Sample records for intracellular glutathione levels

  1. Anethole dithiolethione, a putative neuroprotectant, increases intracellular and extracellular glutathione levels during starvation of cultured astroglial cells.

    PubMed

    Dringen, R; Hamprecht, B; Drukarch, B

    1998-12-01

    Astroglial cells protect neurons against oxidative damage. The antioxidant glutathione plays a pivotal role in the neuroprotective action of astroglial cells which is impaired following loss of glutathione. Anethole dithiolethione (ADT), a sulfur-containing compound which is used in humans as a secretagogue, increases glutathione levels in cultured astroglial cells under "physiological" conditions and is thought thereby to protect against oxidative damage. Presently, we report the effect of ADT (3-100 microM) on glutathione content of and efflux from rat primary astroglia-rich cultures under "pathological" conditions, i.e., extended deprivation of glucose and amino acids. Although cellular viability was not affected significantly, starvation of these cultures for 24 h in a bicarbonate buffer lacking glucose and amino acids led to a decrease in glutathione and protein content of approximately 43% and 40%, respectively. Although no effect on the protein loss occurred, the presence of ADT during starvation counteracted the starvation-induced loss of intracellular glutathione in a concentration-dependent way. At a concentration of 100 microM ADT even a significant increase in astroglial glutathione content was noted after 24 h of starvation. Alike intracellular glutathione levels, the amount of glutathione found in the buffer was elevated substantially if ADT was present during starvation. This ADT-mediated, apparent increase in glutathione efflux was additive to the stimulatory effect on extracellular glutathione levels of acivicin (100 microM), an inhibitor of extracellular enzymatic glutathione breakdown. However, the ADT-induced elevation of both intra- and extracellular glutathione content during starvation was prevented completely by coincubation with buthionine sulfoximine (10 microM), an inhibitor of glutathione synthesis. These results demonstrate that, most likely through stimulation of glutathione synthesis, ADT enables astroglial cells to maintain higher

  2. Effect of asoka on the intracellular glutathione levels and skin tumour promotion in mice.

    PubMed

    Varghese, C D; Nair, S C; Panikkar, B; Panikkar, K R

    1993-04-15

    The bark of Saraka asoca (asoka) is commonly used to treat various diseases by the Indian system of medicine and in Sri Lanka. Further purification and chemical analysis of the active compound from the bark extract of asoka showed that (-)-epicatechin was responsible for the observed antitumour/anticarcinogenic activity. Papilloma formation in mice initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted using croton oil was inhibited by the topical application of 100 mg/kg body weight (b.w.) of (-)-epicatechin isolated from asoka bark extract. Oral administration of the same dose restricted the growth of s.c. injected 20 methylcholanthrene (MCA) induced soil tissue fibrosarcomas significantly in mice. Elevations of almost 2-4-fold in the intracellular reduced glutathione and related enzymes viz., glutathione reductase and glutathione S-transferase of sarcoma-180 tumour cells were noted in the presence of 1 microgram/ml of (-)-epicatechin, further highlighting its antiproliferative effect.

  3. Farnesol-Induced Apoptosis in Candida albicans Is Mediated by Cdr1-p Extrusion and Depletion of Intracellular Glutathione

    PubMed Central

    Zhu, Jingsong; Krom, Bastiaan P.; Sanglard, Dominique; Intapa, Chaidan; Dawson, Clinton C.; Peters, Brian M.; Shirtliff, Mark E.; Jabra-Rizk, Mary Ann

    2011-01-01

    Farnesol is a key derivative in the sterol biosynthesis pathway in eukaryotic cells previously identified as a quorum sensing molecule in the human fungal pathogen Candida albicans. Recently, we demonstrated that above threshold concentrations, farnesol is capable of triggering apoptosis in C. albicans. However, the exact mechanism of farnesol cytotoxicity is not fully elucidated. Lipophilic compounds such as farnesol are known to conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. Glutathione conjugates act as substrates for ATP-dependent ABC transporters and are extruded from the cell. To that end, this current study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with Cdr1p-mediated extrusion of glutathione conjugates, results in total glutathione depletion, oxidative stress and ultimately fungal cell death. The combined findings demonstrated a significant decrease in intracellular glutathione levels concomitant with up-regulation of CDR1 and decreased cell viability. However, addition of exogenous reduced glutathione maintained intracellular glutathione levels and enhanced viability. In contrast, farnesol toxicity was decreased in a mutant lacking CDR1, whereas it was increased in a CDR1-overexpressing strain. Further, gene expression studies demonstrated significant up-regulation of the SOD genes, primary enzymes responsible for defense against oxidative stress, with no changes in expression in CDR1. This is the first study describing the involvement of Cdr1p-mediated glutathione efflux as a mechanism preceding the farnesol-induced apoptotic process in C. albicans. Understanding of the mechanisms underlying farnesol-cytotoxicity in C. albicans may lead to the development of this redox-cycling agent as an alternative antifungal agent. PMID:22205973

  4. Detection of intracellular glutathione using ThiolTracker violet stain and fluorescence microscopy.

    PubMed

    Mandavilli, Bhaskar S; Janes, Michael S

    2010-07-01

    Glutathione plays an important role in protecting mammalian cells from oxidative stress and cell death. Because reduced glutathione (GSH) represents the large majority of intracellular free thiols, cell-permeant, thiol-reactive fluorescent probes represent potentially useful indicators of intracellular GSH. The ThiolTracker Violet stain (a registered trademark of Invitrogen) is a bright fluorescent probe that is highly reactive to thiols and can be used as a convenient and effective indicator of intracellular GSH and general redox status by a variety of detection modalities. While this probe has been validated in flow cytometry and microplate fluorimetry assays, the following method will describe details on the use of the ThiolTracker Violet dye in traditional fluorescence microscopy, as well as high-content imaging and analysis.

  5. Radiosensitization of Hypoxic Tumor Cells by Depletion of Intracellular Glutathione

    NASA Astrophysics Data System (ADS)

    Bump, Edward A.; Yu, Ning Y.; Brown, J. Martin

    1982-08-01

    Depletion of glutathione in Chinese hamster ovary cells in vitro by diethyl maleate resulted in enhancement of the effect of x-rays on cell survival under hypoxic conditions but not under oxygenated conditions. Hypoxic EMT6 tumor cells were similarly sensitized in vivo. The action of diethyl maleate is synergistic with the effect of the electron-affinic radiosensitizer misonidazole, suggesting that the effectiveness of misonidazole in cancer radiotherapy may be improved by combining it with drugs that deplete intracellular glutathione.

  6. Radiosensitization of hypoxic tumor cells by depletion of intracellular glutathione

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

    Bump, E.A.; Yu, N.Y.; Brown, J.M.

    1982-08-06

    Depletion of glutathione in Chinese hamster ovary cells in vitro by diethyl maleate resulted in enhancement of the effect of x-rays on cell survival under hypoxic conditions but not under oxygenated conditions. Hypoxic EMT6 tumor cells were similarly sensitized in vivo. The action of diethyl maleate is synergistic with the effect of the electron-affinic radiosensitizer misonidazole, suggesting that the effectiveness of misonidazole in cancer radiotherapy may be improved by combining it with drugs that deplete intracellular glutathione.

