptothecin in a mouse breast cancer model. This work provides strong evidence for MOF-2 as a promising new PDT candidate and anticancer drug. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulation of Cell Physiology and Pathology by Protein S-Glutathionylation: Lessons Learned from the Cardiovascular System

PubMed Central

Pimentel, David; Haeussler, Dagmar Johanna; Matsui, Reiko; Burgoyne, Joseph Robert; Cohen, Richard Alan

2012-01-01

Abstract Significance: Reactive oxygen and nitrogen species contributing to homeostatic regulation and the pathogenesis of various cardiovascular diseases, including atherosclerosis, hypertension, endothelial dysfunction, and cardiac hypertrophy, is well established. The ability of oxidant species to mediate such effects is in part dependent on their ability to induce specific modifications on particular amino acids, which alter protein function leading to changes in cell signaling and function. The thiol containing amino acids, methionine and cysteine, are the only oxidized amino acids that undergo reduction by cellular enzymes and are, therefore, prime candidates in regulating physiological signaling. Various reports illustrate the significance of reversible oxidative modifications on cysteine thiols and their importance in modulating cardiovascular function and physiology. Recent Advances: The use of mass spectrometry, novel labeling techniques, and live cell imaging illustrate the emerging importance of reversible thiol modifications in cellular redox signaling and have advanced our analytical abilities. Critical Issues: Distinguishing redox signaling from oxidative stress remains unclear. S-nitrosylation as a precursor of S-glutathionylation is controversial and needs further clarification. Subcellular distribution of glutathione (GSH) may play an important role in local regulation, and targeted tools need to be developed. Furthermore, cellular redundancies of thiol metabolism complicate analysis and interpretation. Future Directions: The development of novel pharmacological analogs that specifically target subcellular compartments of GSH to promote or prevent local protein S-glutathionylation as well as the establishment of conditional gene ablation and transgenic animal models are needed. Antioxid. Redox Signal. 16, 524–542. PMID:22010840

Comparison of antioxidant activity between green and roasted coffee beans using molecular methods

PubMed Central

PRIFTIS, ALEXANDROS; STAGOS, DIMITRIOS; KONSTANTINOPOULOS, KONSTANTINOS; TSITSIMPIKOU, CHRISTINA; SPANDIDOS, DEMETRIOS A.; TSATSAKIS, ARISTIDES M.; TZATZARAKIS, MANOLIS N.; KOURETAS, DEMETRIOS

2015-01-01

Coffee is one of the most popular and widely consumed beverages worldwide due to its pleasant taste and aroma. A number of studies have been performed to elucidate the possible beneficial effects of coffee consumption on human health and have shown that coffee exhibits potent antioxidant activity, which may be attributed mainly to its polyphenolic content. However, there is also evidence to suggest that coffee roasting (the procedure which turns green coffee beans to the dark, roasted ones from which the beverage derives) may alter the polyphenolic profile of the beans (e.g., via the Maillard reaction) and, concomitantly, their antioxidant activity. In the present study, the antioxidant activity of 13 coffee varieties was examined in both green and roasted coffee bean extracts using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-eth-ylbenzothiazoline-6-sulfonic acid) (ABTS•+) radical scavenging assays. In addition, 5 selected varieties were also examined for their protective effects against peroxyl and hydroxyl radical-induced DNA strand cleavage. Finally, C2C12 murine myoblasts were treated with non-cytotoxic concentrations of the most potent extract in order to examine its effects on the cellular redox status by measuring the glutathione (GSH) and reactive oxygen species (ROS) levels by flow cytometry. Our results revealed that, in 8 out of the 13 coffee varieties, roasting increased free radical scavenging activity as shown by DPPH and ABTS•+ assays. Moreover, we found that when one coffee variety was roasted for different amounts of time, the increase in the antioxidant activity depended on the roasting time. By contrast, in 5 varieties, roasting reduced the antioxidant activity. Similar differences between the roasted and green beans were also observed in the free radical-induced DNA strand cleavage assay. The observed differences in the antioxidant activity between the different coffee varieties may be attributed to their varying polyphenolic content and composition, as well as to the different molecules produced during roasting. In addition, in the cell culture assay, the tested coffee extract led to increased GSH levels in a dose-dependent manner, indicating the enhancement of cellular antioxidant mechanisms. PMID:26458565

Effects of triclosan (TCS) on fecundity, the antioxidant system, and oxidative stress-mediated gene expression in the copepod Tigriopus japonicus.

PubMed

Park, Jun Chul; Han, Jeonghoon; Lee, Min-Chul; Seo, Jung Soo; Lee, Jae-Seong

2017-08-01

Triclosan (TCS) is an antimicrobial agent that has been widely dispersed and detected in the marine environment. However, the effects of TCS in marine invertebrates are poorly understood. In this study, the effects of TCS on life cycle history (e.g. mortality and fecundity) along with cellular reactive oxygen species (ROS) levels, GSH content, antioxidant enzymatic activities, and mRNA expression levels of oxidative stress-mediated genes were measured in the copepod Tigriopus japonicus. The no observed effect concentration (NOEC) and median lethal concentration (LC50) of TCS in the adult stage were determined to be 300μg/L and 437.476μg/L, respectively, while in the nauplius stages the corresponding values were 20μg/L, and 51.76μg/L, respectively. Fecundity was significantly reduced (P<0.05) in response to TCS at 100μg/L. Concentration- and time-dependent analysis of ROS, GSH content (%), and antioxidant enzymatic activities (e.g. GST, GPx, and SOD) were significantly increased (P<0.05) in response to TCS exposure. Additionally, mRNA expression of detoxification (e.g., CYPs) and antioxidant (e.g., glutathione S-transferase-sigma isoforms, Cu/Zn superoxide dismutase, catalase) genes was modulated in response to TCS exposure at different concentrations over a 24h period. Our results revealed that TCS can induce reduced fecundity and oxidative stress with transcriptional regulation of oxidative stress-mediated genes with activation of the antioxidant system in the copepod T. japonicus. Copyright © 2017 Elsevier B.V. All rights reserved.

Sertraline-induced potentiation of the CYP3A4-dependent neurotoxicity of carbamazepine: an in vitro study.

PubMed

Ghosh, Chaitali; Hossain, Mohammad; Spriggs, Addison; Ghosh, Arnab; Grant, Gerald A; Marchi, Nicola; Perucca, Emilio; Janigro, Damir

2015-03-01

Drug toxicity is a hurdle to drug development and to clinical translation of basic research. Antiepileptic drugs such as carbamazepine (CBZ) and selective serotonin reuptake inhibitors such as sertraline (SRT) are commonly co-prescribed to patients with epilepsy and comorbid depression. Because SRT may interfere with cytochrome P450 (CYP) enzyme activity and CYPs have been implicated in the conversion of CBZ to reactive cytotoxic metabolites, we investigated in vitro models to determine whether SRT affects the neurotoxic potential of CBZ and the mechanisms involved. Human fetal brain-derived dopaminergic neurons, human brain microvascular endothelial cells (HBMECs), and embryonic kidney (HEK) cells were used to evaluate cytotoxicity of CBZ and SRT individually and in combination. Nitrite and glutathione (GSH) levels were measured with drug exposure. To validate the role of CYP3A4 in causing neurotoxicity, drug metabolism was compared to cell death in HEK CYP3A4 overexpressed and cells pretreated with the CYP3A4 inhibitor ketoconazole. In all cellular systems tested, exposure to CBZ (127 μM) or SRT (5 μM) alone caused negligible cytotoxicity. By contrast CBZ, tested at a much lower concentration (17 μM) in combination with SRT (5 μM), produced prominent cytotoxicity within 15 min exposure. In neurons and HBMECs, cytotoxicity was associated with increased nitrite levels, suggesting involvement of free radicals as a pathogenetic mechanism. Pretreatment of HBMECs with reduced GSH or with the GSH precursor N-acetyl-L-cysteine prevented cytotoxic response. In HEK cells, the cytotoxic response to the CBZ + SRT combination correlated with the rate of CBZ biotransformation and production of 2-hydroxy CBZ, further suggesting a causative role of reactive metabolites. In the same system, cytotoxicity was potentiated by overexpression of CYP3A4, and prevented by CYP3A4 inhibitor. These results demonstrate an unexpected neurotoxic interaction between CBZ and SRT, apparently related to increased CYP3A4-mediated production of reactive CBZ metabolites. The potential clinical implications of these findings are discussed. Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.

Whey Protein Supplementation Improves Nutritional Status, Glutathione Levels, and Immune Function in Cancer Patients: A Randomized, Double-Blind Controlled Trial.

PubMed

Bumrungpert, Akkarach; Pavadhgul, Patcharanee; Nunthanawanich, Pornpimon; Sirikanchanarod, Anchalee; Adulbhan, Araya

2018-06-01

Clinical side effects from medical therapy play an important role in causing malnutrition among cancer patients. Whey protein isolates (WPIs) have the potential to improve the nutritional status of cancer patients. The present study determined the effects of whey protein supplementation on nutritional status, glutathione (GSH) levels, immunity, and inflammatory markers in cancer patients in Thailand. A total of 42 cancer patients (41-63 years old) who received intravenous chemotherapy were randomized in a double-blind controlled trial at the National Cancer Institute in Thailand. Patients received 40 g of WPI plus zinc and selenium (intervention group, n = 23) or a maltodextrin oral snack (control group, n = 19) every day during the daytime for 12 weeks. Nutritional status, GSH levels, immunity, and inflammatory markers were assessed at baseline, 6, and 12 weeks. Whey protein supplementation significantly increased albumin (2.9%) and immunoglobulin G (4.8%) levels compared to the control group at week 12. Controls showed a significantly lower percent change in GSH levels (6.0%), whereas there was a significant time-dependent increase in the intervention group (11.7%). Whey protein supplementation improved nutrition status scores in the intervention group compared to the control. These data indicate that whey protein supplementation can increase GSH levels and improve nutritional status and immunity in cancer patients undergoing chemotherapy. These results will facilitate implementation of malnutrition risk prevention strategies and improve protein status, including immune function, during chemotherapy.

[Effect of occupational stress on oxidation/antioxidant capacity in nurses].

PubMed

Cao, Lili; Tian, Honger; Zhang, Qingdong; Zhu, Xinyun; Zhan, Yongguo; Su, Jingguo; Xu, Tian; Zhu, Huabin; Liu, Ling

2014-02-01

To investigate the effect of occupational stress on the oxidation/antioxidant capacity in nurses. A total of 131 nurses were included as study subjects. The occupational health information collection system (based on the Internet of things) was used for measurement of occupational stress. Levels of hydroxyl free radicals and antioxidant enzymes were determined. The serum level of superoxide dismutase (SOD) was the highest in nurses under the age of 30 and the lowest in those over 45 (P < 0.05). The serum levels of glutathione peroxidase (GSH-Px) and peroxidase (POD) were the highest in nurses of working age less than 5 years, followed by those of 5-15 years, and nurses with more than 25 years' working experience showed the lowest GSH-Px and POD levels (P < 0.05). Furthermore, nurses with a university (college) degree had a higher GSH-Px level and a lower POD level compared with those with junior and senior high school degrees (P < 0.05). Job prospects and job control were positive occupational stress factors for SOD. Job hazards were negative occupational stress factors for POD. Psychological satisfaction was negative occupational stress reaction for hydroxyl free radicals. Calmness was positive occupational stress reaction for SOD, and daily stress was a negative one. The positive occupational stress reactions for GSH-Px were psychological satisfaction and job satisfaction, and daily stress was negative reaction. Nurses with higher occupational stress have stronger oxidation and weaker antioxidant capacity, which intensifies oxidant-antioxidant imbalance and leads to oxidative stress damage.

Crucial yet divergent roles of mitochondrial redox state in skeletal muscle vs. brown adipose tissue energetics.

PubMed

Mailloux, Ryan J; Adjeitey, Cyril Nii-Klu; Xuan, Jian Ying; Harper, Mary-Ellen

2012-01-01

Reduced glutathione (GSH) is the major determinant of redox balance in mitochondria and as such is fundamental in the control of cellular bioenergetics. GSH is also the most important nonprotein antioxidant molecule in cells. Surprisingly, the effect of redox environment has never been examined in skeletal muscle and brown adipose tissue (BAT), two tissues that have exceptional dynamic range and that are relevant to the development of obesity and related diseases. Here, we show that the redox environment plays crucial, yet divergent, roles in modulating mitochondrial bioenergetics in skeletal muscle and BAT. Skeletal muscle mitochondria were found to naturally have a highly reduced environment (GSH/GSSG≈46), and this was associated with fairly high (∼40%) rates of state 4 (nonphosphorylating) respiration and decreased reactive oxygen species (ROS) emission. The deglutathionylation of uncoupling protein 3 (UCP3) following an increase in the reductive potential of mitochondria results in a further increase in nonphosphorylating respiration (∼20% in situ). BAT mitochondria were found to have a much more oxidized status (GSH/GSSG≈13) and had basal reactive oxygen species emission that was higher (∼250% increase in ROS generation) than that in skeletal muscle mitochondria. When redox status was subsequently increased (i.e., more reduced), UCP1-mediated uncoupling was more sensitive to GDP inhibition. Surprisingly, BAT was found to be devoid of glutaredoxin-2 (Grx2) expression, while there was abundant expression in skeletal muscle. Taken together, these findings reveal the importance of redox environment in controlling bioenergetic functions in both tissues, and the highly unique characteristics of BAT in this regard.

Changes in metabolites, antioxidant system, and gene expression in Microcystis aeruginosa under sodium chloride stress.

PubMed

Chen, Lei; Mao, Feijian; Kirumba, George Chira; Jiang, Cheng; Manefield, Mike; He, Yiliang

2015-12-01

Microcystis (M.) aeruginosa, one of the most common bloom-forming cyanobacteria, occurs worldwide. The Qingcaosha (QCS) Reservoir is undergoing eutrophication and faces the problem of saltwater intrusion. The aim of this study was to investigate the effects of sudden salinity changes on physiological parameters and related gene transcription in M. aeruginosa under controlled laboratory conditions. The results showed that sodium chloride (50, 200 and 500 mg L(-1) NaCl) inhibited the algal growth and decreased pigment concentrations (chlorophyll a, carotenoid and phycocyanin). Sodium chloride increased both the intracellular and extracellular microcystin contents and elevated the mcyD transcript level in M. aeruginosa. It also increased the malondialdehyde (MDA) content and caused cytomembrane damage. This damage caused the release of intracellular toxins into the culture medium. In addition, NaCl decreased the maximum electron transport rate, increased the levels of reactive oxygen species (ROS) and changed the cellular redox status. Consequently, NaCl inhibited the expression of cpcB, psbA and rbcL. Furthermore, NaCl increased the activities of superoxide dismutases (SOD), catalase (CAT), glutathione reductase (GR), and total glutathione peroxidase (GPx). The transcript levels of sod and reduced glutathione (gsh) were also increased after exposure to NaCl. Our results indicate that a sudden increase in salinity increases the production and excretion of microcystin, changes the cellular redox status, enhances the activities of antioxidant enzymes, inhibits photosynthesis, and affects transcript levels of related genes in M. aeruginosa. Copyright © 2015 Elsevier Inc. All rights reserved.

Anti-apoptotic effect of caspase inhibitors on H₂O₂-treated HeLa cells through early suppression of its oxidative stress.

PubMed

Park, Woo Hyun

2014-05-01

Oxidative stress-induced cytotoxicity in cervical cancer cells may be of toxicological interest. In the present study, the effects of exogenous H2O2 on cell growth and death in HeLa cervical cancer cells were investigated, and the anti-apoptotic effects of various caspase (pan-caspase, caspase-3, -8 or -9) inhibitors on H2O2-treated HeLa cells were also evaluated with regard to reactive oxygen species (ROS) and glutathione (GSH) levels. Based on MTT assays, H2O2 inhibited the growth of HeLa cells with an IC50 value of ~75 µM at 24 h. H2O2 increased the number of dead cells and Annexin V-FITC-positive cells in the HeLa cells, which was accompanied by the activation of caspase-3 and the loss of mitochondrial membrane potential (MMP; ΔΨm). However, relatively higher doses of H2O2 induced necrosis in HeLa cells. Caspase inhibitors significantly prevented H2O2-induced HeLa cell death. H2O2 increased ROS including O2•- at 24 h and increased the activity of catalase in HeLa cells. H2O2 also increased the ROS level at 1 h, and several caspase inhibitors attenuated the increased level at 1 h but not at 6, 12 and 24 h. H2O2 decreased the GSH level in HeLa cells at 1 h, and several caspase inhibitors attenuated the decreased level of GSH at this time. H2O2 induced GSH depletion at 24 h. In conclusion, H2O2 inhibited the growth of HeLa cells via apoptosis and/or necrosis, which was accompanied by intracellular increases in ROS levels and GSH depletion. Caspase inhibitors are suggested to suppress H2O2-induced oxidative stress to rescue HeLa cells at the early time point of 1 h.

Prefrontal cortical glutathione-dependent defense and proinflammatory mediators in chronically isolated rats: Modulation by fluoxetine or clozapine.