  7. Role of {alpha}{sub v}{beta}{sub 5} integrin receptor in endocytosis of crocidolite and its effect on intracellular glutathione levels in human lung epithelial (A549) cells

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

    Pande, Priyadarshini; Mosleh, Tariq A.; Aust, Ann E.

    Crocidolite, containing 27% iron by weight, is the most carcinogenic form of asbestos. Crocidolite fibers are endocytized by {alpha}{sub v}{beta}{sub 5} integrin receptors in rabbit pleural mesothelial cells. We show here that crocidolite fibers are endocytized in human lung epithelial (A549) cells and in primary small airway epithelial (SAEC) cells. Presence of the integrin {alpha}{sub v}{beta}{sub 5} blocking antibody, P1F6, significantly reduced the uptake of crocidolite fibers in A549 cells. Thus, the integrin {alpha}{sub v}{beta}{sub 5} receptor is involved in endocytosis of crocidolite fibers in A549 cells as well. Previously, it has been observed that asbestos fibers lead to changesmore » in the intracellular redox environment, i.e. a marked decrease in intracellular glutathione concentrations and an increase in the extracellular glutathione in A549 cells. In addition, the decrease in intracellular glutathione was found to be largely independent of iron present on the surface of the fiber. A549 cells were treated with crocidolite in the presence of endocytosis inhibitor cytochalasin D. Our data indicate that, upon preventing endocytosis, we were able to reverse the decrease in total intracellular glutathione. The decrease in total intracellular glutathione could also be prevented in the presence of the monoclonal antibody P1F6. Thus, we observed that endocytosis of crocidolite fibers via integrin {alpha}{sub v}{beta}{sub 5} receptor is linked to the marked decrease in total intracellular glutathione in A549 cells.« less

  8. 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.

  9. 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

  10. 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.

  11. Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide.

    PubMed

    Abegg, Maxwel Adriano; Alabarse, Paulo Vinícius Gil; Schüller, Artur Krumberg; Benfato, Mara Silveira

    2012-10-01

    The capacity to overcome the oxidative stress imposed by phagocytes seems to be critical for Candida species to cause invasive candidiasis. To better characterize the oxidative stress response (OSR) of 8 clinically relevant Candida sp., glutathione, a vital component of the intracellular redox balance, was measured using the 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB)-glutathione disulfide (GSSG) reductase reconversion method; the total antioxidant capacity (TAC) was measured using a modified method based on the decolorization of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid radical cation (ABTS*+). Both methods were used with cellular Candida sp. extracts treated or not with hydrogen peroxide (0.5 mM). Oxidative stress induced by hydrogen peroxide clearly reduced intracellular glutathione levels. This depletion was stronger in Candida albicans and the levels of glutathione in untreated cells were also higher in this species. The TAC demonstrated intra-specific variation. Glutathione levels did not correlate with the measured TAC values, despite this being the most important non-enzymatic intracellular antioxidant molecule. The results indicate that the isolated measurement of TAC does not give a clear picture of the ability of a given Candida sp. to respond to oxidative stress.

  12. Repression of adenosine triphosphate-binding cassette transporter ABCG2 by estrogen increases intracellular glutathione in brain endothelial cells following ischemic reperfusion injury.

    PubMed

    Shin, Jin A; Jeong, Sae Im; Kim, Hye Won; Jang, Gyeonghui; Ryu, Dong-Ryeol; Ahn, Young-Ho; Choi, Ji Ha; Choi, Youn-Hee; Park, Eun-Mi

    2018-06-01

    The adenosine triphosphate-binding cassette efflux transporter ABCG2, which is located in the blood-brain barrier limits the entry of endogenous compounds and xenobiotics into the brain, and its expression and activity are regulated by estrogen. This study was aimed to define the role of ABCG2 in estrogen-mediated neuroprotection against ischemic injury. ABCG2 protein levels before and after ischemic stroke were increased in the brain of female mice by ovariectomy, which were reversed by estrogen replacement. In brain endothelial cell line bEnd.3, estrogen reduced the basal ABCG2 protein level and efflux activity and protected cells from ischemic injury without inducing ABCG2 expression. When bEnd.3 cells were transfected with ABCG2 small interfering RNA, ischemia-induced cell death was reduced, and the intracellular concentration of glutathione, an antioxidant that is transported by ABCG2, was increased. In addition, after ischemic stroke in ovariectomized mice, estrogen prevented the reduction of intracellular glutathione level in brain microvessels. These data suggested that the suppression of ABCG2 by estrogen is involved in neuroprotection against ischemic injury by increasing intracellular glutathione, and that the modulation of ABCG2 activity offers a therapeutic target for brain diseases in estrogen-deficient aged women. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Characterization of a Novel Dithiocarbamate Glutathione Reductase Inhibitor and Its Use as a Tool to Modulate Intracellular Glutathione*

    PubMed Central

    Seefeldt, Teresa; Zhao, Yong; Chen, Wei; Raza, Ashraf S.; Carlson, Laura; Herman, Jocqueline; Stoebner, Adam; Hanson, Sarah; Foll, Ryan; Guan, Xiangming

    2009-01-01

    Thiol redox state (TRS) is an important parameter to reflect intracellular oxidative stress and is associated with various normal and abnormal biochemical processes. Agents that can be used to increase intracellular TRS will be valuable tools in TRS-related research. Glutathione reductase (GR) is a critical enzyme in the homeostasis of TRS. The enzyme catalyzes the reduction of GSSG to GSH to maintain a high GSH:GSSG ratio. Inhibition of the enzyme can be used to increase TRS. Despite the reports of various GR inhibitors, N,N-bis(2-chloroethyl)-N-nitrosourea, an anticancer drug with IC50 = 647 μm against yeast GR, remains the most commonly used GR inhibitor in the literature. However, the toxicity caused by nonspecific interactions, as well as inhibition of DNA synthesis, complicates the use of N,N-bis(2-chloroethyl)-N-nitrosourea as a GR inhibitor. We report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a novel irreversible GR inhibitor. 2-AAPA was prepared by one-step synthesis from commercially available reagents. The Ki and kinact of 2-AAPA against yeast GR were determined to be 56 μm and 0.1 min–1, respectively. At the concentration that produced >80% yeast GR inhibition, 2-AAPA showed no inhibition against glutamylcysteine synthetase, glutathione synthetase, catalase, and superoxide dismutase, but minimal inhibition against glutathione S-transferase and glutathione peroxidase. In CV-1 cells, 2-AAPA (0.1 mm) produced 97% GR inhibition, 25% GSH reduction, and a 5-fold increase in GSSG in 20 min. The compound can be a useful tool in TRS-related research. PMID:19049979