PubMed

Todorović, Nevena; Filipović, Dragana

2017-07-04

Chronic psychosocial stress modulates brain antioxidant systems and causes neuroinflammation that plays a role in the pathophysiology of depression. Although the antidepressant fluoxetine (FLX) represents the first-line treatment for depression and the atypical antipsychotic clozapine (CLZ) is considered as a second-line treatment for psychotic disorders, the downstream mechanisms of action of these treatments, beyond serotonergic or dopaminergic signaling, remain elusive. We examined behavioral changes, glutathione (GSH)-dependent defense and levels of proinflammatory mediators in the prefrontal cortex (PFC) of adult male Wistar rats exposed to 21days of chronic social isolation (CSIS). We also tested the ability of FLX (15mg/kg/day) or CLZ (20mg/kg/day), applied during CSIS, to prevent stress-induced changes. CSIS caused depressive- and anxiety-like behaviors, compromised GSH-dependent defense, and induced nuclear factor-kappa B (NF-κB) activation with a concomitant increase in cytosolic levels of proinflammatory mediators cyclooxigenase-2, interleukin-1beta and tumor necrosis factor-alpha in the PFC. NF-κB activation and proinflammatory response in the PFC were not found in CSIS rats treated with FLX or CLZ. In contrast, only FLX preserved GSH content in CSIS rats. CLZ not only failed to protect against CSIS-induced GSH depletion, but it diminished its levels when applied to non-stressed rats. In conclusion, prefrontal cortical GSH depletion and the proinflammatory response underlying depressive- and anxiety-like states induced by CSIS were prevented by FLX. The protective effect of CLZ, which was equally effective as FLX on the behavioral level, was limited to proinflammatory components. Hence, different mechanisms underlie the protective effects of these two drugs in CSIS rats. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

The protective effects of N-Acetl-cysteine, oxo-thiazolidine-carboxylate, acetaminophen and their combinations against sulfur mustard cytotoxicity on human skin fibroblast cell line (HF2FF).

PubMed

Saberi, Mehdi; Zaree Mahmodabady, Ali

2009-10-01

Using human skin-fibroblast cell line HF2FF, the efficacy of some drugs was evaluated against sulfur mustard (SM) cytotoxicity. The drugs were the sulfhydryl containing molecule including N-acetylcysteine (NAC), 2-oxo-thiazolidine-4-carboxylate (OTC) and acetaminophen as glutathione (GSH) stimulator pathway. The protective effects of NAC (0.1 mM), OTC (1.8 mM), and acetaminophen (25 mM) alone or in combination with each other were evaluated on SM (180 M)-induced cytotoxicity. NAC and OTC were applied with SM simultaneously and acetaminophen 30 min before SM exposure, incubated for 1 h and then were rinsed and incubated with fresh medium. The efficacy was evaluated by determination of cells viability, intracellular GSH level and catalase activity 1 and 24 h post SM exposure or co-treatments. The cells viability was decreased 21.8% and 55.2%, respectively for 1 and 24 h post SM (1 h exposure) incubation. So, the 1-h SM exposure and 24-h treatment incubation were selected for evaluation. While, NAC alone treatment increased the cells viability (25%), GSH level (320%) and catalase activity (18%), the most effective combination was NAC plus OTC and acetaminophen which increased more significantly the cells viability (about 40%), GSH level (470%) and catalase activity (100%). The most effective combination was NAC (0.1 mM) plus OTC (1.8 mM) and acetaminophen (25 mM) which should be used before or concomitant with SM exposure. These drugs may reduce SM toxicity possibly by increment of GSH level and catalase activity. This efficacy needs to be confirmed by in vivo study.

Modulation of endothelial GSH concentrations: effect of exogenous GSH and GSH monoethyl ester

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsan, M.F.; White, J.E.; Rosano, C.L.

We studied the effects of exogenous glutathione (GSH) and GSH monoethyl ester (GSH-MEE) on the enhancement of endothelial GSH concentrations. The preparation of GSH-MEE used contained 91% GSH-MEE, approximately 9% GSH diethyl ester (GSH-DEE) and a trace amount of GSH. Both GSH and GSH-MEE markedly stimulated the intracellular concentrations of GSH in endothelial cells. GSH-MEE was more potent than GSH. The enhancement of endothelial GSH concentration by exogenous GSH was completely inhibited by buthionine sulfoximine (BSO), a potent inhibitor of gamma-glutamylcysteine synthase, or acivicin (AT-125), an inhibitor of gamma-glutamyl transpeptidase, suggesting that it was due to the extracellular breakdown andmore » subsequent intracellular resynthesis of GSH. In contrast, the effect of GSH-MEE was largely resistant to BSO and acivicin, suggesting that it was primarily due to transport of GSH-MEE followed by intracellular hydrolysis. The GSH-MEE preparation, which contained 9% GSH-DEE, at concentrations of 2 mM or higher caused vacuolization of endothelial cells. The enhancement of GSH concentrations by exogenous GSH, but not by GSH-MEE, protected endothelial cells against H/sub 2/O/sub 2/-induced injury.« less

Methionine-supplemented diet affects the expression of cardiovascular disease-related genes and increases inflammatory cytokines in mice heart and liver.

PubMed

Aissa, Alexandre Ferro; Amaral, Catia Lira do; Venancio, Vinicius Paula; Machado, Carla da Silva; Hernandes, Lívia Cristina; Santos, Patrick Wellington da Silva; Curi, Rui; Bianchi, Maria de Lourdes Pires; Antunes, Lusânia Maria Greggi

2017-01-01

Some important environmental factors that influence the development of cardiovascular diseases (CVD) include tobacco, excess alcohol, and unhealthy diet. Methionine obtained from the diet participates in the synthesis of DNA, proteins, lipids and affects homocysteine levels, which is associated with the elevated risk for CVD development. Therefore, the aim of this study was to investigate the manner in which dietary methionine might affect cellular mechanisms underlying CVD occurrence. Swiss albino mice were fed either control (0.3% DL-methionine), methionine-supplemented (2% DL-methionine), or a methionine-deprived diet (0% DL-methionine) over a 10-week period. The parameters measured included plasma homocysteine concentrations, oxidative stress by reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, levels of inflammatory cytokines IL-1ß, TNF-α, and IL-6, as well as expression of genes associated with CVD. The levels of apolipoprotein A5 (APOA5), a regulator of plasma triglycerides, were measured. The methionine-supplemented diet increased oxidative stress by lowering the GSH/GSSG ratio in heart tissues and decreased expression of the genes Apob, Ctgf, Serpinb2, Spp1, Il1b, and Sell, but elevated expression of Thbs4, Tgfb2, Ccr1, and Vegfa. Methionine-deprived diet reduced expression of Col3a1, Cdh5, Fabp3, Bax, and Hbegf and increased expression of Sell, Ccl5, Itga2, Birc3, Msr1, Bcl2a1a, Il1r2, and Selp. Methionine-deprived diet exerted pro-inflammatory consequences as evidenced by elevated levels of cytokines IL-1ß, TNF-α, and IL-6 noted in liver. Methionine-supplemented diet increased hepatic IL-6 and cardiac TNF-α. Both methionine supplementation and deprivation lowered hepatic levels of APOA5. In conclusion, data demonstrated that a methionine-supplemented diet modulated important biological processes associated with high risk of CVD development.

Molecular and histological changes in cerebral cortex and lung tissues under the effect of tramadol treatment.

PubMed

Awadalla, Eatemad A; Salah-Eldin, Alaa-Eldin

2016-08-01

Tramadol abuse is one of the most frequent health problems in Egypt and worldwide. In most cases, tramadol abused by men face a problem with premature ejaculation. Tramadol like other opioids induces a decrease in plasma antioxidant levels, which may reflect a failure of the antioxidant defense mechanism against oxidative damage. The present work aimed to study the possible deleterious effects of oral administration of tramadol on brain and lung tissues in rats. Twenty adult male albino rats were divided into two groups; a control administered with normal saline and tramadol-treated (40mg/kg b.w.) group for 20 successive days. At the end of experimental period, blood was collected and specimens from brains and lungs were taken for histopathological and molecular studies. Malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activities were measured in serum of control and tramadol-treated groups. Brain and lung specimens were histopathological evaluated using light microscopy. The expression levels of apoptotic related genes; Bcl-2, Bax and Caspase-3 were study in brain and lung tissues using RT-PCR analysis. We recorded a significant increase MDA level, while antioxidant enzymes; GSH, SOD and CAT were significantly decreased after tramadol-treatment. The obtained results revealed that tramadol induced a remarkable histomorphological changes in rats' brains (cerebral cortex and hippocampus) and severe histopathological changes in rats' lung when compared to that of control. On molecular level, the expression of the pro-apoptotic Bax and Caspase-3 showed a significant increase whereas the anti-apoptotic Bcl-2 decreased markedly indicating that tramadol is harmful at cellular level and can induce apoptotic changes in brain tissues. Our data confirmed the risk of increased oxidative stress, neuronal and pulmonary damage due to tramadol abuse. Although tramadol is reported to be effective in pain management, its toxicity should be kept in mind. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

The Effect of L-Carnitine Treatment on Levels of Malondialdehyde and Glutathione in Patients with Age Related Macular Degeneration

PubMed Central

Ates, Orhan; Alp, H. Hakan; Mumcu, Ugur; Azizi, Sedat; Cinici, Emine; Kiziltunc, Ahmet; Baykal, Orhan

2008-01-01

Objective: The aim of this study was to determine the antioxidant properties of the L-carnitine (LC) in the treatment of patients with age-related macular degeneration (AMD). Materials and Methods: This study involved 60 patients diagnosed with early AMD. The patients were divided into two groups. Group I was the study group that received LC supplementation for 3 months. Group II was the control group and did not consent to LC supplementation over the 3 months. At the end of the 3-month period, markers of lipid peroxidation, malondialdehyde (MDA) and reduced glutathione (GSH) were measured in the two groups. Results: In the study group, the MDA level was significantly reduced, while the GSH level was significantly increased at the end of the 3-month period (P<0.001). Conclusion: Our results suggest that LC may protect against oxidative damage by decreasing the MDA level, a marker of lipid peroxidation, and increasing GSH. PMID:25610013

Hepatoprotective Flavonoids in Opuntia ficus-indica Fruits by Reducing Oxidative Stress in Primary Rat Hepatocytes.

PubMed

Kim, Jung Wha; Kim, Tae Bum; Kim, Hyun Woo; Park, Sang Wook; Kim, Hong Pyo; Sung, Sang Hyun

2017-01-01

Liver disorder was associated with alcohol consumption caused by hepatic cellular damages. Opuntia ficus-indica fruit extracts (OFIEs), which contain betalain pigments and polyphenols including flavonoids, have been introduced as reducing hangover symptoms and liver protective activity. To evaluate hepatoprotective activity of OFIEs and isolated compounds by high-speed countercurrent chromatography (HSCCC). The extract of O. ficus-indica fruits was fractionated into methylene chloride and n -butanol. The n -butanol fraction was isolated by HSCCC separation (methylene chloride-methanol- n -butanol-water, 5:4:3:5, v/v/v/v). The hepatoprotective activity of OFIEs and isolated compounds was evaluated on rat primary hepatocytes against ethanol-induced toxicity. Antioxidative parameters such as glutathione reductase and glutathione peroxidase (GSH-P x ) enzymes and the GSH content were measured. Two flavonoids, quercetin 3- O -methyl ester (1) and (+)-taxifolin, and two flavonoid glycosides, isorhamnetin 3- O -β- d -glucoside (3) and narcissin (4), were isolated from the n -butanol fraction by HSCCC separation. Among them, compound 2 significantly protected rat primary hepatocytes against ethanol exposure by preserving antioxidative properties of GR and GSH-P x . OFIEs and (+)-taxifolin were suggested to reduce hepatic damage by alcoholic oxidative stress. Hepatoprotective Flavonoids were isolated from Opuntia ficus-indica by high -speed countercurrent chromatography (HSCCC).

[Different effects of anticancer drugs on two human thyroid cell lines with different stages of differentiation].

PubMed

Yamanaka, T; Hishinuma, A

1995-01-20

We established two human thyroid tumor cell lines. One cell line (hPTC) was established from the tissue of a papillary thyroid carcinoma surgically excised from a 27-year-old female patient. The other cell line (hAG) was established from the tissue of an adenomatous goiter excised from a 59-year old female patient. Synthesis of cAMP by hPTC and hAG increased when they were stimulated by TSH. hPTC and hAG continued to divide as a monolayer in a tissue culture for three years and two years, respectively. We assessed the efficacy of anticancer drugs (doxorubicin:ADR, cisplatin:CDDP, nimustine:ACNU, bleomycin:BLM, cyclophosphamide:CPA, aclarubicin:ACR) with resard to hPTC. The hPTC cells were cultured in 24-well plates in the presence of the anticancer drugs for 48 hours, and the cellular DNA of the live cells was measured with diaminobenzoic acid. ADR had the lowest ED50 (0.029 mu g/ml) and the clinical blood concentration was 13.8 times that of the ED50. The clinical blood concentration divided by ED50 for the other anticancer drugs are, in order of higher values, 2.3 for CPA, 1.7 for BLM, 1.2 for CDDP, 0.5 for ACR, and less than 0.1 for ACNU. ADR showed time-independent effects since a 2-hour exposure of ADR to the hPTC cells resulted in the significant reduction of the cellular DNA content of the live cells even after 48 hours. The effects of the other anticancer drugs were time-dependent. We then studied the difference of the effects of ADR on hPTC and hAG. ED50 for hPTC was significantly low (0.035 mu g/ml) compared to that for hAG (0.460 mu g/ml). Since free radical formation is one of the major anticancer mechanisms of ADR the effects of free radicals on ED50's for hPTC and hAG were measured by adding glutathione (GSH), N-acetylcystein (NAC), buthionine sulfoximine (BSO), and alpha-tocopherol (alpha-toco) into the culture media. GSH catches up with free radicals in the extracellular fluid. NAC promotes production of GSH in the cytoplasm, but BSO interferes with the production of GSH in the cytoplasm. alpha-toco catches up with free radicals on the plasma membrane. GSH and alpha-toco did not effect ED50 for hPTC and hAG. However, NAC increased ED50 for hPTC and hAG, and BSO reduced ED50 for hPTC and hAG. The effects of NAC and BSO on ED50 for hPTC were greater than those for hAG.(ABSTRACT TRUNCATED AT 400 WORDS)

The evolution of glutathione metabolism in phototrophic microorganisms

NASA Technical Reports Server (NTRS)

Fahey, Robert C.; Buschbacher, Ralph M.; Newton, Gerald L.

1988-01-01

The low molecular weight thiol composition of a variety of phototropic microorganisms is examined in order to ascertain how evolution of glutathione (GSH) production is related to the evolution of oxygenic photosynthesis. Cells were extracted in the presence of monobromobimane (mBBr) to convert thiols (RSH) to fluorescent derivatives (RSmB) which were analyzed by high performance liquid chromatography (HPLC). Significant levels of GSH were not found in green sulfur bacteria. Substantial levels were present in purple bacteria, cyanobacteria, and eukaryotic algae. Other thiols measured included cysteine, gamma-glutamylcysteine, thiosulfate, coenzyme A, and sulfide. Many of the organisms also exhibited a marked ability to reduce mBBr to syn-(methyl,methyl)bimane, an ability which was quenched by treatment with 2-pyridyl disulfide or 5,5 prime-bisdithio - (2-nitrobenzoic acid) prior to reaction with mBBr. These observations indicate the presence of a reducing system capable of electron transfer to mBBr and reduction of reactive disulfides. The distribution of GSH in phototropic eubacteria indicates that GSH synthesis evolved at or around the time that oxygenic photosynthesis evolved.

Enhanced Neuroprotective Effects of Coadministration of Tetrandrine with Glutathione in Preclinical Model of Parkinson's Disease

PubMed Central

Li, Xiang-Yun; Mei, Guang-Hai; Dong, Qiang; Zhang, Yu; Guo, Zhuang-Li; Su, Jing-Jing; Tang, Yu-Ping; Jin, Xue-Hong; Zhou, Hou-Guang; Huang, Yan-Yan

2015-01-01

Aim. In this study we examined the influence of tetrandrine (Tet) on the neuroprotective effects of glutathione (GSH) in the 6-hydroxydopamine- (6-OHDA-) lesioned rat model of Parkinson's disease (PD). Methods. Levels in the redox system, dopamine (DA) metabolism, dopaminergic neuronal survival, and apoptosis of the substantia nigra (SN) and striatum, as well as the rotational behavior of animals were examined after a 50-day administration of GSH + Tet (or GSH) and/or L-3,4-dihydroxyphenylalanine (L-dopa) to PD rats. Ethics Committee of Huashan Hospital, Fudan University approved the protocol (number SYXK2009-0082). Results. Administration of GSH or Tet alone did not show any significant effects on the factors evaluated in the PD rats. However, in the GSH + Tet group, we observed markedly decreased oxidative damage, inhibition of DA metabolism and enhanced DA synthesis, increased tyrosine hydroxylase- (TH-) immunopositive neuronal survival, and delayed apoptosis of dopaminergic neurons in the SN. Animal rotational behavior was improved in the GSH + Tet group. Additionally, coadministration of GSH + Tet appeared to offset the possible oxidative neurotoxicity induced by L-dopa. Conclusion. In this study, we demonstrated that tetrandrine allowed occurrence of the neuroprotective effect of glutathione probably due to inhibition of P-glycoprotein on 6-hydroxydopamine-lesioned rat models of Parkinson's disease, including rats undergoing long-term L-dopa treatment. PMID:26664824

Supramolecular interactions of oxidative stress biomarker glutathione with fluorone black.

PubMed

Hepel, Maria; Stobiecka, Magdalena

2018-03-05

Oxidative stress biomarkers, including glutathione (GSH) and related compounds, are involved in a variety of interactions enabling redox potential maintenance in living cells and protection against radicals. Since the oxidative stress is promoting and, in many cases, inducing serious illnesses, monitoring of GSH levels can aid in diagnostics and disease prevention. Herein, we report on the discovery of the formation of a supramolecular ensemble of GSH with fluorone black (9-phenyl fluorone, FB) which is optically active and enables sensitive determination of GSH by resonance elastic light scattering (RELS). We have found that supramolecular interactions of GSH with FB can be probed with spectroscopic, RELS, and electrochemical methods. Our investigations show that RELS intensity for FB solutions increases with GSH concentration while fluorescence emission of FB is not affected, as quenching begins only above 0.8mM GSH. The UV-Vis difference spectra show a positive peak at 383nm and a negative peak at 458nm, indicating a higher-energy absorbing complex in comparison to the non-bonded FB host. Supramolecular interactions of FB with GSH have also been corroborated by electrochemical measurements involving two configurations of FB-GSH ensembles on electrodes: (i) an inverted orientation on Au-coated quartz crystal piezoelectrode (Au@SG-FB), with strong thiolate bonding to gold, and (ii) a non-inverted orientation on glassy carbon electrode (GCE@FB-GS), with weak π-π stacking attachment and efficient charge mediation through the ensemble. The formation of a supramolecular ensemble with hydrogen bonding has also been confirmed by quantum mechanical calculations. The discovery of supramolecular FB-GSH ensemble formation enables elucidating the mechanisms of strong RELS responses, changes in UV-Vis absorption spectra, and the electrochemical reactivity. Also, it provides new insights to the understanding of the efficient charge-transfer in redox potential homeostasis which is likely based on an intermediate formation of a similar type of supramolecular ensembles. Copyright © 2017 Elsevier B.V. All rights reserved.

Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells

PubMed Central

Freitas, Hercules R.; Ferraz, Gabriel; Ferreira, Gustavo C.; Ribeiro-Resende, Victor T.; Chiarini, Luciana B.; do Nascimento, José Luiz M.; Matos Oliveira, Karen Renata H.; Pereira, Tiago de Lima; Ferreira, Leonardo G. B.; Kubrusly, Regina C.; Faria, Robson X.

2016-01-01

Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1–10mM) showed that 5–10mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50mM KCl (labeled as βIII tubulin positive cells). BBG 100nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70μM and MK-801 20μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit. PMID:27078878

Supramolecular interactions of oxidative stress biomarker glutathione with fluorone black

NASA Astrophysics Data System (ADS)

Hepel, Maria; Stobiecka, Magdalena

2018-03-01

Oxidative stress biomarkers, including glutathione (GSH) and related compounds, are involved in a variety of interactions enabling redox potential maintenance in living cells and protection against radicals. Since the oxidative stress is promoting and, in many cases, inducing serious illnesses, monitoring of GSH levels can aid in diagnostics and disease prevention. Herein, we report on the discovery of the formation of a supramolecular ensemble of GSH with fluorone black (9-phenyl fluorone, FB) which is optically active and enables sensitive determination of GSH by resonance elastic light scattering (RELS). We have found that supramolecular interactions of GSH with FB can be probed with spectroscopic, RELS, and electrochemical methods. Our investigations show that RELS intensity for FB solutions increases with GSH concentration while fluorescence emission of FB is not affected, as quenching begins only above 0.8 mM GSH. The UV-Vis difference spectra show a positive peak at 383 nm and a negative peak at 458 nm, indicating a higher-energy absorbing complex in comparison to the non-bonded FB host. Supramolecular interactions of FB with GSH have also been corroborated by electrochemical measurements involving two configurations of FB-GSH ensembles on electrodes: (i) an inverted orientation on Au-coated quartz crystal piezoelectrode (Au@SG-FB), with strong thiolate bonding to gold, and (ii) a non-inverted orientation on glassy carbon electrode (GCE@FB-GS), with weak π-π stacking attachment and efficient charge mediation through the ensemble. The formation of a supramolecular ensemble with hydrogen bonding has also been confirmed by quantum mechanical calculations. The discovery of supramolecular FB-GSH ensemble formation enables elucidating the mechanisms of strong RELS responses, changes in UV-Vis absorption spectra, and the electrochemical reactivity. Also, it provides new insights to the understanding of the efficient charge-transfer in redox potential homeostasis which is likely based on an intermediate formation of a similar type of supramolecular ensembles.

Differences in oxidative stress between young Canada geese and mallards exposed to lead-contaminated sediment

USGS Publications Warehouse

Mateo, R.; Hoffman, D.J.

2001-01-01

Lead (Pb) exposure causes an increase in tissue lipid peroxides and variation in glutathione (GSH) concentration, which can be related to peroxidative damage of cell membranes in Pb poisoned animals. Species and individual variation in sensitivity to Pb poisoning among animals may be due to differential resistance to oxidative stress. We compared the effects of oxidative stress caused by Pb exposure (1.7, 414 and 828 ig/g of diet) for the first six weeks in growing young of two species of waterfowl, Canada geese (Branta canadensis) and mallards (Anas platyrhynchos), with the first species being possibly more sensitive to Pb poisoning based on previous field and laboratory observations. Blood and liver Pb concentrations increased more in mallards than in geese; this may be explained on the basis of body weight, being 3.2 times higher in geese, and hepatic metabolism where GSH-S-transferase activity is 2.9 fold higher in geese and presumably has a role in the binding of Pb to GSH and subsequent biliary excretion. In contrast, mallards showed higher hepatic levels of GSH and activities of GSH peroxidase (GPX) and GSH reductase (GR). Although both species showed an increase in hepatic GSH concentration with Pb exposure, the increase of lipid peroxidation with Pb exposure was more significant in geese. Within treatment groups, hepatic GSH concentrations were inversely related to liver Pb concentration in both species, which may correspond to the role of GSH in Pb excretion. Hepatic GSH was also inversely related to hepatic lipid peroxidation, but only in mallards and in agreement with the differences observed in GPX and GR activities. The lower resistance to lipid peroxidation of Canada geese may explain why birds of this species found dead in the field by Pb shot ingestion often have a lower number of shot in the gizzard and lower liver Pb concentrations than mallards.

Haloperidol, a sigma receptor 1 antagonist, promotes ferroptosis in hepatocellular carcinoma cells.

PubMed

Bai, Tao; Wang, Shuai; Zhao, Yipu; Zhu, Rongtao; Wang, Weijie; Sun, Yuling

2017-09-30

Ferroptosis is a novel form of cell death, which is characterized by accumulation of reactive oxygen species (ROS). Sigma 1 receptor (S1R) has been suggested to function in oxidative stress metabolism. Both erastin and sorafenib significantly induced S1R protein expression. Haloperidol strongly promoted erastin- and sorafenib-induced cell death, which was blocked by ferrostatin-1 but not ZVAD-FMK or necrosulfonamide. During ferroptosis, haloperidol substantially increased the cellular levels of Fe 2+ , GSH and lipid peroxidation. Furthermore, several ferroptosis-related protein targets were up-regulated in the absence of haloperidol. Thus, Our study identified an association between haloperidol and ferroptosis for the first time. Our analyses of a combination of drugs may provide a novel strategy of hepatocellular carcinoma (HCC) therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

The glutathione defense system in the pathogenesis of rheumatoid arthritis.

PubMed

Hassan, M Q; Hadi, R A; Al-Rawi, Z S; Padron, V A; Stohs, S J

2001-01-01

In order to assess a possible role of the natural glutathione defense system in the pathogenesis of rheumatoid arthritis (RA), serum reduced glutathione levels (GSH), glutathione reductase (GSR), glutathione S-transferase (GST), glutathione peroxidase (GSH-Px) and alkaline phosphatase (ALP) activities, lipid peroxidation (MDA content) and indexes of inflammation were evaluated in 58 rheumatic patients. Rheumatoid athritis was associated with significant depletion (ca. 50%) in GSH levels compared with normal control subjects. Serum levels of the detoxifying enzymes GSR and GSH-Px decreased by ca. 50% and 45%, respectively, whereas a threefold increase in the activity of GST was observed. A 1.2-fold increase in ALP was observed in patients with RA. These effects were accompanied by a 3.1-fold increase in serum MDA content. The MDA content was higher in RA patients who were seropositive for rheumatoid factor as well as positive for C-reactive proteins. The erythrocyte sedimentation rate for all patients with RA was approximately 13.8-fold higher than for the control group, and was higher among RA patients who were positive for C-reactive proteins and exhibited seropositivity for rheumatoid factor. Patients with RA receiving gold therapy exhibited significantly lower MDA levels whereas all other factors that were measured were not effected. The results support a hypothesis that defense mechanisms against reactive oxygen species are impaired in RA. Copyright 2001 John Wiley & Sons, Ltd.

Low Nourishment of Vitamin C Induces Glutathione Depletion and Oxidative Stress in Healthy Young Adults.

PubMed

Waly, Mostafa I; Al-Attabi, Zahir; Guizani, Nejib

2015-09-01

The present study was conducted to assess the status of vitamin C among healthy young adults in relation to serum antioxidant parameters [glutathione (GSH), thiols, and total antioxidant capacity, (TAC)], and oxidative stress markers [malondialdehyde (MDA), and nitrites plus nitrates (NN)]. A prospective study included 200 young adults, and their dietary intake was assessed by using food diaries. Fasting plasma vitamin C, serum levels of GSH, thiols, TAC, MDA, and NN were measured using biochemical assays. It was observed that 38% of the enrolled subjects, n=76, had an adequate dietary intake of vitamin C (ADI group). Meanwhile, 62%, n=124, had a low dietary intake of vitamin C (LDI group) as compared to the recommended dietary allowances. The fasting plasma level of vitamin C was significantly higher in the ADI group as compared to the LDI group. Oxidative stress in the sera of the LDI group was evidenced by depletion of GSH, low thiols levels, impairment of TAC, an elevation of MDA, and increased NN. In the ADI group, positive correlations were found between plasma vitamin C and serum antioxidant parameters (GSH, thiols, and TAC). Meanwhile, the plasma vitamin C was negatively correlated with serum MDA and NN levels. This study reveals a significant increase of oxidative stress status and reduced antioxidant capacity in sera from healthy young adults with low intake of the dietary antioxidant, vitamin C.

Transcriptional profile of genes involved in ascorbate glutathione cycle in senescing leaves for an early senescence leaf (esl) rice mutant.

PubMed

Li, Zhaowei; Su, Da; Lei, Bingting; Wang, Fubiao; Geng, Wei; Pan, Gang; Cheng, Fangmin

2015-03-15

To clarify the complex relationship between ascorbate-glutathione (AsA-GSH) cycle and H2O2-induced leaf senescence, the genotype-dependent difference in some senescence-related physiological parameters and the transcript levels and the temporal patterns of genes involved in the AsA-GSH cycle during leaf senescence were investigated using two rice genotypes, namely, the early senescence leaf (esl) mutant and its wild type. Meanwhile, the triggering effect of exogenous H2O2 on the expression of OsAPX genes was examined using detached leaves. The results showed that the esl mutant had higher H2O2 level than its wild type at the initial stage of leaf senescence. At transcriptional level, the association of expression of various genes involved in the AsA-GSH cycle with leaf senescence was isoform dependent. For OsAPXs, the transcripts of two cytosolic OsAPX genes (OsAPX1 and OsAPX2), thylakoid-bound OsAPX8, chloroplastic OsAPX7 and peroxisomal OsAPX4 exhibited remarkable genotype-dependent variation in their expression levels and temporal patterns during leaf senescence, there were significantly increasing transcripts of OsAXP1 and OsAPX7, severely repressed transcripts of OsAPX4 and OsAPX8 for the esl rice at the initial leaf senescence. In contrast, the repressing transcript of OsAPX8 was highly sensitive to the increasing H2O2 level in the senescing rice leaves, while higher H2O2 concentration resulted in the enhancing transcripts of two cytosolic OsAPX genes, OsAPX7 transcript was greatly variable with different H2O2 concentrations and incubating duration, suggesting that the different OsAPXs isoforms played a complementary role in perceiving and scavenging H2O2 accumulation at various H2O2 concentrations during leaf senescence. Higher H2O2 level, increased AsA level, higher activities of APX and glutathione reductase (GR), and relatively stable GSH content during the entire sampling period in the leaves of esl mutant implied that a close interrelationship existed between AsA level and APX activity in the ongoing senescence of rice leaves. The GSH supply in rice leaves was not the limiting factor for the efficient maintenance of AsA-GSH cycle, despite the senescence-related change in GR activity between the two rice genotypes. Copyright © 2014 Elsevier GmbH. All rights reserved.

Glutathione-deficient Plasmodium berghei parasites exhibit growth delay and nuclear DNA damage.

PubMed

Padín-Irizarry, Vivian; Colón-Lorenzo, Emilee E; Vega-Rodríguez, Joel; Castro, María Del R; González-Méndez, Ricardo; Ayala-Peña, Sylvette; Serrano, Adelfa E

2016-06-01

Plasmodium parasites are exposed to endogenous and exogenous oxidative stress during their complex life cycle. To minimize oxidative damage, the parasites use glutathione (GSH) and thioredoxin (Trx) as primary antioxidants. We previously showed that disruption of the Plasmodium berghei gamma-glutamylcysteine synthetase (pbggcs-ko) or the glutathione reductase (pbgr-ko) genes resulted in a significant reduction of GSH in intraerythrocytic stages, and a defect in growth in the pbggcs-ko parasites. In this report, time course experiments of parasite intraerythrocytic development and morphological studies showed a growth delay during the ring to schizont progression. Morphological analysis shows a significant reduction in size (diameter) of trophozoites and schizonts with increased number of cytoplasmic vacuoles in the pbggcs-ko parasites in comparison to the wild type (WT). Furthermore, the pbggcs-ko mutants exhibited an impaired response to oxidative stress and increased levels of nuclear DNA (nDNA) damage. Reduced GSH levels did not result in mitochondrial DNA (mtDNA) damage or protein carbonylations in neither pbggcs-ko nor pbgr-ko parasites. In addition, the pbggcs-ko mutant parasites showed an increase in mRNA expression of genes involved in oxidative stress detoxification and DNA synthesis, suggesting a potential compensatory mechanism to allow for parasite proliferation. These results reveal that low GSH levels affect parasite development through the impairment of oxidative stress reduction systems and damage to the nDNA. Our studies provide new insights into the role of the GSH antioxidant system in the intraerythrocytic development of Plasmodium parasites, with potential translation into novel pharmacological interventions. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Thiols decrease cytokine levels and down-regulate the expression of CD30 on human allergen-specific T helper (Th) 0 and Th2 cells

PubMed Central

Bengtsson, Å; Lundberg, M; Avila-Cariño, J; Jacobsson, G; Holmgren, A; Scheynius, A

2001-01-01

The thiol antioxidant N-acetyl-l-cysteine (NAC), known as a precursor of glutathione (GSH), is used in AIDS treatment trials, as a chemoprotectant in cancer chemotherapy and in treatment of chronic bronchitis. In vitro, GSH and NAC are known to enhance T cell proliferation, production of IL-2 and up-regulation of the IL-2 receptor. The 120-kD CD30 surface antigen belongs to the tumour necrosis factor (TNF) receptor superfamily. It is expressed by activated T helper (Th) cells and its expression is sustained in Th2 cells. We have analysed the effect of GSH and NAC on the cytokine profile and CD30 expression on human allergen-specific T cell clones (TCC). TCC were stimulated with anti-CD3 antibodies in the presence of different concentrations of GSH and NAC. Both thiols caused a dose dependent down-regulation of IL-4, IL-5 and IFN-γ levels in Th0 and Th2 clones, with the most pronounced decrease of IL-4. Furthermore, they down-regulated the surface expression of CD30, and the levels of soluble CD30 (sCD30) in the culture supernatants were decreased. In contrast, the surface expression of CD28 or CD40 ligand (CD40L) was not significantly changed after treatment with 20 mm NAC. These results indicate that GSH and NAC favour a Th1 response by a preferential down-regulation of IL-4. In addition, the expression of CD30 was down regulated by GSH and NAC, suggesting that CD30 expression is dependent on IL-4, or modified by NAC. In the likely event that CD30 and its soluble counterpart prove to contribute to the pathogenesis in Th2 related diseases such as allergy, NAC may be considered as a future therapeutic agent in the treatment of these diseases. PMID:11298119

H2O2-induced mild stress in relation with in vitro ovine oocyte developmental competence: implications for blastocyst apoptosis and related genes expression.