  14. Multifaceted Roles of Glutathione and Glutathione-Based Systems in Carcinogenesis and Anticancer Drug Resistance.

    PubMed

    Hatem, Elie; El Banna, Nadine; Huang, Meng-Er

    2017-11-20

    Glutathione is the most abundant antioxidant molecule in living organisms and has multiple functions. Intracellular glutathione homeostasis, through its synthesis, consumption, and degradation, is an intricately balanced process. Glutathione levels are often high in tumor cells before treatment, and there is a strong correlation between elevated levels of intracellular glutathione/sustained glutathione-mediated redox activity and resistance to pro-oxidant anticancer therapy. Recent Advances: Ample evidence demonstrates that glutathione and glutathione-based systems are particularly relevant in cancer initiation, progression, and the development of anticancer drug resistance. This review highlights the multifaceted roles of glutathione and glutathione-based systems in carcinogenesis, anticancer drug resistance, and clinical applications. The evidence summarized here underscores the important role played by glutathione and the glutathione-based systems in carcinogenesis and anticancer drug resistance. Future studies should address mechanistic questions regarding the distinct roles of glutathione in different stages of cancer development and cancer cell death. It will be important to study how metabolic alterations in cancer cells can influence glutathione homeostasis. Sensitive approaches to monitor glutathione dynamics in subcellular compartments will be an indispensible step. Therapeutic perspectives should focus on mechanism-based rational drug combinations that are directed against multiple redox targets using effective, specific, and clinically safe inhibitors. This new strategy is expected to produce a synergistic effect, prevent drug resistance, and diminish doses of single drugs. Antioxid. Redox Signal. 27, 1217-1234.

  15. 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

  16. Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells.

    PubMed Central

    Schraufstätter, I U; Hinshaw, D B; Hyslop, P A; Spragg, R G; Cochrane, C G

    1985-01-01

    Exposure of target cells to a bolus of H2O2 induced cell lysis after a latent period of several hours, which was prevented only when the H2O2 was removed within the first 30 min of injury by addition of catalase. This indicated that early metabolic events take place that are important in the fate of the cell exposed to oxidants. In this study, we described two early and independent events of H2O2-induced injury in P388D1 macrophagelike tumor cells: activation of the glutathione cycle and depletion of cellular NAD. Glutathione cycle and hexose monophosphate shunt (HMPS) were activated within seconds after the addition of H2O2. High HMPS activity maintained glutathione that was largely reduced. However, when HMPS activity was inhibited--by glucose depletion or by incubation at 4 degrees C--glutathione remained in the oxidized state. Total pyridine nucleotide levels were diminished when cells were exposed to H2O2, and the breakdown product, nicotinamide, was recovered in the extracellular medium. Intracellular NAD levels fell by 80% within 20 min of exposure of cells to H2O2. The loss of NADP(H) and stimulation of the HMPS could be prevented when the glutathione cycle was inhibited by either blocking glutathione synthesis with buthionine sulfoximine (BSO) or by inhibiting glutathione reductase with (1,3-bis) 2 chlorethyl-1-nitrosourea. The loss of NAD developed independently of glutathione cycle and HMPS activity, as it also occurred in BSO-treated cells. PMID:3840176

  17. 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.

  18. Glutathione Levels in Human Tumors

    PubMed Central

    Gamcsik, Michael P.; Kasibhatla, Mohit S.; Teeter, Stephanie D.; Colvin, O. Michael

    2013-01-01

    This review summarizes clinical studies in which glutathione was measured in tumor tissue from patients with brain, breast, gastrointestinal, gynecological, head and neck and lung cancer. Glutathione tends to be elevated in breast, ovarian, head and neck and lung cancer and lower in brain and liver tumors compared to disease-free tissue. Cervical, colorectal, gastric and esophageal cancers show both higher and lower levels of tumor glutathione. Some studies show an inverse relationship between patient survival and tumor glutathione. Based on this survey, we recommend approaches that may improve the clinical value of glutathione as a biomarker. PMID:22900535

  19. [Alternative nutrition and glutathione levels].

    PubMed

    Krajcovicová-Kudlácková, M; Simoncic, R; Béderová, A; Brtková, A; Magálová, T; Barteková, S

    1999-08-30

    Low protein quality and quantity is reported to be a possible risk of alternative nutrition. Pulses contain 18-41% of methionine in relation to reference protein, moreover, its content in cereals is by one half lower. Therefore vegetarians and vegans may have an insufficient intake of sulphur-containing amino acids that may subsequently affect glutathione values (precursors of its synthesis). In groups of adults on an alternative diet--lactoovovegetarians (n = 47) and vegans (n = 44) aged 19-62 years with average duration on a vegetarian or vegan diet of 7.6 and 4.9 years, respectively, glutathione levels (GSH) were measured in erythrocytes (spectrophotometrically), as well as the activity of GSH-dependent enzymes. As nutritional control (n = 42) served an average sample of omnivores selected from a group of 489 examined, apparently healthy subjects of the same age range living in the same region. One to low protein intake (56% of RDA) exclusively of plant origin significantly lower levels of total proteins were observed in vegans with a 16% frequency of hypoproteinaemia (vs 0% in omnivores). In comparison to omnivores a significantly lower glutathione level was found (4.28 +/- 0.12 vs 4.84 +/- 0.14 mumol/g Hb, P < 0.01). Lactoovovegetarians because of their protein intake in adequate amounts with a 27% proportion of animal proteins (dairy products, eggs) consume a balanced mixture of amino acids, which is reflected in total protein levels similar to omnivores and significantly higher values of glutathione -5.26 +/- 0.12 mumol/g Hb, P < 0.05 (intake of glutathione in diet, higher consumption of fruit and vegetable in comparison to omnivores). A sufficient supply of glutathione as the substrate for enzymatic reactions of hydrogen peroxide or lipid hydroperoxide catabolism, as well as for detoxication of xenobiotics, was reflected in lactoovovegetarians in a significantly higher activity of glutathione-peroxidase and glutathione-S-transferase in erythrocytes. Low

  20. 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.

  1. Effect of T-2 Toxin, Fasting, and 2-Methyl-thiazolidine-4-carboxylate, a Glutathione Prodrug, on Hepatic Glutathione Levels1,2

    DTIC Science & Technology

    1986-11-14

    Iftnvi fnr fard mirn: O Fo, M73 3 r0tiON OF I MOV 6s IS OBSOLETE SECURITY CLASSIFICATION OF THIS PACE (When Does Entoeed) , I I I I I 4.5± 0.39...Glende, 1973). An important cellular defense against peroxida- tive damage is the presence of glutathione and its use as an enzyme substrate or...cofactor. Even though intracellular glutathione concentration is in the millimolar range (Kosower and Kosower, 1978), there are conditions which lead to

  2. Quercetin affects glutathione levels and redox ratio in human aortic endothelial cells not through oxidation but formation and cellular export of quercetin-glutathione conjugates and upregulation of glutamate-cysteine ligase.

    PubMed

    Li, Chuan; Zhang, Wei-Jian; Choi, Jaewoo; Frei, Balz

    2016-10-01

    Endothelial dysfunction due to vascular inflammation and oxidative stress critically contributes to the etiology of atherosclerosis. The intracellular redox environment plays a key role in regulating endothelial cell function and is intimately linked to cellular thiol status, including and foremost glutathione (GSH). In the present study we investigated whether and how the dietary flavonoid, quercetin, affects GSH status of human aortic endothelial cells (HAEC) and their response to oxidative stress. We found that treating cells with buthionine sulfoximine to deplete cellular GSH levels significantly reduced the capacity of quercetin to inhibit lipopolysaccharide (LPS)-induced oxidant production. Furthermore, incubation of HAEC with quercetin caused a transient decrease and then full recovery of cellular GSH concentrations. The initial decline in GSH was not accompanied by a corresponding increase in glutathione disulfide (GSSG). To the contrary, GSSG levels, which were less than 0.5% of GSH levels at baseline (0.26±0.01 vs. 64.7±1.9nmol/mg protein, respectively), decreased by about 25% during incubation with quercetin. As a result, the GSH: GSSG ratio increased by about 70%, from 253±7 to 372±23. These quercetin-induced changes in GSH and GSSG levels were not affected by treating HAEC with 500µM ascorbic acid phosphate for 24h to increase intracellular ascorbate levels. Incubation of HAEC with quercetin also led to the appearance of extracellular quercetin-glutathione conjugates, which was paralleled by upregulation of the multidrug resistance protein 1 (MRP1). Furthermore, quercetin slightly but significantly increased mRNA and protein levels of glutamate-cysteine ligase (GCL) catalytic and modifier subunits. Taken together, our results suggest that quercetin causes loss of GSH in HAEC, not because of oxidation but due to formation and cellular export of quercetin-glutathione conjugates. Induction by quercetin of GCL subsequently restores GSH levels, thereby

  3. 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.

  4. Glutathione and growth inhibition of Mycobacterium tuberculosis in healthy and HIV infected subjects

    PubMed Central

    Venketaraman, Vishwanath; Rodgers, Tatanisha; Linares, Rafael; Reilly, Nancy; Swaminathan, Shobha; Hom, David; Millman, Ariel C; Wallis, Robert; Connell, Nancy D

    2006-01-01

    Intracellular levels of glutathione are depleted in patients with acquired immunodeficiency syndrome in whom the risk of tuberculosis, particularly disseminated disease is many times that of healthy individuals. In this study, we examined the role of glutathione in immunity against tuberculosis infection in samples derived from healthy and human immunodeficiency virus infected subjects. Our studies confirm that glutathione levels are reduced in peripheral blood mononuclear cells and in red blood cells isolated from human immunodeficiency virus-infected subjects (CD4>400/cumm). Furthermore, treatment of blood cultures from human immunodeficiency virus infected subjects with N-acetyl cysteine, a glutathione precursor, caused improved control of intracellular M. tuberculosis infection. N-acetyl cysteine treatment decreased the levels of IL-1, TNF-α, and IL-6, and increased the levels of IFN-γ in blood cultures derived from human immunodeficiency virus-infected subjects, promoting the host immune responses to contain M. tuberculosis infection successfully. PMID:16504020

  5. Fully glutathione degradable waterborne polyurethane nanocarriers: Preparation, redox-sensitivity, and triggered intracellular drug release.

    PubMed

    Omrani, Ismail; Babanejad, Niloofar; Shendi, Hasan Kashef; Nabid, Mohammad Reza

    2017-01-01

    Polyurethanes are important class of biomaterials that are extensively used in medical devices. In spite of their easy synthesis, polyurethanes that are fully degradable in response to the intracellular reducing environment are less explored for controlled drug delivery. Herein, a novel glutathione degradable waterborne polyurethane (WPU) nanocarrier for redox triggered intracellular delivery of a model lipophilic anticancer drug, doxorubicin (DOX) is reported. The WPU was prepared from polyaddition reaction of isophorone diisocyanate (IPDI) and a novel linear polyester polyol involving disulfide linkage, disulfide labeled chain extender, dimethylolpropionic acid (DMPA) using dibutyltin dilaurate (DBTDL) as a catalyst. The resulting polyurethane self-assembles into nanocarrier in water. The dynamic light scattering (DLS) measurements and scanning electron microscope (SEM) revealed fast swelling and disruption of nanocarriers under an intracellular reduction-mimicking environment. The in vitro release studies showed that DOX was released in a controlled and redox-dependent manner. MTT assays showed that DOX-loaded WPU had a high in vitro antitumor activity in both HDF noncancer cells and MCF- 7 cancer cells. In addition, it is found that the blank WPU nanocarriers are nontoxic to HDF and MCF-7 cells even at a high concentration of 2mg/mL. Hence, nanocarriers based on disulfide labeled WPU have appeared as a new class of biocompatible and redox-degradable nanovehicle for efficient intracellular drug delivery. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Defects in a New Class of Sulfate/Anion Transporter Link Sulfur Acclimation Responses to Intracellular Glutathione Levels and Cell Cycle Control1[W][OPEN

    PubMed Central

    Fang, Su-Chiung; Chung, Chin-Lin; Chen, Chun-Han; Lopez-Paz, Cristina; Umen, James G.

    2014-01-01

    We previously identified a mutation, suppressor of mating type locus3 15-1 (smt15-1), that partially suppresses the cell cycle defects caused by loss of the retinoblastoma tumor suppressor-related protein encoded by the MAT3 gene in Chlamydomonas reinhardtii. smt15-1 single mutants were also found to have a cell cycle defect leading to a small-cell phenotype. SMT15 belongs to a previously uncharacterized subfamily of putative membrane-localized sulfate/anion transporters that contain a sulfate transporter domain and are found in a widely distributed subset of eukaryotes and bacteria. Although we observed that smt15-1 has a defect in acclimation to sulfur-limited growth conditions, sulfur acclimation (sac) mutants, which are more severely defective for acclimation to sulfur limitation, do not have cell cycle defects and cannot suppress mat3. Moreover, we found that smt15-1, but not sac mutants, overaccumulates glutathione. In wild-type cells, glutathione fluctuated during the cell cycle, with highest levels in mid G1 phase and lower levels during S and M phases, while in smt15-1, glutathione levels remained elevated during S and M. In addition to increased total glutathione levels, smt15-1 cells had an increased reduced-to-oxidized glutathione redox ratio throughout the cell cycle. These data suggest a role for SMT15 in maintaining glutathione homeostasis that impacts the cell cycle and sulfur acclimation responses. PMID:25361960

  7. 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.

  8. Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

    PubMed

    Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna

    2015-09-01

    Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. High concentration of antioxidants N-acetylcysteine and mitoquinone-Q induces intercellular adhesion molecule 1 and oxidative stress by increasing intracellular glutathione.

    PubMed

    Mukherjee, Tapan K; Mishra, Anurag K; Mukhopadhyay, Srirupa; Hoidal, John R

    2007-02-01

    In endothelial cells, the intracellular level of glutathione is depleted during offering protection against proinflammatory cytokine TNF-alpha-induced oxidative stress. Administration of anti-inflammatory drugs, i.e., N-acetylcysteine (NAC) or mitoquinone-Q (mito-Q) in low concentrations in the human pulmonary aortic endothelial cells offered protection against depletion of reduced glutathione and oxidative stress mediated by TNF-alpha. However, this study addressed that administration of NAC or mito-Q in high concentrations resulted in a biphasic response by initiating an enhanced generation of both reduced glutathione and oxidized glutathione and enhanced production of reactive oxygen species, along with carbonylation and glutathionylation of the cellular proteins. This study further addressed that IkappaB kinase (IKK), a phosphorylation-dependent regulator of NF-kappaB, plays an important regulatory role in the TNF-alpha-mediated induction of the inflammatory cell surface molecule ICAM-1. Of the two catalytic subunits of IKK (IKKalpha and IKKbeta), low concentrations of NAC and mito-Q activated IKKalpha activity, thereby inhibiting the downstream NF-kappaB and ICAM-1 induction by TNF-alpha. High concentrations of NAC and mito-Q instead caused glutathionylation of IKKalpha, thereby inhibiting its activity that in turn enhanced the downstream NF-kappaB activation and ICAM-1 expression by TNF-alpha. Thus, establishing IKKalpha as an anti-inflammatory molecule in endothelial cells is another focus of this study. This is the first report that describes a stressful situation in the endothelial cells created by excess of antioxidative and anti-inflammatory agents NAC and mito-Q, resulting in the generation of reactive oxygen species, carbonylation and glutathionylation of cellular proteins, inhibition of IKKalpha activity, and up-regulation of ICAM-1expression.