PubMed

Nikdel, K; Aminafshar, M; Mohammadi-Sangcheshmeh, A; EmamJomeh-Kashan, N; Seyedjafari, E

2017-05-20

In this study, in vitro maturation was performed in presence of various concentrations (0, 10, 100, or 1000 µM) of H2O2. The intracellular glutathione (GSH) level, fertilization, cleavage, and blastocyst rates, total cell number, and apoptotic cell number and expression of Bax, Bcl-2, and p53 genes in blastocyst-stage embryos were studied. At 10 μM H2O2 concentration, a higher GSH level was detected in comparison to the other groups while oocytes exposed to 1000 μM H2O2 had the lowest GSH level. Treatment of oocytes with 1000 μM H2O2 decreased the rate of two pronuclei formation as compared with other groups. A higher rate of blastocyst formation was seen in 100 μM H2O2 group as compared with the control group. However, exogenous H2O2 in maturation medium did not affect total cell numbers and apoptotic cell ratio at the blastocyst stage. Moreover, mRNA transcript abundance of Bax, Bcl-2, and p53 genes was similar between blastocysts derived from H2O2-induced oocytes and control blastocysts. Treatment of oocytes with H2O2 at mild level during in vitro maturation had a positive effect on GSH level and this, in turn, may lead to improvement in preimplantation embryonic development.

Metabolomics changes in a rat model of obstructive jaundice: mapping to metabolism of amino acids, carbohydrates and lipids as well as oxidative stress.

PubMed

Long, Yue; Dong, Xin; Yuan, Yawei; Huang, Jinqiang; Song, Jiangang; Sun, Yumin; Lu, Zhijie; Yang, Liqun; Yu, Weifeng

2015-07-01

The study examined the global metabolic and some biochemical changes in rats with cholestasis induced by bile duct ligation (BDL). Serum samples were collected in male Wistar rats with BDL (n = 8) and sham surgery (n = 8) at day 3 after surgery for metabolomics analysis using a combination of reversed phase chromatography and hydrophilic interaction chromatography (HILIC) and quadrupole-time-of-flight mass spectrometry (Q-TOF MS). The serum levels of malondialdehyde (MDA), total antioxidative capacity (T-AOC), glutathione (GSH) and glutathione disulfide (GSSG), the activities of superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px) were measured to estimate the oxidative stress state. Key changes after BDL included increased levels of l-phenylalanine, l-glutamate, l-tyrosine, kynurenine, l-lactic acid, LysoPC(c) (14:0), glycine and succinic acid and decreased levels of l-valine, PC(b) (19:0/0:0), taurine, palmitic acid, l-isoleucine and citric acid metabolism products. And treatment with BDL significantly decreased the levels of GSH, T-AOC as well as SOD, GSH-Px activities, and upregulated MDA levels. The changes could be mapped to metabolism of amino acids and lipids, Krebs cycle and glycolysis, as well as increased oxidative stress and decreased antioxidant capability. Our study indicated that BDL induces major changes in the metabolism of all 3 major energy substances, as well as oxidative stress.

Hepatic reduction of carbamoyl-PROXYL in ferric nitrilotriacetate induced iron overloaded mice: an in vivo ESR study.

PubMed

Morales, Noppawan Phumala; Yamaguchi, Yumiko; Murakami, Kimiyo; Kosem, Nuttavut; Utsumi, Hideo

2012-01-01

Reduction of a nitroxyl radical, carbamoyl-PROXYL in association of free radical production and hepatic glutathione (GSH) was investigated in iron overloaded mice using an in vivo L-band electron spin resonance (ESR) spectrometer. Significant increases in hepatic iron, lipid peroxidation and decrease in hepatic GSH were observed in mice intraperitoneally (i.p.) administrated with ferric nitrilotriacetate (Fe(III)-NTA, a total 45 µmol/mouse over a period of 3 weeks). Free radical production in iron overloaded mice was evidenced by significantly enhanced rate constant of ESR signal decay of carbamoyl-PROXYL, which was slightly reduced by treatment with iron chelator, deferoxamine. Moreover, the rate constant of ESR signal decay was negatively correlated with hepatic GSH level (r=-0.586, p<0.001). On the other hand, hepatic GSH-depletion (>80%) in mice through daily i.p. injection and drinking water supplementation of L-buthionine-[S,R]-sulfoximine (BSO) significantly retarded ESR signal decay, while there were no changes in serum aspartate aminotransferase and liver thiobarbituric acid-reactive substances levels. In conclusion, GSH plays two distinguish roles on ESR signal decay of carbamoyl-PROXYL, as an antioxidant and as a reducing agent, dependently on its concentration. Therefore, it should be taken into account in the interpretation of free radical production in each specific experimental setting.

Organophosphate pesticides-induced changes in the redox status of rat tissues and protective effects of antioxidant vitamins.

PubMed

Mishra, Vibhuti; Srivastava, Nalini

2015-04-01

Organophosphates (OPs) pesticides are among the most toxic synthetic chemicals purposefully added in the environment. The common use of OP insecticides in public health and agriculture results in an environmental pollution and a number of acute and chronic poisoning events. Present study was aimed to evaluate the potential of monocrotophos and quinalphos to effect the redox status and glutathione (GSH) homeostasis in rat tissues and find out whether antioxidant vitamins have some protection on the pesticide-induced alterations. The results showed that these pesticides alone or in combination, caused decrease in the levels of GSH and the corresponding increase in the levels of GSSG, decreasing the GSH/GSSG ratio. The results also showed that NADPH/NADP(+) and NADH/NAD(+) ratios were decreased in the liver and brain of rats on exposure with mococrotophos, quinalphos, and their mixture. These pesticides, alone or in combination, caused alterations in the activities of GSH reductase and glucose-6-phosphate dehydrogenase in the rat tissues. However, the expression of the GSH recycling enzymes did not show significant alterations as compared to control. From the results, it can be concluded that these pesticides generate oxidative stress but their effects were not synergistic when given together and prior feeding of antioxidant vitamins tend to reduce the toxicities of these pesticides. Copyright © 2013 Wiley Periodicals, Inc.

Transient glutathione depletion in the substantia nigra compacta is associated with neuroinflammation in rats.

PubMed

Díaz-Hung, Mei-Li; Yglesias-Rivera, Arianna; Hernández-Zimbrón, Luis Fernando; Orozco-Suárez, Sandra; Ruiz-Fuentes, Jenny Laura; Díaz-García, Alexis; León-Martínez, Rilda; Blanco-Lezcano, Lisette; Pavón-Fuentes, Nancy; Lorigados-Pedre, Lourdes

2016-10-29

Glutathione (GSH) deficiency has been identified as an early event in the progression of Parkinson's disease. However, the role of GSH in the etiology and pathogenesis of this neurodegenerative disorder is not well established. The aim of this study is to assess the effect of transient GSH depletion in the substantia nigra pars compacta (SNpc) on neuroinflammation after the injection of a single dose of l-buthionine sulfoximine (BSO) into the SNpc of male Sprague-Dawley rats. The results showed that BSO treatment stimulates microglia (p<0.01) and astroglial response (p<0.01), c-Jun N-terminal kinase and inducible nitric oxide synthase (iNOS) (p<0.001) in the SNpc, accompanied by dopaminergic dysfunction. In addition, high levels of tumor necrosis factor α (p<0.01), interleukins IL-1β p<0.01), IL-6 p<0.001) and nitric oxide p<0.01) were found in the treated animals compared to control groups, while no significant differences were found in IL-10 levels. These results suggest that transient GSH depletion can increase the susceptibility of SNpc to degeneration by promoting an inflammatory response and nitrosative stress, reinforcing the possible role of GSH unbalance, oxygen/nitrogen reactive species and neuroinflammation as causal factors on the degeneration of the SNpc. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Oxidation of DJ-1 Induced by 6-Hydroxydopamine Decreasing Intracellular Glutathione

PubMed Central

Miyama, Akiko; Saito, Yoshiro; Yamanaka, Kazunori; Hayashi, Kojiro; Hamakubo, Takao; Noguchi, Noriko

2011-01-01

DJ-1, the causative gene of a familial form of Parkinson's disease (PD), has been reported to undergo preferential oxidation of the cysteine residue at position 106 (Cys-106) under oxidative stress; however, details of the molecular mechanisms are not well known. In the present study, mechanisms of DJ-1 oxidation induced by 6-hydroxydopamine (6-OHDA) were investigated by using SH-SY5Y cells. The treatment of these cells with 6-OHDA caused an obvious acidic spot sift of DJ-1 due to its oxidation. However, when catalase, which is an hydrogen peroxide (H2O2)-removing enzyme, was added during the treatment, it failed to prevent the oxidation induced by 6-OHDA, suggesting that electrophilic p-quinone formed from 6-OHDA, but not H2O2, was responsible for the DJ-1 oxidation. Benzoquinone, another electrophilic p-quinone, also induced DJ-1 oxidation. The intracellular glutathione (GSH) levels were significantly decreased by 6-OHDA, irrespective of the presence or absence of catalase. The inhibition of GSH synthesis by buthionine sulfoximine resulted in a decrease in GSH levels and enhancement of DJ-1 oxidation. The pretreatment of cells with N-acetyl-cysteine prevented the loss of intracellular GSH and subsequently DJ-1 oxidation induced by 6-OHDA. Collectively, these results suggest that electrophilic p-quinone formed from 6-OHDA induces DJ-1 oxidation by decreasing intracellular GSH. PMID:22132160

Reduced glutathione and procaine hydrochloride protect the nucleoprotein structure of boar spermatozoa during freeze-thawing by stabilising disulfide bonds.

PubMed

Yeste, Marc; Flores, Eva; Estrada, Efrén; Bonet, Sergi; Rigau, Teresa; Rodríguez-Gil, Joan E

2013-01-01

One important change the head of boar spermatozoa during freeze-thawing is the destabilisation of its nucleoprotein structure due to a disruption of disulfide bonds. With the aim of better understanding these changes in frozen-thawed spermatozoa, two agents, namely reduced glutathione (GSH) and procaine hydrochloride (ProHCl), were added at different concentrations to the freezing media at different concentrations and combinations over the range 1-2mM. Then, 30 and 240 min after thawing, cysteine-free residue levels of boar sperm nucleoproteins, DNA fragmentation and other sperm functional parameters were evaluated. Both GSH and ProHCl, at final concentrations of 2mM, induced a significant (P<0.05) increase in the number of non-disrupted sperm head disulfide bonds 30 and 240 min after thawing compared with the frozen-thawed control. This effect was accompanied by a significant (P<0.05) decrease in DNA fragmentation 240 min after thawing. Concomitantly, 1 and 2mM GSH, but not ProHCl at any of the concentrations tested, partially counteracted the detrimental effects caused by freeze-thawing on sperm peroxide levels, motility patterns and plasma membrane integrity. In conclusion, the results show that both GSH and ProHCl have a stabilising effect on the nucleoprotein structure of frozen-thawed spermatozoa, although only GSH exerts an appreciable effect on sperm viability.

Cytotoxicity Mechanism of Two Naphthoquinones (Menadione and Plumbagin) in Saccharomyces cerevisiae

PubMed Central

Castro, Frederico Augusto Vieira; Mariani, Diana; Panek, Anita Dolly; Eleutherio, Elis Cristina Araújo; Pereira, Marcos Dias

2008-01-01

Background Quinones are compounds extensively used in studies of oxidative stress due to their role in plants as chemicals for defense. These compounds are of great interest for pharmacologists and scientists, in general, because several cancer chemotherapeutic agents contain the quinone nucleus. However, due to differences in structures and diverse pharmacological effects, the exact toxicity mechanisms exerted by quinones are far from elucidatation. Methodology/Principal Findings Using Saccharomyces cerevisiae, we evaluated the main mechanisms of toxicity of two naphthoquinones, menadione and plumbagin, by determining tolerance and oxidative stress biomarkers such as GSH and GSSG, lipid peroxidation levels, as well as aconitase activity. The importance of glutathione transferases (GST) in quinone detoxification was also addressed. The GSSG/GSH ratio showed that menadione seemed to exert its toxicity mainly through the generation of ROS while plumbagin acted as an electrophile reacting with GSH. However, the results showed that, even by different pathways, both drugs were capable of generating oxidative stress through their toxic effects. Our results showed that the control strain, BY4741, and the glutathione transferase deficient strains (gtt1Δ and gtt2Δ) were sensitive to both compounds. With respect to the role of GST isoforms in cellular protection against quinone toxicity, we observed that the Gtt2 deficient strain was unable to overcome lipid peroxidation, even after a plumbagin pre-treatment, indicating that this treatment did not improve tolerance when compared with the wild type strain. Cross-tolerance experiments confirmed distinct cytotoxicity mechanisms for these naphthoquinones since only a pre-treatment with menadione was able to induce acquisition of tolerance against stress with plumbagin. Conclusions/Significance These results suggest different responses to menadione and plumbagin which could be due to the fact that these compounds use different mechanisms to exert their toxicity. In addition, the Gtt2 isoform seemed to act as a general protective factor involved in quinone detoxification. PMID:19098979

N-acetylcysteine attenuates TNF-α-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells

PubMed Central

Hashimoto, Shu; Gon, Yasuhiro; Matsumoto, Ken; Takeshita, Ikuko; Horie, Takashi

2001-01-01

We have previously shown that tumour necrosis factor-α (TNF-α) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H2O2 generated by TNF-α can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-α-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-α-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-α and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. Intracellular GSH levels increased in NAC-treated cells. NAC attenuated TNF-α-induced activation of p38 MAP kinase and MKK3/MKK6. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-α-stimulated cells. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-α-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury. PMID:11156586

N-acetylcysteine attenuates TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells.

PubMed

Hashimoto, S; Gon, Y; Matsumoto, K; Takeshita, I; Horie, T

2001-01-01

1. We have previously shown that tumour necrosis factor-alpha (TNF-alpha) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H(2)O(2) generated by TNF-alpha can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-alpha-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. 2. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-alpha and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. 3. Intracellular GSH levels increased in NAC-treated cells. 4. NAC attenuated TNF-alpha-induced activation of p38 MAP kinase and MKK3/MKK6. 5. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-alpha-stimulated cells. 6. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury.

Role of aramchol in steatohepatitis and fibrosis in mice

PubMed Central

Iruarrizaga‐Lejarreta, Marta; Varela‐Rey, Marta; Fernández‐Ramos, David; Martínez‐Arranz, Ibon; Delgado, Teresa C; Simon, Jorge; Gutiérrez‐de Juan, Virginia; delaCruz‐Villar, Laura; Azkargorta, Mikel; Lavin, José L.; Mayo, Rebeca; Van Liempd, Sebastiaan M.; Aurrekoetxea, Igor; Buqué, Xabier; Delle Cave, Donatella; Peña, Arantza; Rodríguez‐Cuesta, Juan; Aransay, Ana M.; Elortza, Felix; Falcón‐Pérez, Juan M.; Aspichueta, Patricia; Hayardeny, Liat; Noureddin, Mazen; Sanyal, Arun J.; Alonso, Cristina; Anguita, Juan; Martínez‐Chantar, María Luz; Lu, Shelly C.

2017-01-01

Nonalcoholic steatohepatitis (NASH) is the advanced form of nonalcoholic fatty liver disease (NAFLD) that sets the stage for further liver damage. The mechanism for the progression of NASH involves multiple parallel hits, including oxidative stress, mitochondrial dysfunction, inflammation, and others. Manipulation of any of these pathways may be an approach to prevent NASH development and progression. Arachidyl‐amido cholanoic acid (Aramchol) is presently in a phase IIb NASH study. The aim of the present study was to investigate Aramchol's mechanism of action and its effect on fibrosis using the methionine‐ and choline‐deficient (MCD) diet model of NASH. We collected liver and serum from mice fed an MCD diet containing 0.1% methionine (0.1MCD) for 4 weeks; these mice developed steatohepatitis and fibrosis. We also collected liver and serum from mice receiving a control diet, and metabolomes and proteomes were determined for both groups. The 0.1MCD‐fed mice were given Aramchol (5 mg/kg/day for the last 2 weeks), and liver samples were analyzed histologically. Aramchol administration reduced features of steatohepatitis and fibrosis in 0.1MCD‐fed mice. Aramchol down‐regulated stearoyl‐coenyzme A desaturase 1, a key enzyme involved in triglyceride biosynthesis and the loss of which enhances fatty acid β‐oxidation. Aramchol increased the flux through the transsulfuration pathway, leading to a rise in glutathione (GSH) and the GSH/oxidized GSH ratio, the main cellular antioxidant that maintains intracellular redox status. Comparison of the serum metabolomic pattern between 0.1MCD‐fed mice and patients with NAFLD showed a substantial overlap. Conclusion: Aramchol treatment improved steatohepatitis and fibrosis by 1) decreasing stearoyl‐coenyzme A desaturase 1 and 2) increasing the flux through the transsulfuration pathway maintaining cellular redox homeostasis. We also demonstrated that the 0.1MCD model resembles the metabolic phenotype observed in about 50% of patients with NAFLD, which supports the potential use of Aramchol in NASH treatment. (Hepatology Communications 2017;1:911–927) PMID:29159325

Oxidative stress, thiols, and redox profiles.