  10. Cellular copper homeostasis: current concepts on its interplay with glutathione homeostasis and its implication in physiology and human diseases.

    PubMed

    Bhattacharjee, Ashima; Chakraborty, Kaustav; Shukla, Aditya

    2017-10-18

    Copper is a trace element essential for almost all living organisms. But the level of intracellular copper needs to be tightly regulated. Dysregulation of cellular copper homeostasis leading to various diseases demonstrates the importance of this tight regulation. Copper homeostasis is regulated not only within the cell but also within individual intracellular compartments. Inactivation of export machinery results in excess copper being redistributed into various intracellular organelles. Recent evidence suggests the involvement of glutathione in playing an important role in regulating copper entry and intracellular copper homeostasis. Therefore interplay of both homeostases might play an important role within the cell. Similar to copper, glutathione balance is tightly regulated within individual cellular compartments. This review explores the existing literature on the role of glutathione in regulating cellular copper homeostasis. On the one hand, interplay of glutathione and copper homeostasis performs an important role in normal physiological processes, for example neuronal differentiation. On the other hand, perturbation of the interplay might play a key role in the pathogenesis of copper homeostasis disorders.

  11. 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

  12. 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.

  13. Cellular glutathione levels in HL-60 cells during respiratory burst are not correlated with ultra-weak photon emission.

    PubMed

    Burgos, Rosilene Cristina Rossetto; Zhang, Wei; van Wijk, Eduard P A; Hankemeier, Thomas; Ramautar, Rawi; van der Greef, Jan

    2017-10-01

    Recently, ultra-weak photon emission (UPE) was developed as a novel tool for measuring oxidative metabolic processes, as its generation is related to reactive oxygen species (ROS). Both an imbalance in ROS or the uncontrolled production of ROS can lead to oxidative stress, which is commonly associated with many diseases. In addition to playing several biological functions, the thiol amino acid glutathione has an important antioxidant function in the body's defense against ROS. Specifically, glutathione is an important endogenous antioxidant that helps maintain oxidant levels. At the cellular level, glutathione is present in its reduced form (GSH) at relatively high concentrations (in the millimolar range) and in its oxidized form (GSSG) at low concentrations (in the micromolar range). Thus, the GSH/GSSG ratio is often used as an indicator of cellular redox state. Here, we used the HL-60 cell line as a model system in order to determine whether UPE is correlated with intracellular GSH and GSSG levels. HL-60 cells were differentiated into neutrophil-like cells and then stimulated to undergo respiratory burst. We then recorded UPE in real time for 9000 seconds and used capillary electrophoresis coupled to mass spectrometry to measure GSH and GSSG levels in cell extracts. We found that although respiratory burst significantly decreased the GSH/GSSG ratio, this change was not significantly correlated with the UPE profile. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. 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

  15. Glutathione-related enzymes and the eye.

    PubMed

    Ganea, Elena; Harding, John J

    2006-01-01

    Glutathione and the related enzymes belong to the defence system protecting the eye against chemical and oxidative stress. This review focuses on GSH and two key enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase in lens, cornea, and retina. Lens contains a high concentration of reduced glutathione, which maintains the thiol groups in the reduced form. These contribute to lens complete transparency as well as to the transparent and refractive properties of the mammalian cornea, which are essential for proper image formation on the retina. In cornea, gluthatione also plays an important role in maintaining normal hydration level, and in protecting cellular membrane integrity. In retina, glutathione is distributed in the different types of retinal cells. Intracellular enzyme, glutathione reductase, involved in reducing the oxidized glutathione has been found at highest activity in human and primate lenses, as compared to other species. Besides the enzymes directly involved in maintaining the normal redox status of the cell, glucose-6-phosphate dehydrogenase which catalyzes the first reaction of the pentose phosphate pathway, plays a key role in protection of the eye against reactive oxygen species. Cornea has a high activity of the pentose phosphate pathway and glucose-6-phosphate dehydrogenase activity. Glycation, the non-enzymic reaction between a free amino group in proteins and a reducing sugar, slowly inactivates gluthathione-related and other enzymes. In addition, glutathione can be also glycated. The presence of glutathione, and of the related enzymes has been also reported in other parts of the eye, such as ciliary body and trabecular meshwork, suggesting that the same enzyme systems are present in all tissues of the eye to generate NADPH and to maintain gluthatione in the reduced form. Changes of glutathione and related enzymes activity in lens, cornea, retina and other eye tissues, occur with ageing, cataract, diabetes, irradiation and

  16. 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.

  17. A Glutathione Peroxidase, Intracellular Peptidases and the TOR Complexes Regulate Peptide Transporter PEPT-1 in C. elegans

    PubMed Central

    Benner, Jacqueline; Daniel, Hannelore; Spanier, Britta

    2011-01-01

    The intestinal peptide transporter PEPT-1 in Caenorhabditis elegans is a rheogenic H+-dependent carrier responsible for the absorption of di- and tripeptides. Transporter-deficient pept-1(lg601) worms are characterized by impairments in growth, development and reproduction and develop a severe obesity like phenotype. The transport function of PEPT-1 as well as the influx of free fatty acids was shown to be dependent on the membrane potential and on the intracellular pH homeostasis, both of which are regulated by the sodium-proton exchanger NHX-2. Since many membrane proteins commonly function as complexes, there could be proteins that possibly modulate PEPT-1 expression and function. A systematic RNAi screening of 162 genes that are exclusively expressed in the intestine combined with a functional transport assay revealed four genes with homologues existing in mammals as predicted PEPT-1 modulators. While silencing of a glutathione peroxidase surprisingly caused an increase in PEPT-1 transport function, silencing of the ER to Golgi cargo transport protein and of two cytosolic peptidases reduced PEPT-1 transport activity and this even corresponded with lower PEPT-1 protein levels. These modifications of PEPT-1 function by gene silencing of homologous genes were also found to be conserved in the human epithelial cell line Caco-2/TC7 cells. Peptidase inhibition, amino acid supplementation and RNAi silencing of targets of rapamycin (TOR) components in C. elegans supports evidence that intracellular peptide hydrolysis and amino acid concentration are a part of a sensing system that controls PEPT-1 expression and function and that involves the TOR complexes TORC1 and TORC2. PMID:21980510

  18. Glutathione Is Involved in Environmental Stress Responses in Rhizobium tropici, Including Acid Tolerance

    PubMed Central

    Riccillo, Pablo M.; Muglia, Cecilia I.; de Bruijn, Frans J.; Roe, Andrew J.; Booth, Ian R.; Aguilar, O. Mario

    2000-01-01

    The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance. Rhizobium tropici strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn5-luxAB insertion in a gene showing a high degree of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments. PMID:10692382

  19. Involvement of glutathione/glutathione S-transferase antioxidant system in butyrate-inhibited vascular smooth muscle cell proliferation.

    PubMed

    Ranganna, Kasturi; Mathew, Omana P; Yatsu, Frank M; Yousefipour, Zivar; Hayes, Barbara E; Milton, Shirlette G

    2007-11-01

    Vascular smooth muscle cell (VSMC) proliferation is an important etiological factor in vascular proliferative diseases such as primary atherosclerosis, hypertension, arterial and in-stent restenosis, and transplant vasculopathy. Our studies established that butyrate, a bacterial fermentation product of dietary fiber and a chromatin modulator, is a potent inhibitor of VSMC proliferation. The cardiovascular health benefits of a high-fiber diet, the principle source of butyrate in the body, have been known for a long time, however, very little is known about the antiatherogenic potential of butyrate. Because oxidative stress plays an important role in the pathogenesis of atherosclerosis, we examined involvement of the glutathione/glutathione S-transferase (GST) antioxidant system in butyrate's inhibition of VSMC proliferation. Treatment of proliferating VSMCs with butyrate leads to the induction of several GSTs. Interestingly, our study also demonstrated the nuclear localization of GST-P1 (GST-7-7), which is considered to be a cytosolic protein; this was demonstrated using immunostaining and was corroborated by western blotting. Also, the butyrate-induced antiproliferative action, and the induction of GST-P1 and its nuclear localization are downregulated when butyrate is withdrawn. Furthermore, assessment of intracellular glutathione levels reveals their augmentation by butyrate. Conversely, butyrate treatment reduces the levels of reactive oxygen species in VSMCs. Collectively, the butyrate-treatment-related increase in glutathione content, the reduction in reactive oxygen species, the upregulation of GST and the nuclear localization of GST-P1 in growth-arrested VSMCs imply that butyrate's antiproliferative action involves modulation of the cellular redox state. Thus, induction of the glutathione/GST antioxidant system appears to have other regulatory role(s) besides detoxification and regulation of the cellular redox state, for example, cell-cycle control and cell

  20. A mathematical modelling approach to assessing the reliability of biomarkers of glutathione metabolism.

    PubMed

    Geenen, Suzanne; du Preez, Franco B; Reed, Michael; Nijhout, H Frederik; Kenna, J Gerry; Wilson, Ian D; Westerhoff, Hans V; Snoep, Jacky L

    2012-07-16

    One of the main pathways for the detoxification of reactive metabolites in the liver involves glutathione conjugation. Metabolic profiling studies have shown paradoxical responses in glutathione-related biochemical pathways. One of these is the increase in 5-oxoproline and ophthalmic acid concentrations with increased dosage of paracetamol. Experimental studies have thus far failed to resolve these paradoxes and the robustness of how these proposed biomarkers correlate with liver glutathione levels has been questioned. To better understand how these biomarkers behave in the glutathione system a kinetic model of this pathway was made. By using metabolic control analysis and by simulating biomarker levels under a variety of conditions, we found that 5-oxoproline and ophthalmic acid concentrations may not only depend on the glutathione but also on the methionine status of the cell. We show that neither of the two potential biomarkers are reliable on their own since they need additional information about the methionine status of the system to relate them uniquely to intracellular glutathione concentration. However, when both biomarkers are measured simultaneously a direct inference of the glutathione concentration can be made, irrespective of the methionine concentration in the system. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. The Incomplete Glutathione Puzzle: Just Guessing at Numbers and Figures?

    PubMed

    Deponte, Marcel

    2017-11-20

    Glutathione metabolism is comparable to a jigsaw puzzle with too many pieces. It is supposed to comprise (i) the reduction of disulfides, hydroperoxides, sulfenic acids, and nitrosothiols, (ii) the detoxification of aldehydes, xenobiotics, and heavy metals, and (iii) the synthesis of eicosanoids, steroids, and iron-sulfur clusters. In addition, glutathione affects oxidative protein folding and redox signaling. Here, I try to provide an overview on the relevance of glutathione-dependent pathways with an emphasis on quantitative data. Recent Advances: Intracellular redox measurements reveal that the cytosol, the nucleus, and mitochondria contain very little glutathione disulfide and that oxidative challenges are rapidly counterbalanced. Genetic approaches suggest that iron metabolism is the centerpiece of the glutathione puzzle in yeast. Furthermore, recent biochemical studies provide novel insights on glutathione transport processes and uncoupling mechanisms. Which parts of the glutathione puzzle are most relevant? Does this explain the high intracellular concentrations of reduced glutathione? How can iron-sulfur cluster biogenesis, oxidative protein folding, or redox signaling occur at high glutathione concentrations? Answers to these questions not only seem to depend on the organism, cell type, and subcellular compartment but also on different ideologies among researchers. A rational approach to compare the relevance of glutathione-dependent pathways is to combine genetic and quantitative kinetic data. However, there are still many missing pieces and too little is known about the compartment-specific repertoire and concentration of numerous metabolites, substrates, enzymes, and transporters as well as rate constants and enzyme kinetic patterns. Gathering this information might require the development of novel tools but is crucial to address potential kinetic competitions and to decipher uncoupling mechanisms to solve the glutathione puzzle. Antioxid. Redox Signal

  2. 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.

  3. The mechanism of improved intracellular organic selenium and glutathione contents in selenium-enriched Candida utilis by acid stress.

    PubMed

    Zhang, Gao-Chuan; Wang, Da-Hui; Wang, Dong-Hua; Wei, Gong-Yuan

    2017-03-01

    Batch culture of Candida utilis CCTCC M 209298 for the preparation of selenium (Se)-enriched yeast was carried out under different pH conditions, and maximal intracellular organic Se and glutathione (GSH) contents were obtained in a moderate acid stress environment (pH 3.5). In order to elucidate the physiological mechanism of improved performance of Se-enriched yeast by acid stress, assays of the key enzymes involved in GSH biosynthesis and determinations of energy supply and regeneration were performed. The results indicated that moderate acid stress increased the activity of γ-glutamylcysteine synthetase and the ratios of NADH/NAD + and ATP/ADP, although no significant changes in intracellular pH were observed. In addition, the molecular mechanism of moderate acid stress favoring the improvement of Se-yeast performance was revealed by comparing whole transcriptomes of yeast cells cultured at pH 3.5 and 5.5. Comparative analysis of RNA-Seq data indicated that 882 genes were significantly up-regulated by moderate acid stress. Functional annotation of the up-regulated genes based on gene ontology and the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway showed that these genes are involved in ATP synthesis and sulfur metabolism, including the biosynthesis of methionine, cysteine, and GSH in yeast cells. Increased intracellular ATP supply and more amounts of sulfur-containing substances in turn contributed to Na 2 SeO 3 assimilation and biotransformation, which ultimately improved the performance of the Se-enriched C. utilis.