PubMed

Harris, Craig; Hansen, Jason M

2012-01-01

Oxidative stress has been recognized as a contributing factor in the toxicity of a large number of developmental toxicants. Traditional definitions of oxidative stress state that a shift in the balance between reduced and oxidized biomolecules within cells, in favor of the latter, result in changes that are deleterious to vital cell functions and can culminate in malformations and death. The glutathione (GSH)/glutathione disulfide (GSSG) redox couple has been the traditional marker of choice for characterization of oxidative stress because of its high concentrations and direct roles as antioxidant and cellular protectant. Steady state depletion of GSH through conjugation, oxidation, or export has often been reported as the sole criteria for invoking oxidative stress and a myriad of associated deleterious consequences. Numerous other, mostly qualitative, observations have also been reported to suggest oxidative stress has occurred but it is not always clear how well they reflect the state of a cell or its functions. Our emerging understanding of redox signaling and the roles of reactive oxygen species (ROS), thiols, oxidant molecules, and cellular antioxidants, all acting as second messengers, has prompted a redefinition of oxidative stress based on changes in the real posttranslational protein thiol modifications that are central to redox regulation and control. Thiol-based redox couples such as GSH/GSSG, cysteine/cystine (cys/cySS), thioredoxin-reduced/thioredoxin-oxidized (TRX(red)/TRX(ox)) form independent signaling nodes that selectively regulate developmental events and are closely linked to changes in intracellular redox potentials. Accurate assessment of the consequences of increased free radicals in developing conceptuses should best be made using a battery of measurements including the quantitative assessment of intracellular redox potential, ROS, redox status of biomolecules, and induced changes in specific redox signaling nodes. Methods are presented for a determination of ROS production, soluble thiol oxidation, redox potential, and a proteomic approach to evaluate the thiol oxidation state of specific proteins.

Improving the cytoplasmic maturation of bovine oocytes matured in vitro with intracellular and/or extracellular antioxidants is not associated with increased rates of embryo development.

PubMed

Rocha-Frigoni, Nathália A S; Leão, Beatriz C S; Dall'Acqua, Priscila Chediek; Mingoti, Gisele Z

2016-11-01

The production of reactive oxygen species (ROS) is a normal process that occurs in the cellular mitochondrial respiratory chain. However, an increase in ROS levels during in vitro production of bovine embryos induces oxidative stress, leading to failed embryonic development. Therefore, we investigated whether supplementation of IVM medium with intracellular (cysteine and cysteamine; C + C) and/or extracellular (catalase; CAT) antioxidants improves the culture system, affects the mitochondrial membrane potential, affects the intracellular levels of ROS and glutathione (GSH) in the bovine oocytes at the end of maturation, and thereby affects the subsequent embryonic development. At the end of IVM, the metaphase II rates were unaffected by the treatments (76.7 ± 1.7% to 80.6 ± 5.2%; P > 0.05). The intracellular ROS levels, expressed in arbitrary fluorescence units, found in the oocytes treated with intracellular antioxidants (C + C and C + C + CAT groups; 1.06, averaged) were as low as those observed in immature oocytes (0 hour: 1.00 ± 0.12). Among mature oocytes, higher (P < 0.05) ROS levels were found in the control group (1.91 ± 0.10) when compared to the ROS levels found in oocytes treated with antioxidants. Intracellular GSH levels in all groups were lower (0.17 ± 0.09 to 0.51 ± 0.05; P < 0.05) than those in immature oocytes (1.00 ± 0.08), although GSH levels in the C + C group (0.51 ± 0.05) were greater (P < 0.05) than in the control, CAT, and C + C + CAT groups (0.23; averaged). The mitochondrial membrane potential in all groups was improved (1.6; averaged; P < 0.05) compared to the membrane potential observed in the immature oocytes (1.00 ± 0.05), with the exception of the C + C group (0.94 ± 0.03). There was no effect (P > 0.05) of antioxidant supplementation on embryonic development to the blastocyst stage (36.1%; averaged); however, there was an increased tendency (P = 0.0689) to obtain a higher blastocyst rate for the C + C + CAT group (47.5 ± 5.6%) compared to the control group (29.9 ± 4.8%). In conclusion, despite improvements in specific parameters of cytoplasmic maturation, the addition of intracellular and/or extracellular antioxidants during IVM did not affect embryo development. Copyright © 2016 Elsevier Inc. All rights reserved.

MG132 as a proteasome inhibitor induces cell growth inhibition and cell death in A549 lung cancer cells via influencing reactive oxygen species and GSH level.

PubMed

Han, Yong Hwan; Park, Woo Hyun

2010-07-01

Carbobenzoxy-Leu-Leu-leucinal (MG132) as a proteasome inhibitor has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). In the present study, we evaluated the effects of MG132 on the growth of A549 lung cancer cells in relation to cell growth, ROS and glutathione (GSH) levels. Treatment with MG132 inhibited the growth of A549 cells with an IC(50) of approximately 20 microM at 24 hours. DNA flow cytometric analysis indicated that 0.5 approximately 30 microM MG132 induced a G1 phase arrest of the cell cycle in A549 cells. Treatment with 10 or 30 microM MG132 also induced apoptosis, as evidenced by sub-G1 cells and annexin V staining cells. This was accompanied by the loss of mitochondrial membrane potential (MMP; Delta psi m). The intracellular ROS levels including O(2) (*-) were strongly increased in 10 or 30 microM MG132-treated A549 cells but were down-regulated in 0.1, 0.5 or 1 microM MG132-treated cells. Furthermore, 10 or 30 microM MG132 increased mitochondrial O(2) (*- ) level but 0.1, 0.5 or 1 microM MG132 decreased that. In addition, 10 or 30 microM MG132 induced GSH depletion in A549 cells. In conclusion, MG132 inhibited the growth of human A549 cells via inducing the cell cycle arrest as well as triggering apoptosis, which was in part correlated with the changes of ROS and GSH levels. Our present data provide important information on the anti-growth mechanisms of MG132 in A549 lung cancer cells in relation to ROS and GSH.

Antioxidant effects of melatonin in heart tissue after induction of experimental periodontitis in rats.

PubMed

Özdem, Muhsin; Kırzıoğlu, Fatma Y; Yılmaz, Hacı R; Vural, Hüseyin; Fentoğlu, Özlem; Uz, Efkan; Koçak, Ahmet; Yiğit, Ayşe

2017-01-01

The aim of this study was to evaluate the effects of melatonin on the oxidative stress in heart tissues after induction of experimental periodontitis in rats. Thirty Wistar Albino male rats were divided into four groups as follows: healthy + saline solution (Hs, n = 7), healthy + melatonin (Hm, n = 7), periodontitis + saline solution (Ps, n = 8), and periodontitis + melatonin (Pm, n = 8). Experimental periodontitis was induced using a ligature placed at the gingival margin of the maxillary second molars. Melatonin was applied intraperitoneally (10 mg/kg) every day for 2 weeks. After sacrificing the rats, serum levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) levels, and melatonin levels were evaluated. The Pm group exhibited lower alveolar bone loss than the Ps group. Melatonin levels increased in the periodontitis groups, and the Pm group had lower MDA levels and higher GSH-Px levels than the Ps group. These findings suggest that melatonin administration reduces MDA and increases GSH-Px levels in heart tissue, and these effects may be due to its antioxidant properties. Further studies are needed to understand the effects of melatonin on the association between periodontitis and cardiovascular disease.

Analysis of the influences of short-term levosimendan exposure on oxidant/antioxidant status and trace-element levels in the physiological status of the thoracic aorta of rats.

PubMed

Aydin, Cemalettin; Ay, Yasin; Basel, Halil; Kavak, Servet; Inan, Bekir; Bektaş, Hava; Gümrükçüoğlu, Hasan Ali; Ekim, Hasan; Demir, Halit

2012-12-01

The objective of this study was to evaluate the effect of levosimendan (chemical formula C₁₄H₁₂N₆O) exposure on oxidant/antioxidant status and trace-element levels in the thoracic aorta of rats. Eighteen male Wistar albino rats were randomly divided into two groups of eight animals each. Group 1 was not exposed to levosimendan and served as a control. Levosimendan (12 μg/kg) diluted in 10 ml 0.5 % dextrose was administered intraperitoneally to group 2. Animals of both groups were killed after 3 days, and their thoracic aortae were harvested for determination of changes in tissue oxidant/antioxidant status and trace-element levels. The animals in both groups were killed 72 h after levosimendan exposure, and thoracic aortae were harvested for determination of the lipid peroxidation product MDA and antioxidant GSH levels and the activities of antioxidant enzymes such as SOD, GSH-Px and CAT. It was found that MDA, GSH and CAT enzyme levels increased in thoracic aortae of rats after levosimendan administration. SOD and CA enzyme activities and the level of antioxidant GSH decreased in thoracic aortae of rats after levosimendan treatment. Pb, Cd and Fe levels of thoracic aortae were significantly higher (P < 0.001) and Mg, Mn, Zn and Cu were significantly lower (P < 0.001) in the levosimendan group compared to the control group. These results suggest that short-term levosimendan treatment caused an increase in free radical production and a decrease in antioxidant enzyme activity in thoracic aortae of levosimendan-treated rats. It also causes a decrease or increase in many mineral levels of the thoracic aorta, which is an undesirable condition for normal pharmacological function.

Comparison of in Vitro Bioactivation of Flutamide and Its Cyano Analogue: Evidence for Reductive Activation by Human NADPH:Cytochrome P450 Reductase

PubMed Central

Wen, Bo; Coe, Kevin J.; Rademacher, Peter; Fitch, William L.; Monshouwer, Mario; Nelson, Sidney D.

2009-01-01

Flutamide (FLU), a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with idiosyncratic hepatotoxicity in patients. It is proposed that bioactivation of FLU and subsequent binding of reactive metabolite(s) to cellular proteins play a causative role. A toxicogenomic study comparing FLU and its nitro to cyano analogue (CYA) showed that the nitroaromatic group of FLU enhanced cytotoxicity to hepatocytes, indicating that reduction of the nitroaromatic group may represent a potential route of FLU-induced hepatotoxicity [Coe et al. (2007) Chem. Res. Toxicol. 20, 1277–1290]. In the current study, we compared in vitro bioactivation of FLU and CYA in human liver microsomes and cryopreserved human hepatocytes. A nitroreduction metabolite FLU-6 was formed in liver microsomal incubations of FLU under atmospheric oxygen levels and, to a greater extent, under anaerobic conditions. Seven glutathione (GSH) adducts of FLU, FLU-G1–7, were tentatively identified in human liver microsomal incubations using liquid chromatography–tandem mass spectrometry (LC/MS/MS), while CYA formed only four corresponding GSH adducts, CYA-G1–4, under the same conditions. Of particular interest was the formation of FLU-G5–7 from FLU, where the nitroaromatic group of FLU was reduced to an amino group. A tentative pathway is that upon nitroreduction, the para-diamines undergo cytochrome P450 (P450)-catalyzed two-electron oxidations to form corresponding para-diimine intermediates that react with GSH to form GSH adducts FLU-G5–7, respectively. The identities of FLU-G5–7 were further confirmed by LC/MS/MS analyses of microsomal incubations of a synthesized standard FLU-6. In an attempt to identify enzymes involved in the nitroreduction of FLU, NADPH:cytochrome P450 reductase (CPR) was shown to reduce FLU to FLU-6 under both aerobic and anaerobic conditions. Furthermore, the formation of FLU-G5–7 was completely blocked by the addition of a reversible CPR inhibitor, α-lipoic acid, to the incubations of FLU under aerobic conditions. In summary, these results clearly demonstrate that nitroreduction of FLU by CPR contributes to bioactivation and potentially to hepatotoxicity of FLU. PMID:19548358

Toxicity evaluation of surface water treated with different disinfectants in HepG2 cells.

PubMed

Marabini, Laura; Frigerio, Silvia; Chiesara, Enzo; Radice, Sonia

2006-01-01

It is well known that water disinfection through chlorination causes the formation of a mixture of disinfection by-products (DBPs), many of which are genotoxic and carcinogenic. To demonstrate the formation of such compounds, a pilot water plant supplied with water from Lake Trasimeno was set up at the waterworks of Castiglione del Lago (PG, Italy). The disinfectants, continuously added to pre-filtered lake water flowing into three different basins, were sodium hypochlorite, chlorine dioxide and peracetic acid, an alternative disinfectant used until now for disinfecting waste waters, but not yet studied for a possible use in drinking water treatment. The aim of this study was to evaluate the formation during the disinfection processes of some toxic compounds that could explain the genotoxic effects of drinking waters. Differently treated waters were concentrated by solid-phase adsorption on silica C(18) columns and toxicity was assessed in a line of human hepatoma cells (HepG2), a metabolically competent cellular line very useful for human risk evaluation. The seasonal variability of the physical-chemical water characteristics (AOX, UV 254 nm, potential formation of THM, pH and temperature) made indispensable experimentation with water samples taken during the various seasons. Autumn waters cause greater toxicity compared to those of other seasons, in particular dilution of the concentrate at 0.5l equivalent of disinfected waters with chlorine dioxide and peracetic acid causes a 55% reduction in cellular vitality while the cellular vitality is over 80% with the all other water concentrates. Moreover it is very interesting underline that non-cytotoxic quantities of the autumnal water concentrates cause, after 2h treatment, a decrease in GSH and a statistically significant increase in oxygen radicals, while after prolonged treatment (24h) cause a GSH increase, without variations in the oxygen radical content. This phenomenon could be interpreted as the cellular adaptation response to an initial oxidative stress.

Metabolic cooperation of ascorbic acid and glutathione in normal and vitamin C-deficient ODS rats.

PubMed

Wang, Y; Kashiba, M; Kasahara, E; Tsuchiya, M; Sato, E F; Utsumi, K; Inoue, M

2001-01-01

Although the coordination of various antioxidants is important for the protection of organisms from oxidative stress, dynamic aspects of the interaction of endogenous antioxidants in vivo remain to be elucidated. We studied the metabolic coordination of two naturally occurring water-soluble antioxidants, ascorbic acid (AA) and reduced glutathione (GSH), in liver, kidney and plasma of control and scurvy-prone osteogenic disorder Shionogi (ODS) rats that hereditarily lack the ability to synthesize AA. When supplemented with AA, its levels in liver and kidney of ODS rats increased to similar levels of those in control rats. Hepato-renal levels of glutathione were similar with the two animal groups except for the slight increase in its hepatic levels in AA-supplemented ODS rats. Administration of L-buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, rapidly decreased the hepato-renal levels of glutathione in a biphasic manner, a rapid phase followed by a slower phase. Kinetic analysis revealed that glutathione turnover was enhanced significantly in liver mitochondria and renal cytosol of ODS rats. Administration of BSO significantly increased AA levels in the liver and kidney of control rats but decreased them in AA-supplemented ODS rats. Kinetic analysis revealed that AA is synthesized by control rat liver by some BSO-enhanced mechanism and the de novo synthesized AA is transferred to the kidney. Such a coordination of the metabolism of GSH and AA in liver and kidney is suppressed in AA-deficient ODS rats. These and other results suggest that the metabolism of AA and GSH forms a compensatory network by which oxidative stress can be decreased.

Glutathionylation state of uncoupling protein-2 and the control of glucose-stimulated insulin secretion.

PubMed

Mailloux, Ryan J; Fu, Accalia; Robson-Doucette, Christine; Allister, Emma M; Wheeler, Michael B; Screaton, Robert; Harper, Mary-Ellen

2012-11-16

The role of reactive oxygen species (ROS) in glucose-stimulated insulin release remains controversial because ROS have been shown to both amplify and impede insulin release. In regard to preventing insulin release, ROS activates uncoupling protein-2 (UCP2), a mitochondrial inner membrane protein that negatively regulates glucose-stimulated insulin secretion (GSIS) by uncoupling oxidative phosphorylation. With our recent discovery that the UCP2-mediated proton leak is modulated by reversible glutathionylation, a process responsive to small changes in ROS levels, we resolved to determine whether glutathionylation is required for UCP2 regulation of GSIS. Using Min6 cells and pancreatic islets, we demonstrate that induction of glutathionylation not only deactivates UCP2-mediated proton leak but also enhances GSIS. Conversely, an increase in mitochondrial matrix ROS was found to deglutathionylate and activate UCP2 leak and impede GSIS. Glucose metabolism also decreased the total amount of cellular glutathionylated proteins and increased the cellular glutathione redox ratio (GSH/GSSG). Intriguingly, the provision of extracellular ROS (H(2)O(2), 10 μM) amplified GSIS and also activated UCP2. Collectively, our findings indicate that the glutathionylation status of UCP2 contributes to the regulation of GSIS, and different cellular sites and inducers of ROS can have opposing effects on GSIS, perhaps explaining some of the controversy surrounding the role of ROS in GSIS.

Glutathionylation State of Uncoupling Protein-2 and the Control of Glucose-stimulated Insulin Secretion*

PubMed Central

Mailloux, Ryan J.; Fu, Accalia; Robson-Doucette, Christine; Allister, Emma M.; Wheeler, Michael B.; Screaton, Robert; Harper, Mary-Ellen

2012-01-01

The role of reactive oxygen species (ROS) in glucose-stimulated insulin release remains controversial because ROS have been shown to both amplify and impede insulin release. In regard to preventing insulin release, ROS activates uncoupling protein-2 (UCP2), a mitochondrial inner membrane protein that negatively regulates glucose-stimulated insulin secretion (GSIS) by uncoupling oxidative phosphorylation. With our recent discovery that the UCP2-mediated proton leak is modulated by reversible glutathionylation, a process responsive to small changes in ROS levels, we resolved to determine whether glutathionylation is required for UCP2 regulation of GSIS. Using Min6 cells and pancreatic islets, we demonstrate that induction of glutathionylation not only deactivates UCP2-mediated proton leak but also enhances GSIS. Conversely, an increase in mitochondrial matrix ROS was found to deglutathionylate and activate UCP2 leak and impede GSIS. Glucose metabolism also decreased the total amount of cellular glutathionylated proteins and increased the cellular glutathione redox ratio (GSH/GSSG). Intriguingly, the provision of extracellular ROS (H2O2, 10 μm) amplified GSIS and also activated UCP2. Collectively, our findings indicate that the glutathionylation status of UCP2 contributes to the regulation of GSIS, and different cellular sites and inducers of ROS can have opposing effects on GSIS, perhaps explaining some of the controversy surrounding the role of ROS in GSIS. PMID:23035124

Oral supplementation with whey proteins increases plasma glutathione levels of HIV-infected patients.