  4. The Incomplete Glutathione Puzzle: Just Guessing at Numbers and Figures?

    PubMed Central

    2017-01-01

    Abstract Significance: Glutathione metabolism is comparable to a jigsaw puzzle with too many pieces. It is supposed to comprise (i) the reduction of disulfides, hydroperoxides, sulfenic acids, and nitrosothiols, (ii) the detoxification of aldehydes, xenobiotics, and heavy metals, and (iii) the synthesis of eicosanoids, steroids, and iron–sulfur clusters. In addition, glutathione affects oxidative protein folding and redox signaling. Here, I try to provide an overview on the relevance of glutathione-dependent pathways with an emphasis on quantitative data. Recent Advances: Intracellular redox measurements reveal that the cytosol, the nucleus, and mitochondria contain very little glutathione disulfide and that oxidative challenges are rapidly counterbalanced. Genetic approaches suggest that iron metabolism is the centerpiece of the glutathione puzzle in yeast. Furthermore, recent biochemical studies provide novel insights on glutathione transport processes and uncoupling mechanisms. Critical Issues: Which parts of the glutathione puzzle are most relevant? Does this explain the high intracellular concentrations of reduced glutathione? How can iron–sulfur cluster biogenesis, oxidative protein folding, or redox signaling occur at high glutathione concentrations? Answers to these questions not only seem to depend on the organism, cell type, and subcellular compartment but also on different ideologies among researchers. Future Directions: A rational approach to compare the relevance of glutathione-dependent pathways is to combine genetic and quantitative kinetic data. However, there are still many missing pieces and too little is known about the compartment-specific repertoire and concentration of numerous metabolites, substrates, enzymes, and transporters as well as rate constants and enzyme kinetic patterns. Gathering this information might require the development of novel tools but is crucial to address potential kinetic competitions and to decipher uncoupling

  5. Dorsal Anterior Cingulate Lactate and Glutathione Levels in Euthymic Bipolar I Disorder: 1H-MRS Study.

    PubMed

    Soeiro-de-Souza, Márcio Gerhardt; Pastorello, Bruno F; Leite, Cláudia da Costa; Henning, Anke; Moreno, Ricardo A; Garcia Otaduy, Maria Concepción

    2016-08-01

    Oxidative stress and mitochondrial dysfunction are 2 closely integrated processes implicated in the physiopathology of bipolar disorder. Advanced proton magnetic resonance spectroscopy techniques enable the measurement of levels of lactate, the main marker of mitochondrial dysfunction, and glutathione, the predominant brain antioxidant. The objective of this study was to measure brain lactate and glutathione levels in bipolar disorder and healthy controls. Eighty-eight individuals (50 bipolar disorder and 38 healthy controls) underwent 3T proton magnetic resonance spectroscopy in the dorsal anterior cingulate cortex (2x2x4.5cm(3)) using a 2-D JPRESS sequence. Lactate and glutathione were quantified using the ProFit software program. Bipolar disorder patients had higher dorsal anterior cingulate cortex lactate levels compared with controls. Glutathione levels did not differ between euthymic bipolar disorder and controls. There was a positive correlation between lactate and glutathione levels specific to bipolar disorder. No influence of medications on metabolites was observed. This is the most extensive magnetic resonance spectroscopy study of lactate and glutathione in bipolar disorder to date, and results indicated that euthymic bipolar disorder patients had higher levels of lactate, which might be an indication of altered mitochondrial function. Moreover, lactate levels correlated with glutathione levels, indicating a compensatory mechanism regardless of bipolar disorder diagnosis. © The Author 2016. Published by Oxford University Press on behalf of CINP.

  6. Dorsal Anterior Cingulate Lactate and Glutathione Levels in Euthymic Bipolar I Disorder: 1H-MRS Study

    PubMed Central

    Pastorello, Bruno F.; Leite, Cláudia da Costa; Henning, Anke; Moreno, Ricardo A.; Garcia Otaduy, Maria Concepción

    2016-01-01

    Objective: Oxidative stress and mitochondrial dysfunction are 2 closely integrated processes implicated in the physiopathology of bipolar disorder. Advanced proton magnetic resonance spectroscopy techniques enable the measurement of levels of lactate, the main marker of mitochondrial dysfunction, and glutathione, the predominant brain antioxidant. The objective of this study was to measure brain lactate and glutathione levels in bipolar disorder and healthy controls. Methods: Eighty-eight individuals (50 bipolar disorder and 38 healthy controls) underwent 3T proton magnetic resonance spectroscopy in the dorsal anterior cingulate cortex (2x2x4.5cm3) using a 2-D JPRESS sequence. Lactate and glutathione were quantified using the ProFit software program. Results: Bipolar disorder patients had higher dorsal anterior cingulate cortex lactate levels compared with controls. Glutathione levels did not differ between euthymic bipolar disorder and controls. There was a positive correlation between lactate and glutathione levels specific to bipolar disorder. No influence of medications on metabolites was observed. Conclusion: This is the most extensive magnetic resonance spectroscopy study of lactate and glutathione in bipolar disorder to date, and results indicated that euthymic bipolar disorder patients had higher levels of lactate, which might be an indication of altered mitochondrial function. Moreover, lactate levels correlated with glutathione levels, indicating a compensatory mechanism regardless of bipolar disorder diagnosis. PMID:27207914

  7. Phytochelatin synthesis and glutathione levels in response to heavy metals in tomato cells.

    PubMed

    Scheller, H V; Huang, B; Hatch, E; Goldsbrough, P B

    1987-12-01

    Cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, produce phytochelatins (poly[gamma-glutamylcysteinyl]glycines) when exposed to cadmium. The synthesis of these peptides is accompanied by a decline in cellular levels of glutathione. Buthionine sulfoximine, an inhibitor of glutathione synthesis, inhibits the sustained production of phytochelatins. However, phytochelatin synthesis can occur in the presence of buthionine sulfoximine provided that sufficient glutathione is available. These results indicate that glutathione is a substrate for phytochelatin synthesis. The protein synthesis inhibitor cycloheximide does not affect the initial production of phytochelatin.

  8. 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

  9. 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

  10. 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.

  11. Semen Quality of Post-Thawed Local Ram’s in Tris-Egg Yolk Extender with Different Glutathione Level

    NASA Astrophysics Data System (ADS)

    Solihati, N.; Rasad, S. D.; Setiawan, R.; Foziah, E. N.; Wigiyanti, E. T.

    2018-02-01

    The aims of this research were to find out the effect of glutathione level on semen quality of local ram and to find out the glutathione level that provide the best quality of local ram semen. This research use Completely Randomized Design (CRD) with five treatments of glutathione level (0 mM, 4 mM, 5 mM, 6 mM and 7mM) and was repeated for 10 times. Data were analyzed using analysis of varians (ANOVA) and differences between treatment was analyzed using Duncan test. The parameter were semen quality consist of motility, intact plasma membrane (IPM), abnormality, and recovery rate. Result of this research showed that glutathione level significantly (p<0.05) affect on motility, IPM, abnormality and recovery rate. Duncan test result showed that recovery rate from treatment of 5 mM glutathione level have significantly higher than other treatment, whereas for motility, IPM and abnormality were not significant different with 4 mM, and 6 mM. It is concluded that semen quality of local ram was affected by glutathione level, and the level of 5 mM in egg yolk tris extender provide the best quality of local ram semen.