PubMed

Micke, P; Beeh, K M; Schlaak, J F; Buhl, R

2001-02-01

HIV infection is characterized by an enhanced oxidant burden and a systemic deficiency of the tripeptide glutathione (GSH), a major antioxidant. The semi-essential amino acid cysteine is the main source of the free sulfhydryl group of GSH and limits its synthesis. Therefore, different strategies to supplement cysteine supply have been suggested to increase glutathione levels in HIV-infected individuals. The aim of this study was to evaluate the effect of oral supplementation with two different cysteine-rich whey protein formulas on plasma GSH levels and parameters of oxidative stress and immune status in HIV-infected patients. In a prospective double blind clinical trial, 30 patients (25 male, 5 female; mean age (+/- SD) 42 +/- 9.8 years) with stable HIV infection (221 +/- 102 CD4 + lymphocytes L-1) were randomized to a supplemental diet with a daily dose of 45 g whey proteins of either Protectamin (Fresenius Kabi, Bad Hamburg, Germany) or Immunocal (Immunotec, Vandreuil, Canada) for two weeks. Plasma concentrations of total, reduced and oxidized GSH, superoxide anion (O2-) release by blood mononuclear cells, plasma levels of TNF-alpha and interleukins 2 and 12 were quantified with standard methods at baseline and after therapy. Pre-therapy, plasma GSH levels (Protectamin: 1.92 +/- 0.6 microM; Immunocal: 1.98 +/- 0.9 microM) were less than normal (2.64 +/- 0.7 microM, P = 0.03). Following two weeks of oral supplementation with whey proteins, plasma GSH levels increased in the Protectamin group by 44 +/- 56% (2.79 +/- 1.2 microM, P = 0.004) while the difference in the Immunocal group did not reach significance (+ 24.5 +/- 59%, 2.51 +/- 1.48 microM, P = 0.43). Spontaneous O2- release by blood mononuclear cells was stable (20.1 +/- 14.2 vs. 22.6 +/- 16.1 nmol h-1 10-6 cells, P = 0.52) whereas PMA-induced O2- release decreased in the Protectamin group (53.7 +/- 19 vs. 39.8 +/- 18 nmol h-1 10-6 cells, P = 0.04). Plasma concentrations of TNF-alpha and interleukins 2 and 12 (P > 0.08, all comparisons) as well as routine clinical parameters remained unchanged. Therapy was well tolerated. In glutathione-deficient patients with advanced HIV-infection, short-term oral supplementation with whey proteins increases plasma glutathione levels. A long-term clinical trial is clearly warranted to see if this "biochemical efficacy" of whey proteins translates into a more favourable course of the disease.

Creatine supplementation and oxidative stress in rat liver

PubMed Central

2013-01-01

Background The objective of this study was to determine the effects of creatine supplementation on liver biomarkers of oxidative stress in exercise-trained rats. Methods Forty 90-day-old adult male Wistar rats were assigned to four groups for the eight-week experiment. Control group (C) rats received a balanced control diet; creatine control group (CCr) rats received a balanced diet supplemented with 2% creatine; trained group (T) rats received a balanced diet and intense exercise training equivalent to the maximal lactate steady state phase; and supplemented-trained (TCr) rats were given a balanced diet supplemented with 2% creatine and subjected to intense exercise training equivalent to the maximal lactate steady state phase. At the end of the experimental period, concentrations of creatine, hydrogen peroxide (H2O2) and thiobarbituric acid reactive substances (TBARS) were measured as well as the enzyme activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-GPx) and catalase (CAT). Liver tissue levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and the GSH/GSSG ratio were also determined. Results Hepatic creatine levels were highest in the CCr and TCr groups with increased concentration of H2O2 observed in the T and TCr animal groups. SOD activity was decreased in the TCr group. GSH-GPx activity was increased in the T and TCr groups while CAT was elevated in the CCr and TCr groups. GSH, GGS and the GSH/GSSG ratio did not differ between all animal subsets. Conclusions Our results demonstrate that creatine supplementation acts in an additive manner to physical training to raise antioxidant enzymes in rat liver. However, because markers of liver oxidative stress were unchanged, this finding may also indicate that training-induced oxidative stress cannot be ameliorated by creatine supplementation. PMID:24325803

Effects of exercise training on the glutathione antioxidant system.

PubMed

Elokda, Ahmed S; Nielsen, David H

2007-10-01

The glutathione (GSH) antioxidant system has been shown to play an important role in the maintenance of good health and disease prevention. Various approaches have been used to enhance GSH availability including diet, nutritional supplementation, and drug administration, with minor to moderate success. Exercise training has evolved as a new approach. The purpose of this study was to investigate the effects of aerobic exercise training (AET), circuit weight training (CWT), and combined training (AET+CWT) on general adaptations, and resistance to acutely induced oxidative stress, as assessed by changes in the GSH antioxidant system. Eighty healthy sedentary volunteers participated in the study who were randomly assigned to four groups: control (no exercise); AET, CWT, and AET+CWT. Exercise training programs were designed to simulate outpatient cardiac rehabilitation (40 min x 3 days x 6 weeks). Venous blood sampling was taken at rest and post maximal graded exercise test (GXT). A new improved spectrophotometric venous assay analysis technique was used. A mixed model repeated measures analysis of variance design was used with t-tests for preplanned comparisons evaluated at Bonferroni-adjusted alpha levels. Effectiveness of the exercise training programs was demonstrated by significant between-group (exercise group versus control) comparisons. AET, CWT, and AET+CWT showed significant pretraining-posttraining increases in resting GSH and glutathione-glutathione disulfide ratio (GSH:GSSG), and significant decreases in GSSG levels (P<0.005). AET+CWT showed the most pronounced effect compared with AET or CWT alone (P<0.025). This study represents the first longitudinal investigation involving the effects of multiple modes of exercise training on the GSH antioxidant system with evidence, suggesting the GHS:GSSG ratio as the most sensitive change marker. The significant findings of this study have potential clinical implications to individuals involved in cardiac and pulmonary rehabilitation.

Infusion of Hibiscus sabdariffa L. Modulates Oxidative Stress in Patients with Marfan Syndrome.

PubMed

Soto, María Elena; Zuñiga-Muñoz, Alejandra; Guarner Lans, Verónica; Duran-Hernández, Erendira Janet; Pérez-Torres, Israel

2016-01-01

Marfan syndrome (MFS) is associated with progressive aortic dilatation, endothelial dysfunction, and oxidative stress that contribute to the early acute dissection of the vessel and can end up in rupture of the aorta and sudden death. Many studies have described that the organic acids from Hibiscus sabdariffa Linne (HSL) calyces increase cellular antioxidant capacity and decrease oxidative stress. Here we evaluate if the antioxidant properties of HSL infusion improve oxidative stress in MFS patients. Activities of extra cellular super oxide dismutase (ECSOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GSSG-R), glutathione (GSH), lipid peroxidation (LPO) index, total antioxidant capacity (TAC), and ascorbic acid were determined in plasma from MFS patients. Values before and after 3 months of the treatment with 2% HSL infusion were compared in control and MFS subjects. After treatment, there was a significant decrease in ECSOD (p = 0.03), EGPx (p = 0.04), GST (p = 0.03), GSH (p = 0.01), and TAC and ascorbic acid (p = 0.02) but GSSG-R activity (p = 0.04) and LPO (p = 0.02) were increased in MFS patients in comparison to patients receiving the HSL treatment and C subjects. Therefore, the infusion of HSL calyces has antioxidant properties that allow an increase in antioxidant capacity of both the enzymatic and nonenzymatic systems, in the plasma of the MSF patients.

Hyaluronic acid oligosaccharide modified redox-responsive mesoporous silica nanoparticles for targeted drug delivery.

PubMed

Zhao, Qinfu; Geng, Hongjian; Wang, Ying; Gao, Yikun; Huang, Jiahao; Wang, Yan; Zhang, Jinghai; Wang, Siling

2014-11-26

A redox-responsive delivery system based on colloidal mesoporous silica (CMS) has been developed, in which 6-mercaptopurine (6-MP) was conjugated to vehicles by cleavable disulfide bonds. The oligosaccharide of hyaluronic acid (oHA) was modified on the surface of CMS by disulfide bonds as a targeting ligand and was able to increase the stability and biocompatibility of CMS under physiological conditions. In vitro release studies indicated that the cumulative release of 6-MP was less than 3% in the absence of glutathione (GSH), and reached nearly 80% within 2 h in the presence of 3 mM GSH. Confocal microscopy and fluorescence-activated cell sorter (FACS) methods were used to evaluate the cellular uptake performance of fluorescein isothiocyanate (FITC) labeled CMS, with and without oHA modification. The CMS-SS-oHA exhibited a higher cellular uptake performance via CD44 receptor-mediated endocytosis in HCT-116 (CD44 receptor-positive) cells than in NIH-3T3 (CD44 receptor-negative) cells. 6-MP loaded CMS-SS-oHA exhibited greater cytotoxicity against HCT-116 cells than NIH-3T3 cells due to the enhanced cell uptake behavior of CMS-SS-oHA. This study provides a novel strategy to covalently link bioactive drug and targeting ligand to the interiors and exteriors of mesoporous silica to construct a stimulus-responsive targeted drug delivery system.

Cellular metabolism and oxidative stress as a possible determinant for longevity in small breed and large breed dogs

PubMed Central

Winward, Josh; Beattie, Ursula; Cipolli, William

2018-01-01

Among species, larger animals tend to live longer than smaller ones, however, the opposite seems to be true for dogs—smaller dogs tend to live significantly longer than larger dogs across all breeds. We were interested in the mechanism that may allow for small breeds to age more slowly compared with large breeds in the context of cellular metabolism and oxidative stress. Primary dermal fibroblasts from small and large breed dogs were grown in culture. We measured basal oxygen consumption (OCR), proton leak, and glycolysis using a Seahorse XF96 oxygen flux analyzer. Additionally, we measured rates of reactive species (RS) production, reduced glutathione (GSH) content, mitochondrial content, lipid peroxidation (LPO) damage and DNA (8-OHdg) damage. Our data suggests that as dogs of both size classes age, proton leak is significantly higher in older dogs, regardless of size class. We found that all aspects of glycolysis were significantly higher in larger breeds compared with smaller breeds. We found significant differences between age classes in GSH concentration, and a negative correlation between DNA damage in puppies and mean breed lifespan. Interestingly, RS production showed no differences across size and age class. Thus, large breed dogs may have higher glycolytic rates, and DNA damage, suggesting a potential mechanism for their decreased lifespan compared with small breed dogs. PMID:29694441

Infusion of Hibiscus sabdariffa L. Modulates Oxidative Stress in Patients with Marfan Syndrome

PubMed Central

Soto, María Elena; Zuñiga-Muñoz, Alejandra; Guarner Lans, Verónica; Duran-Hernández, Erendira Janet; Pérez-Torres, Israel

2016-01-01

Marfan syndrome (MFS) is associated with progressive aortic dilatation, endothelial dysfunction, and oxidative stress that contribute to the early acute dissection of the vessel and can end up in rupture of the aorta and sudden death. Many studies have described that the organic acids from Hibiscus sabdariffa Linne (HSL) calyces increase cellular antioxidant capacity and decrease oxidative stress. Here we evaluate if the antioxidant properties of HSL infusion improve oxidative stress in MFS patients. Activities of extra cellular super oxide dismutase (ECSOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GSSG-R), glutathione (GSH), lipid peroxidation (LPO) index, total antioxidant capacity (TAC), and ascorbic acid were determined in plasma from MFS patients. Values before and after 3 months of the treatment with 2% HSL infusion were compared in control and MFS subjects. After treatment, there was a significant decrease in ECSOD (p = 0.03), EGPx (p = 0.04), GST (p = 0.03), GSH (p = 0.01), and TAC and ascorbic acid (p = 0.02) but GSSG-R activity (p = 0.04) and LPO (p = 0.02) were increased in MFS patients in comparison to patients receiving the HSL treatment and C subjects. Therefore, the infusion of HSL calyces has antioxidant properties that allow an increase in antioxidant capacity of both the enzymatic and nonenzymatic systems, in the plasma of the MSF patients. PMID:27413258

Cardiaprotective effect of crocetin by attenuating apoptosis in isoproterenol induced myocardial infarction rat model.

PubMed

Zhang, Weili; Li, Yuhui; Ge, Zhiming

2017-09-01

Given study evaluates the cardioprotective effect of crocetin in myocardial infracted (MI) rats. MI was produced by administering isoproterenol (90mg/kg/day, i.p.) in rats for two consecutive days. all the animals were divided in to four groups such as control group receives only saline; MI group which receives only isoproterenol and crocetin treated group which receives crocetin (50, 100 and 200mg/kg/day, p.o.) for the duration of 15 days. At the end of dosing left ventricular functions was assessed to estimate its effect on cardiac functions. Catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), creatine kinase (CK-MB), lactate dehydrogenase (LDH) and inflammatory cytokines were determined in the cardiac tissue homogenate. Histopathology study was also carried out using hematoxylin and eosin staining. Immunohistochemistry was done for the estimation of Caspase-3, Bcl-2, Bax and Nrf-2 level in the myocardial tissues of MI rats. Result of the study suggested that GSH, CAT, CK-MB, and LDH were (p<0.01) increased in the tissue homogenate of crocetin treated group than MI group. However crocetin significantly (p<0.01) decreases the level of MDA and activity of SOD in the tissue homogenate than MI group. It was observed that treatment with crocetin attenuates the level of inflammatory cytokines in the myocardial tissues of MI rats. Moreover level of caspase-3, Bax and Nrf-2 significantly reduced and Bcl-2 enhanced in the myocardial tissues of MI rats than MI group. The altered cellular architecture of heart tissue sections in the myocardial infracted rats were reversed by administration of crocetin treatment. Taking all these data together, it may be suggested that the crocetin act as a possible protective agent in myocardial infarction by decreasing oxidative stress and inflammatory cytokines and thereby attenuates the apoptosis of myocardial cells. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Taurine protects against methotrexate-induced toxicity and inhibits leukocyte death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cetiner, Mustafa; Sener, Goeksel; Sehirli, A. Ozer

2005-11-15

The efficacy of methotrexate (MTX), a widely used cytotoxic chemotherapeutic agent, is often limited by severe side effects and toxic sequelae. Regarding the mechanisms of these side effects, several hypotheses have been put forward, among which oxidative stress is noticeable. The present study was undertaken to determine whether taurine, a potent free radical scavenger, could ameliorate MTX-induced oxidative injury and modulate immune response. Following a single dose of methotrexate (20 mg/kg), either saline or taurine (50 mg/kg) was administered for 5 days. After decapitation of the rats, trunk blood was obtained and the ileum, liver, and kidney were removed tomore » measure malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity, and collagen content, as well as histological examination. Our results showed that MTX administration increased the MDA, MPO activity, and collagen contents and decreased GSH levels in all tissues (P < 0.001), while these alterations were reversed in taurine-treated group (P < 0.05-0.01). Elevated (P < 0.001) TNF-{alpha} level observed following MTX treatment was depressed with taurine (P < 0.01). Oxidative burst of neutrophils stimulated by phorbol myristate acetate was reduced in saline-treated MTX group (P < 0.001), while taurine abolished this effect. Similarly, flow cytometric measurements revealed that leukocyte apoptosis and cell death were increased in MTX-treated animals, while taurine reversed these effects (P < 0.05). Reduced cellularity in bone marrow samples of MTX-treated group (P < 0.01) was reversed back to control levels in taurine-treated rats. Severe degeneration of the intestinal mucosa, liver parenchyma, glomerular, and tubular epithelium observed in saline-treated group was improved by taurine treatment. In conclusion, it appears that taurine protects against methotrexate-induced oxidant organ injury and inhibits leukocyte apoptosis and may be of therapeutic potential in alleviating the systemic side effects of chemotherapeutics.« less

Molecular markers of trichloroethylene-induced toxicity in human kidney cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lash, Lawrence H.; Putt, David A.; Hueni, Sarah E.

Difficulties in evaluation of trichloroethylene (TRI)-induced toxicity in humans and extrapolation of data from laboratory animals to humans are due to the existence of multiple target organs, multiple metabolic pathways, sex-, species-, and strain-dependent differences in both metabolism and susceptibility to toxicity, and the lack or minimal amount of human data for many target organs. The use of human tissue for mechanistic studies is thus distinctly advantageous. The kidneys are one target organ for TRI and metabolism by the glutathione (GSH) conjugation pathway is responsible for nephrotoxicity. The GSH conjugate is processed further to produce the cysteine conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC),more » which is the penultimate nephrotoxic species. Confluent, primary cultures of human proximal tubular (hPT) cells were used as the model system. Although cells in log-phase growth, which are undergoing more rapid DNA synthesis, would give lower LD{sub 50} values, confluent cells more closely mimic the in vivo proximal tubule. DCVC caused cellular necrosis only at relatively high doses (>100 {mu}M) and long incubation times (>24 h). In contrast, both apoptosis and enhanced cellular proliferation occurred at relatively low doses (10-100 {mu}M) and early incubation times (2-8 h). These responses were associated with prominent changes in expression of several proteins that regulate apoptosis (Bcl-2, Bax, Apaf-1, Caspase-9 cleavage, PARP cleavage) and cellular growth, differentiation and stress response (p53, Hsp27, NF-{kappa}B). Effects on p53 and Hsp27 implicate function of protein kinase C, the mitogen activated protein kinase pathway, and the cytoskeleton. The precise pattern of expression of these and other proteins can thus serve as molecular markers for TRI exposure and effect in human kidney.« less

Upregulation of cytosolic NADP+-dependent isocitrate dehydrogenase by hyperglycemia protects renal cells against oxidative stress.

PubMed

Lee, Soh-Hyun; Ha, Sun-Ok; Koh, Ho-Jin; Kim, KilSoo; Jeon, Seon-Min; Choi, Myung-Sook; Kwon, Oh-Shin; Huh, Tae-Lin

2010-02-28

Hyperglycemia-induced oxidative stress is widely recognized as a key mediator in the pathogenesis of diabetic nephropathy, a complication of diabetes. We found that both expression and enzymatic activity of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) were upregulated in the renal cortexes of diabetic rats and mice. Similarly, IDPc was induced in murine renal proximal tubular OK cells by high hyperglycemia, while it was abrogated by co-treatment with the antioxidant N-Acetyl-Cysteine (NAC). In OK cells, increased expression of IDPc by stable transfection prevented hyperglycemia-mediated reactive oxygen species (ROS) production, subsequent cellular oxidative stress and extracellular matrix accumulation, whereas these processes were all stimulated by decreased IDPc expression. In addition, production of NADPH and GSH in the cytosol was positively correlated with the expression level of IDPc in OK cells. These results together indicate that upregulation of IDPc in response to hyperglycemia might play an essential role in preventing the progression of diabetic nephropathy, which is accompanied by ROS-induced cellular damage and fibrosis, by providing NADPH, the reducing equivalent needed for recycling reduced glutathione and low molecular weight antioxidant thiol proteins.

[Effects of germanium on cell growth, polysaccharide production and cellular redox status in suspension cultures of protocorm-like bodies of Dendrobium huoshanense].

PubMed

Wei, Ming; Yang, Chaoying; Jiang, Shaotong

2010-03-01

To solve the problem of low growth rate and metabolism level in suspension cultures of protocorm-like bodies (PLBs) of Dendrobium huoshanense. The effects of germanium on PLB proliferation and accumulation of polysaccharides together with nutrient utilization were investigated and the contents of reducing sugars, soluble proteins, the activities of antioxidant enzymes and redox status of the cells of PLB were analyzed. The results indicated that the optimum concentration of germanium dioxide (4.0 mg/L) significantly enhanced the cell growth and accumulation of polysaccharides, greatly improved contents of reducing sugars and soluble proteins, increased the activities of superoxide dismutase (SOD) and catalase (CAT) but decreased the activity of peroxidase(POD). The cell dry weight and production of polysaccharides were 32.6 g/L and 3.78 g/L, respectively. The analysis of cellular redox status showed that the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in cells and the activity of glutathione reductase were significantly increased by the addition of germanium dioxide. The suitable concentration of germanium dioxide was beneficial to the cell growth and the accumulation of polysaccharides.

Role of Glutathione Conjugation in 1-Bromobutane-induced Immunotoxicity in Mice.

PubMed

Lee, Sang Kyu; Lee, Dong Ju; Jeon, Tae Won; Ko, Gyu Sub; Yoo, Se Hyun; Ha, Hyun Woo; Kang, Mi Jeong; Kang, Wonku; Kim, Sang Kyum; Jeong, Tae Cheon

2010-06-01

Halogenated organic compounds, such as 1-bromobutane (1-BB) , have been used as cleaning agents, agents for chemical syntheses or extraction solvents in workplace. In the present study, immunotoxic effects of 1-BB and its conjugation with glutathione (GSH) were investigated in female BALB/c mice. Animals were treated orally with 1-BB at 375, 750 and 1500 mg/kg in corn oil once for dose response or treated orally with 1-BB at 1500 mg/kg for 6, 12, 24 and 48 hr for time course. S-Butyl GSH was identified in spleen by liquid chromatography-electrospray ionization tandem mass spectrometry. Splenic GSH levels were significantly reduced by single treatment with 1-BB. S-Butyl GSH conjugates were detected in spleen from 6 hr after treatment. Oral 1-BB significantly suppressed the antibody response to a T-dependent antigen and the production of splenic intracellular interlukin-2 in response to Con A. Our present results suggest that 1-BB could cause immunotoxicity as well as reduction of splenic GSH content, due to the formation of GSH conjugates in mice. The present results would be useful to understand molecular toxic mechanism of low molecular weight haloalkanes and to develop biological markers for exposure to haloalkanes.

Characterization of bifunctional L-glutathione synthetases from Actinobacillus pleuropneumoniae and Actinobacillus succinogenes for efficient glutathione biosynthesis.

PubMed

Yang, Jianhua; Li, Wei; Wang, Dezheng; Wu, Hui; Li, Zhimin; Ye, Qin

2016-07-01

Glutathione (GSH), an important bioactive substance, is widely applied in pharmaceutical and food industries. In this work, two bifunctional L-glutathione synthetases (GshF) from Actinobacillus pleuropneumoniae (GshFAp) and Actinobacillus succinogenes (GshFAs) were successfully expressed in Escherichia coli BL-21(DE3). Similar to the GshF from Streptococcus thermophilus (GshFSt), GshFAp and GshFAs can be applied for high titer GSH production because they are less sensitive to end-product inhibition (Ki values 33 and 43 mM, respectively). The active catalytic forms of GshFAs and GshFAp are dimers, consistent with those of GshFPm (GshF from Pasteurella multocida) and GshFSa (GshF from Streptococcus agalactiae), but are different from GshFSt (GshF from S. thermophilus) which is an active monomer. The analysis of the protein sequences and three dimensional structures of GshFs suggested that the binding sites of GshFs for substrates, L-cysteine, L-glutamate, γ-glutamylcysteine, adenosine-triphosphate, and glycine are highly conserved with only very few differences. With sufficient supply of the precursors, the recombinant strains BL-21(DE3)/pET28a-gshFas and BL-21(DE3)/pET28a-gshFap were able to produce 36.6 and 34.1 mM GSH, with the molar yield of 0.92 and 0.85 mol/mol, respectively, based on the added L-cysteine. The results showed that GshFAp and GshFAs are potentially good candidates for industrial GSH production.

Interactive toxicity of inorganic mercury and trichloroethylene in rat and human proximal tubules: Effects on apoptosis, necrosis, and glutathione status

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lash, Lawrence H.; Putt, David A.; Hueni, Sarah E.

Simultaneous or prior exposure to one chemical may alter the concurrent or subsequent response to another chemical, often in unexpected ways. This is particularly true when the two chemicals share common mechanisms of action. The present study uses the paradigm of prior exposure to study the interactive toxicity between inorganic mercury (Hg{sup 2+}) and trichloroethylene (TRI) or its metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in rat and human proximal tubule. Pretreatment of rats with a subtoxic dose of Hg{sup 2+} increased expression of glutathione S-transferase-{alpha}1 (GST{alpha}1) but decreased expression of GST{alpha}2, increased activities of several GSH-dependent enzymes, and increased GSH conjugation of TRI.more » Primary cultures of rat proximal tubular (rPT) cells exhibited both necrosis and apoptosis after incubation with Hg{sup 2+}. Pretreatment of human proximal tubular (hPT) cells with Hg{sup 2+} caused little or no changes in GST expression or activities of GSH-dependent enzymes, decreased apoptosis induced by TRI or DCVC, but increased necrosis induced by DCVC. In contrast, pretreatment of hPT cells with TRI or DCVC protected from Hg{sup 2+} by decreasing necrosis and increasing apoptosis. Thus, whereas pretreatment of hPT cells with Hg{sup 2+} exacerbated cellular injury due to TRI or DCVC by shifting the response from apoptosis to necrosis, pretreatment of hPT cells with either TRI or DCVC protected from Hg{sup 2+}-induced cytotoxicity by shifting the response from necrosis to apoptosis. These results demonstrate that by altering processes related to GSH status, susceptibilities of rPT and hPT cells to acute injury from Hg{sup 2+}, TRI, or DCVC are markedly altered by prior exposures.« less

Activation of the hexosamine pathway causes oxidative stress and abnormal embryo gene expression: involvement in diabetic teratogenesis.

PubMed

Horal, Melissa; Zhang, Zhiquan; Stanton, Robert; Virkamäki, Antti; Loeken, Mary R

2004-08-01

Oxidative stress is critical to the teratogenic effects of diabetic pregnancy, yet the specific biochemical pathways responsible for oxidative stress have not been fully elucidated. The hexosamine pathway is activated in many tissues during diabetes and could contribute to oxidative stress by inhibiting the pentose shunt pathway, thereby diminishing production of the cellular antioxidant, reduced glutathione (GSH). To test the hypothesis that activation of the hexosamine pathway might contribute to the teratogenic effects of diabetic pregnancy, pregnant mice were injected with glucose, to induce hyperglycemia, or glucosamine, to directly activate the hexosamine pathway. Embryo tissue fragments were also cultured in physiological glucose, high glucose, or physiological glucose plus glucosamine, to test effects on oxidative stress and embryo gene expression. Glucosamine increased hexosamine synthesis and inhibited pentose shunt activity. There was a trend for transient hyperglycemia to have the same effects, but they did not reach statistical significance. However, both glucose and glucosamine significantly decreased GSH, and increased oxidative stress, as indicated by 2',7'-dichloro-dihydrofluorescein fluorescence. Glucose and glucosamine inhibited expression of Pax-3, a gene required for neural tube closure both in vivo and in vitro, and increased neural tube defects (NTDs) in vivo; these effects were prevented by GSH ethyl ester. High glucose and glucosamine inhibited Pax-3 expression by embryo culture, but culture in glutamine-free media to block the hexosamine pathway prevented the inhibition of Pax-3 expression by high glucose. Activation of the hexosamine pathway causes oxidative stress through depletion of GSH and consequent disruption of embryo gene expression. Activation of this pathway may contribute to diabetic teratogenesis.

Glutathione biosynthesis plays an important role against 4-tert-octylphenol-induced oxidative stress in Ceratophyllum demersum.

PubMed

Cahyanurani, Annisa' Bias; Chiu, Kuo-Hsun; Wu, Tsung-Meng

2017-09-01

4-tert-octylphenol (OP) is a persistent environmental pollutant with an endocrine-disrupting property. In the present study, we examined the effect of various concentrations of OP (0, 0.5, 1, 1.5, 2 and 3 mg L -1 ) applied to an aquatic plant, the submersed macrophyte Ceratophyllum demersum. The toxic effect caused by OP inhibited the plant's growth rate, reduced total chlorophyll content and increased levels of the reactive oxygen species (ROS) O 2 •- and H 2 O 2 . OP treatment significantly increased the activities of antioxidant enzymes including superoxide dismutase, guaiacol peroxidase, glutathione reductase and ascorbate peroxidase. The contents of the non-enzymatic antioxidant glutathione (GSH) and ratio of GSH to glutathione disulfide were markedly increased with OP treatment. Pretreatment with buthionine sulfoximine, a specific and potent inhibitor of GSH biosynthesis, significantly reduced total GSH content and conferred a more severe toxic phenotype on OP exposure. Thus, with OP-induced oxidative stress, C. demersum might actively regulate the antioxidant machinery, especially the biosynthesis and redox state of GSH. Copyright © 2017 Elsevier Ltd. All rights reserved.

[The selenium haemostasis during experimental anaphylaxis reaction in rats treated with reduced glutathione and selenium enriched spirulina].

PubMed

Golubkina, N A; Mazo, V K; Gmoshinskiĭ, I V; Zorin, S N; Tambiev, A Kh; Kirikova, N N

2000-01-01

The main events caused by anaphilaxis in selenium haemostasis in rats include significant increase of selenium excretion with urine (6.36 +/- 1.18 nM Se/18 h., n = 10, compared with 1.72 +/- 0.38 nM Se/18 h., n = 10) and decrease of selenium plasma/selenium erythrocytes ratio from 0.939 to 0.791. Reduced glutathione (G-SH) administration led to 1.5-fold decrease of plasma selenium level and 1.3-fold increase of selenium concentration in intestinal walls of sensitized rats (r = -0.720, P < 0.001). Chromatographic separation of plasma proteins showed that intragastric intubation of G-SH to sensibilized rats significantly decreased the protein P content and did not influence the concentration of Se-GSHPx, thus indicating the local selenium acceptor role of G-SH. G-SH administration did not influence the intestinal permeability in sensitised rats while use of complex additive: G-SH and selenium enriched spirulina--normalized the latter parameter and the ratio of protein P/Se-GSHPx in plasma.

Interleukin-1β protects astrocytes against oxidant-induced injury via an NFκB-dependent upregulation of glutathione synthesis

PubMed Central

He, Yan; Jackman, Nicole A.; L.Thorn, Trista; Vought, Valarie E.; Hewett, Sandra J.

2015-01-01

Astrocytes produce and export the antioxidant glutathione (GSH). Previously, we found that interleukin-1β (IL-1β) enhanced the expression of astrocyte system xc−, the transporter that delivers the rate-limiting substrate for GSH synthesis —cyst(e)ine. Herein, we demonstrate directly that IL-1β mediates a time-dependent increase in extracellular GSH levels in cortical astrocyte cultures, suggesting both enhanced synthesis and export. This increased GSH production was blocked by inhibition of nuclear factor κB (NFκB) activity but not by inhibition of p38 MAPK. To determine whether this increase could provide protection against oxidative stress, the oxidants tert-butyl hydroperoxide (tBOOH) and ferrous sulfate (FeSO4) were employed. IL-1β treatment prevented the increase in reactive oxygen species produced in astrocytes following tBOOH exposure. Additionally, the toxicity induced by tBOOH or FeSO4 exposure was significantly attenuated following treatment with IL-1β, an effect reversed by concomitant exposure to L-buthionine-S,R-sulfoximine (BSO), which prevented the IL-1β-mediated rise in GSH production. IL-1β failed to increase GSH or to provide protection against t-BOOH toxicity in astrocyte cultures derived from IL-1R1 null mutant mice. Overall, our data indicate that under certain conditions IL-1β may be an important stimulus for increasing astrocyte GSH production, and potentially, total antioxidant capacity in brain, via an NFκB-dependent process. PMID:25880604

Redox-based epigenetic status in drug addiction: a potential contributor to gene priming and a mechanistic rationale for metabolic intervention

PubMed Central

Trivedi, Malav S.; Deth, Richard

2015-01-01

Alcohol and other drugs of abuse, including psychostimulants and opioids, can induce epigenetic changes: a contributing factor for drug addiction, tolerance, and associated withdrawal symptoms. DNA methylation is a major epigenetic mechanism and it is one of more than 200 methylation reactions supported by methyl donor S-adenosylmethionine (SAM). Levels of SAM are controlled by cellular redox status via the folate and vitamin B12-dependent enzyme methionine synthase (MS). For example, under oxidative conditions MS is inhibited, diverting its substrate homocysteine (HCY) to the trans sulfuration pathway. Alcohol, dopamine, and morphine, can alter intracellular levels of glutathione (GSH)-based cellular redox status, subsequently affecting SAM levels and DNA methylation status. Here, existing evidence is presented in a coherent manner to propose a novel hypothesis implicating the involvement of redox-based epigenetic changes in drug addiction. Further, we discuss how a “gene priming” phenomenon can contribute to the maintenance of redox and methylation status homeostasis under various stimuli including drugs of abuse. Additionally, a new mechanistic rationale for the use of metabolic interventions/redox-replenishers as symptomatic treatment of alcohol and other drug addiction and associated withdrawal symptoms is also provided. Hence, the current review article strengthens the hypothesis that neuronal metabolism has a critical bidirectional coupling with epigenetic changes in drug addiction exemplified by the link between redox-based metabolic changes and resultant epigenetic consequences under the effect of drugs of abuse. PMID:25657617

Redox-based epigenetic status in drug addiction: a potential contributor to gene priming and a mechanistic rationale for metabolic intervention.

PubMed

Trivedi, Malav S; Deth, Richard

2014-01-01

Alcohol and other drugs of abuse, including psychostimulants and opioids, can induce epigenetic changes: a contributing factor for drug addiction, tolerance, and associated withdrawal symptoms. DNA methylation is a major epigenetic mechanism and it is one of more than 200 methylation reactions supported by methyl donor S-adenosylmethionine (SAM). Levels of SAM are controlled by cellular redox status via the folate and vitamin B12-dependent enzyme methionine synthase (MS). For example, under oxidative conditions MS is inhibited, diverting its substrate homocysteine (HCY) to the trans sulfuration pathway. Alcohol, dopamine, and morphine, can alter intracellular levels of glutathione (GSH)-based cellular redox status, subsequently affecting SAM levels and DNA methylation status. Here, existing evidence is presented in a coherent manner to propose a novel hypothesis implicating the involvement of redox-based epigenetic changes in drug addiction. Further, we discuss how a "gene priming" phenomenon can contribute to the maintenance of redox and methylation status homeostasis under various stimuli including drugs of abuse. Additionally, a new mechanistic rationale for the use of metabolic interventions/redox-replenishers as symptomatic treatment of alcohol and other drug addiction and associated withdrawal symptoms is also provided. Hence, the current review article strengthens the hypothesis that neuronal metabolism has a critical bidirectional coupling with epigenetic changes in drug addiction exemplified by the link between redox-based metabolic changes and resultant epigenetic consequences under the effect of drugs of abuse.

Silica Nanoparticles Induce Oxidative Stress and Autophagy but Not Apoptosis in the MRC-5 Cell Line

PubMed Central

Petrache Voicu, Sorina Nicoleta; Dinu, Diana; Sima, Cornelia; Hermenean, Anca; Ardelean, Aurel; Codrici, Elena; Stan, Miruna Silvia; Zărnescu, Otilia; Dinischiotu, Anca

2015-01-01

This study evaluated the in vitro effects of 62.5 µg/mL silica nanoparticles (SiO2 NPs) on MRC-5 human lung fibroblast cells for 24, 48 and 72 h. The nanoparticles’ morphology, composition, and structure were investigated using high resolution transmission electron microscopy, selected area electron diffraction and X-ray diffraction. Our study showed a decreased cell viability and the induction of cellular oxidative stress as evidenced by an increased level of reactive oxygen species (ROS), carbonyl groups, and advanced oxidation protein products after 24, 48, and 72 h, as well as a decreased concentration of glutathione (GSH) and protein sulfhydryl groups. The protein expression of Hsp27, Hsp60, and Hsp90 decreased at all time intervals, while the level of protein Hsp70 remained unchanged during the exposure. Similarly, the expression of p53, MDM2 and Bcl-2 was significantly decreased for all time intervals, while the expression of Bax, a marker for apoptosis, was insignificantly downregulated. These results correlated with the increase of pro-caspase 3 expression. The role of autophagy in cellular response to SiO2 NPs was demonstrated by a fluorescence-labeled method and by an increased level of LC3-II/LC3-I ratio. Taken together, our data suggested that SiO2 NPs induced ROS-mediated autophagy in MRC-5 cells as a possible mechanism of cell survival. PMID:26690408

Enhancement of ε-poly-L-lysine synthesis in Streptomyces by exogenous glutathione.

PubMed

Yan, Peng; Sun, Haoben; Lu, Pengqi; Liu, Haili; Tang, Lei

2018-01-01

Our previous work indicated that the vigor of Streptomyces decreased at the later stage of ε-poly-L-lysine (ε-PL) fermentation. In this study, we observed that the level of reactive oxygen species (ROS) in vivo increased sharply after 24 h, and the addition of an antioxidant glutathione (GSH) before this increase in ROS stimulated ε-PL synthesis in shake-flask fermentation. The enhancement of ε-PL production by GSH was further verified in batch and fed-batch fermentations. On a 5-l fermenter scale, the highest increasement was 68.8% in batch fermentation and the highest ε-PL level was 46.5 g l - 1 in fed-batch fermentation. The RT-qPCR analysis showed that the transcriptional level of the catalase gene was down-regulated, and the decrease in cell activity was alleviated by the addition of GSH. The results revealed that exogenous antioxidant might maintain the cell vigor by reducing the excess ROS which provided a novel approach to regulate ε-PL synthesis.

Alkylating agent induced NRF2 blocks endoplasmic reticulum stress-mediated apoptosis via control of glutathione pools and protein thiol homeostasis

PubMed Central

Zanotto-Filho, Alfeu; Masamsetti, V. Pragathi; Loranc, Eva; Tonapi, Sonal S.; Gorthi, Aparna; Bernard, Xavier; Gonçalves, Rosângela Mayer; Moreira, José C. F.; Chen, Yidong; Bishop, Alexander J. R.

2016-01-01

Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival; an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine (BSO) caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine (NAC) or glutathione-ethyl-ester (GSH-E), decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 targets overexpression and poor survival. In KEAP1 mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. PMID:27638861

A Cystine-Rich Whey Supplement (Immunocal®) Delays Disease Onset and Prevents Spinal Cord Glutathione Depletion in the hSOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

PubMed Central

Ross, Erika K.; Winter, Aimee N.; Wilkins, Heather M.; Sumner, Whitney A.; Duval, Nathan; Patterson, David; Linseman, Daniel A.

2014-01-01

Depletion of the endogenous antioxidant, glutathione (GSH), underlies progression of the devastating neurodegenerative disease, amyotrophic lateral sclerosis (ALS). Thus, strategies aimed at elevating GSH may yield new therapeutics for ALS. Here, we investigated the effects of a unique non-denatured whey protein supplement, Immunocal®, in the transgenic Gly position 93 to Ala (G93A) mutant hSOD1 (hSOD1G93A) mouse model of ALS. Immunocal® is rich in the GSH precursor, cystine, and is therefore capable of bolstering GSH content. Transgenic hSOD1G93A mice receiving Immunocal® displayed a significant delay in disease onset compared to untreated hSOD1G93A controls. Additionally, Immunocal® treatment significantly decreased the rate of decline in grip strength and prevented disease-associated reductions in whole blood and spinal cord tissue GSH levels in end-stage hSOD1G93A mice. However, Immunocal® did not extend survival, likely due to its inability to preserve the mitochondrial GSH pool in spinal cord. Combination treatment with Immunocal® and the anti-glutamatergic compound, riluzole, delayed disease onset and extended survival in hSOD1G93A mice. These findings demonstrate that sustaining tissue GSH with Immunocal® only modestly delays disease onset and slows the loss of skeletal muscle strength in hSOD1G93A mice. Moreover, the inability of Immunocal® to rescue mitochondrial GSH in spinal cord provides a possible mechanism for its lack of effect on survival and is a limiting factor in the potential utility of this supplement as a therapeutic for ALS. PMID:26785244

Nicotiana tabacum overexpressing γ-ECS exhibits biotic stress tolerance likely through NPR1-dependent salicylic acid-mediated pathway.

PubMed

Ghanta, Srijani; Bhattacharyya, Dipto; Sinha, Ragini; Banerjee, Anindita; Chattopadhyay, Sharmila

2011-05-01

The elaborate networks and the crosstalk of established signaling molecules like salicylic acid (SA), jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), reactive oxygen species (ROS) and glutathione (GSH) play key role in plant defense response. To obtain further insight into the mechanism through which GSH is involved in this crosstalk to mitigate biotic stress, transgenic Nicotiana tabacum overexpressing Lycopersicon esculentum gamma-glutamylcysteine synthetase (LeECS) gene (NtGB lines) were generated with enhanced level of GSH in comparison with wild-type plants exhibiting resistance to pathogenesis as well. The expression levels of non-expressor of pathogenesis-related genes 1 (NPR1)-dependent genes like pathogenesis-related gene 1 (NtPR1), mitogen-activated protein kinase kinase (NtMAPKK), glutamine synthetase (NtGLS) were significantly enhanced along with NtNPR1. However, the expression levels of NPR1-independent genes like NtPR2, NtPR5 and short-chain dehydrogenase/reductase family protein (NtSDRLP) were either insignificant or were downregulated. Additionally, increase in expression of thioredoxin (NtTRXh), S-nitrosoglutathione reductase 1 (NtGSNOR1) and suppression of isochorismate synthase 1 (NtICS1) was noted. Comprehensive analysis of GSH-fed tobacco BY2 cell line in a time-dependent manner reciprocated the in planta results. Better tolerance of NtGB lines against biotrophic Pseudomonas syringae pv. tabaci was noted as compared to necrotrophic Alternaria alternata. Through two-dimensional gel electrophoresis (2-DE) and image analysis, 48 differentially expressed spots were identified and through identification as well as functional categorization, ten proteins were found to be SA-related. Collectively, our results suggest GSH to be a member in cross-communication with other signaling molecules in mitigating biotic stress likely through NPR1-dependent SA-mediated pathway.

Glial dysfunction in parkin null mice: effects of aging.

PubMed

Solano, Rosa M; Casarejos, Maria J; Menéndez-Cuervo, Jamie; Rodriguez-Navarro, Jose A; García de Yébenes, Justo; Mena, Maria A

2008-01-16

Parkin mutations in humans produce parkinsonism whose pathogenesis is related to impaired protein degradation, increased free radicals, and abnormal neurotransmitter release. The role of glia in parkin deficiency is little known. We cultured midbrain glia from wild-type (WT) and parkin knock-out (PK-KO) mice. After 18-20 d in vitro, PK-KO glial cultures had less astrocytes, more microglia, reduced proliferation, and increased proapoptotic protein expression. PK-KO glia had greater levels of intracellular glutathione (GSH), increased mRNA expression of the GSH-synthesizing enzyme gamma-glutamylcysteine synthetase, and greater glutathione S-transferase and lower glutathione peroxidase activities than WT. The reverse happened in glia cultured in serum-free defined medium (EF12) or in old cultures. PK-KO glia was more susceptible than WT to transference to EF12 or neurotoxins (1-methyl-4-phenylpyridinium, blockers of GSH synthesis or catalase, inhibitors of extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3 kinases), aging of the culture, or combination of these insults. PK-KO glia was less susceptible than WT to Fe2+ plus H2O2 and less responsive to protection by deferoxamine. Old WT glia increased the expression of heat shock protein 70, but PK-KO did not. Glia conditioned medium (GCM) from PK-KO was less neuroprotective and had lower levels of GSH than WT. GCM from WT increased the levels of dopamine markers in midbrain neuronal cultures transferred to EF12 more efficiently than GCM from PK-KO, and the difference was corrected by supplementation with GSH. PK-KO-GCM was a less powerful suppressor of apoptosis and microglia in neuronal cultures. Our data prove that abnormal glial function is critical in parkin mutations, and its role increases with aging.

[Antagonism of total flavonoids from Chrysanthemum morifolium against lead induced oxidative injury in mice].

PubMed

Xia, Dao-zong; Lv, Gui-yuan; Yu, Xin-fens; Wang, Hui-ming; Yang, Qing

2008-12-01

To investigate antagonism effects of total flavonoids from Chrysanthemum morifolium. (TFCM) against lead induced oxidative injury. Ninety male mice were randomly divided into 9 groups. Mice except normal control group inject lead acetate every other day for 20 days. In the next 10 d, drugs were orally administrated to mice once a day. After the last aministration, mice were sacrificed and immediately subjected to necropsy. The concentration of lead, zinc and copper in blood, brain, liver and kidney were determined. The body weight, relative organ weight, antioxidant enzyme levels (GSH, GSH-Px, SOD and CAT) and lipid peroxidation products (MDA) were performed. TFCM might antagonize the decrease of body weight and the increase of organ weight/body weight ratio. The combined treatment with TFCM and DMSA can significantly lower the lead levels in blood, brain, liver and kidney. In contrast, lead concentration in mice treated with TFCM alone did not show significant change in these organs. The other trace elements such as zinc and copper had no significant decrease after TFCM or DMSA treatment. Middle and high-dose TFCM was more effective than DMSA in increasing the activity of GSH, GSH-Px, SOD, CAT and decreasing the concentration of MDA in mice brain. In addition, high-dose TFCM was more effective than DMSA in increasing the activity of GSH-Px, CAT and decreasing the concentration of MDA in mice liver and kidney. The combined treatment with TFCM and DMSA also can reverse lipid peroxidation and increase antioxidant enzyme levels in lead poisoning mice dose-dependently, and it had more beneficial effects than treatment with DMSA alone. TFCM might improve antioxidant defense system, reverse lipid peroxidation and protect brain, liver and kidney against lead induced oxidative damage in mice significantly.

Transformation of liver cells by 3-methylcholanthrene potentiates oxidative stress via the downregulation of glutathione synthesis.

PubMed

Kim, Sou Hyun; Yoon, Dahye; Lee, Yun-Hee; Lee, Jaewon; Kim, Nam Deuk; Kim, Suhkmann; Jung, Young-Suk

2017-12-01

Polycyclic aromatic hydrocarbons (PAHs) are widespread contaminants resulting from the incomplete combustion of organic materials in the environment. The primary concern for the hazardous effect of PAHs is their ability to activate the pathway linked to the aryl hydrocarbon receptor (AhR) and lead to carcinogenesis. While previous research has demonstrated that oxidative stress plays a key role in the AhR-dependent toxic response, the effect of PAHs on the biosynthesis of glutathione (GSH), which is a powerful endogenous antioxidant, has not been extensively investigated. In the present study, we utilized a global metabolomic approach, via high resolution magic angle spinning nuclear magnetic resonance spectroscopy, and identified significant metabolome differences between non-tumorigenic liver cells (BNL CL.2; CL2) and transformed liver cells (BNL 1MEA. 7R.1; 1MEA) chronically exposed to 3-methylcholanthrene (3MC), a well‑known carcinogenic PAH. A significant change that was observed, was a lower GSH level in 1MEA cells compared with that in CL2 cells. This was contrasted by increased levels of precursor metabolites for GSH synthesis, such as S-adenosylmethionine and cysteine. These changes were accompanied by a significantly reduced expression of γ-glutamylcysteine ligase (GCL), known to be the rate‑limiting step of GSH synthesis. Furthermore, the protein level of cysteine dioxygenase was downregulated; however, the concentration of taurine was unaltered. Therefore, the present study demonstrated that cells transformed by chronic exposure to 3MC exhibited inhibition of GSH biosynthesis by suppression of GCL protein expression and reduction of cysteine availability, which may subsequently render cells vulnerable to oxidative stress.

Organophosphorus insecticides chlorpyrifos and diazinon and oxidative stress in neuronal cells in a genetic model of glutathione deficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giordano, Gennaro; Afsharinejad, Zhara; Guizzetti, Marina

2007-03-15

Over the past several years evidence has been accumulating from in vivo animal studies, observations in humans, and in vitro studies, that organophosphorus (OP) insecticides may induce oxidative stress. Such effects may contribute to some of the toxic manifestations of OPs, particularly upon chronic or developmental exposures. The aim of this study was to investigate the role of oxidative stress in the neurotoxicity of two commonly used OPs, chlorpyrifos (CPF) and diazinon (DZ), their oxygen analogs (CPO and DZO), and their 'inactive' metabolites (TCP and IMP), in neuronal cells from a genetic model of glutathione deficiency. Cerebellar granule neurons frommore » wild type mice (Gclm +/+) and mice lacking the modifier subunit of glutamate cysteine ligase (Gclm -/-), the first and limiting step in the synthesis of glutathione (GSH), were utilized. The latter display very low levels of GSH and are more susceptible to the toxicity of agents that increase oxidative stress. CPO and DZO were the most cytotoxic compounds, followed by CPF and DZ, while TCP and IMP displayed lower toxicity. Toxicity was significantly higher (10- to 25-fold) in neurons from Gclm (-/-) mice, and was antagonized by various antioxidants. Depletion of GSH from Gclm (+/+) neurons significantly increased their sensitivity to OP toxicity. OPs increased intracellular levels of reactive oxygen species and lipid peroxidation and in both cases the effects were greater in neurons from Gclm (-/-) mice. OPs did not alter intracellular levels of GSH, but significantly increased those of oxidized glutathione (GSSG). Cytotoxicity was not antagonized by cholinergic antagonists, but was decreased by the calcium chelator BAPTA-AM. These studies indicate that cytotoxicity of OPs involves generation of reactive oxygen species and is modulated by intracellular GSH, and suggest that it may involve disturbances in intracellular homeostasis of calcium.« less

Dual-energy precursor and nuclear erythroid-related factor 2 activator treatment additively improve redox glutathione levels and neuron survival in aging and Alzheimer mouse neurons upstream of reactive oxygen species.

PubMed

Ghosh, Debolina; LeVault, Kelsey R; Brewer, Gregory J

2014-01-01

To determine whether glutathione (GSH) loss or increased reactive oxygen species (ROS) are more important to neuron loss, aging, and Alzheimer's disease (AD), we stressed or boosted GSH levels in neurons isolated from aging 3xTg-AD neurons compared with those from age-matched nontransgenic (non-Tg) neurons. Here, using titrating with buthionine sulfoximine, an inhibitor of γ-glutamyl cysteine synthetase (GCL), we observed that GSH depletion increased neuronal death of 3xTg-AD cultured neurons at increasing rates across the age span, whereas non-Tg neurons were resistant to GSH depletion until old age. Remarkably, the rate of neuron loss with ROS did not increase in old age and was the same for both genotypes, which indicates that cognitive deficits in the AD model were not caused by ROS. Therefore, we targeted for neuroprotection activation of the redox sensitive transcription factor, nuclear erythroid-related factor 2 (Nrf2) by 18 alpha glycyrrhetinic acid to stimulate GSH synthesis through GCL. This balanced stimulation of a number of redox enzymes restored the lower levels of Nrf2 and GCL seen in 3xTg-AD neurons compared with those of non-Tg neurons and promoted translocation of Nrf2 to the nucleus. By combining the Nrf2 activator together with the NADH precursor, nicotinamide, we increased neuron survival against amyloid beta stress in an additive manner. These stress tests and neuroprotective treatments suggest that the redox environment is more important for neuron survival than ROS. The dual neuroprotective treatment with nicotinamide and an Nrf2 inducer indicates that these age-related and AD-related changes are reversible. Copyright © 2014 Elsevier Inc. All rights reserved.