  12. [Association between glutathione peroxidase levels and clinical manifestations of dengue].

    PubMed

    Rojas, Elsa Marina; Díaz-Quijano, Fredi Alexander; Coronel-Ruiz, Carolina; Martínez-Vega, Ruth Aralf; Rueda, Ernesto; Villar-Centeno, Luis Angel

    2007-06-01

    Glutathione peroxidase (GP) can be used as a marker of oxidative stress in infectious diseases. To evaluate the association between the levels of glutathione peroxidase (GP) and the manifestations and complications of dengue. Between April 2003 and December 2004, 161 patients with dengue were prospectively evaluated. In the first evaluation, within 48 and 96 hours of disease onset, a plasma sample was obtained to measure the GP levels. The association between GP levels, clinical manifestations and complications was evaluated during the follow up. Mean GP values were 1198 U/L (95% confidence interval 1089-1306). Values greater than 1200 U/L were associated with headache, arthralgias and increased heart rate. There was a negative association between GP levels and serum triglycerides. During follow up, patients with GP >1200 U/L had a higher frequency of spontaneous hemorrhages. In a logistic regression analysis arthralgias, fever and increased heart rate, were independently associated with levels >1200 U/L. GP levels was associated to some of the manifestations of dengue. This finding suggests that the intensity of oxidative stress can influence the clinical presentation of dengue.

  13. ZnO Nanoparticles Treatment Induces Apoptosis by Increasing Intracellular ROS Levels in LTEP-a-2 Cells.

    PubMed

    Wang, Caixia; Hu, Xiaoke; Gao, Yan; Ji, Yinglu

    2015-01-01

    Owing to the wide use of novel nanoparticles (NPs) such as zinc oxide (ZnO) in all aspects of life, toxicological research on ZnO NPs is receiving increasing attention in these days. In this study, the toxicity of ZnO NPs in a human pulmonary adenocarcinoma cell line LTEP-a-2 was tested in vitro. Log-phase cells were exposed to different levels of ZnO NPs for hours, followed by colorimetric cell viability assay using tetrazolium salt and cell survival rate assay using trypan blue dye. Cell morphological changes were observed by Giemsa staining and light microscopy. Apoptosis was detected by using fluorescence microscopy and caspase-3 activity assay. Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method. Results showed that ZnO NPs (≥ 0.01 μg/mL) significantly inhibited proliferation (P < 0.05) and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure. The intracellular ROS level rose up to 30-40% corresponding to significant depletion (approximately 70-80%) in GSH content in LTEP-a-2 cells (P < 0.05), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production. This study elucidates the toxicological mechanism of ZnO NPs in human pulmonary adenocarcinoma cells and provides reference data for application of nanomaterials in the environment.

  14. Cell Proliferation, Reactive Oxygen and Cellular Glutathione

    PubMed Central

    Day, Regina M.; Suzuki, Yuichiro J.

    2005-01-01

    A variety of cellular activities, including metabolism, growth, and death, are regulated and modulated by the redox status of the environment. A biphasic effect has been demonstrated on cellular proliferation with reactive oxygen species (ROS)—especially hydrogen peroxide and superoxide—in which low levels (usually submicromolar concentrations) induce growth but higher concentrations (usually >10–30 micromolar) induce apoptosis or necrosis. This phenomenon has been demonstrated for primary, immortalized and transformed cell types. However, the mechanism of the proliferative response to low levels of ROS is not well understood. Much of the work examining the signal transduction by ROS, including H2O2, has been performed using doses in the lethal range. Although use of higher ROS doses have allowed the identification of important signal transduction pathways, these pathways may be activated by cells only in association with ROS-induced apoptosis and necrosis, and may not utilize the same pathways activated by lower doses of ROS associated with increased cell growth. Recent data has shown that low levels of exogenous H2O2 up-regulate intracellular glutathione and activate the DNA binding activity toward antioxidant response element. The modulation of the cellular redox environment, through the regulation of cellular glutathione levels, may be a part of the hormetic effect shown by ROS on cell growth. PMID:18648617

  15. Immunoregulatory effects of glutathione during mesenchymal stem cell differentiation to hepatocyte-like cells.

    PubMed

    Ahmadi-Ashtiani, Hamid-Reza; Allameh, Abdolamir; Rastegar, Hossein; Mortaz, Esmaeil; Saraf, Zahir

    2012-09-01

    The role of mesenchymal stem cell in cellular therapy is the subject of interest for many researchers. The differentiation potential of MSCs and abilities in modulations of the recipient's immune system makes them important cells in tissue regenerative studies. MSCs by releasing the proinflammatory cytokines play important role in immunomodulatory systems; however the signaling pathways for releasing of these mediators are not well understood. Glutathione has been shown to play a role in modulation of cytokines in hepatogenic differentiation. In the current study we aimed to investigate the effects of buthionine sulfoximine (BSO, inhibitor for glutathione synthesis) and N-acetylecystin (NAC, an inhibitor for ROS generation) on proinflammatory cytokines production in a hepatogenic differentiation model. BSO and NAC significantly decreased IL-6 and TNF-α levels at 14 days of differentiation, whereas, NAC decreased the levels of IL-8 at days 2 and 14 of differentiation. Moreover, intracellular glutathione level during the differentiation was depleted. Our current study suggests a novel role of GSH as an immunopharmacological regulatory molecule during hepatogenic differentiation. Finally, this information may shed some light on the understanding of MSCs responses in transplantation and cell therapy in diseases such as chronic hepatic diseases.

  16. 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.

  17. A synthetic chalcone as a potent inducer of glutathione biosynthesis.

    PubMed

    Kachadourian, Remy; Day, Brian J; Pugazhenti, Subbiah; Franklin, Christopher C; Genoux-Bastide, Estelle; Mahaffey, Gregory; Gauthier, Charlotte; Di Pietro, Attilio; Boumendjel, Ahcène

    2012-02-09

    Chalcones continue to attract considerable interest due to their anti-inflammatory and antiangiogenic properties. We recently reported the ability of 2',5'-dihydroxychalcone (2',5'-DHC) to induce both breast cancer resistance protein-mediated export of glutathione (GSH) and c-Jun N-terminal kinase-mediated increased intracellular GSH levels. Herein, we report a structure-activity relationship study of a series of 30 synthetic chalcone derivatives with hydroxyl, methoxyl, and halogen (F and Cl) substituents and their ability to increase intracellular GSH levels. This effect was drastically improved with one or two electrowithdrawing groups on phenyl ring B and up to three methoxyl and/or hydroxyl groups on phenyl ring A. The optimal structure, 2-chloro-4',6'-dimethoxy-2'-hydroxychalcone, induced both a potent NF-E2-related factor 2-mediated transcriptional response and an increased formation of glutamate cysteine ligase holoenzyme, as shown using a human breast cancer cell line stably expressing a luciferase reporter gene driven by antioxidant response elements.

  18. Glutathione oxidation in response to intracellular H2O2: Key but overlapping roles for dehydroascorbate reductases.

    PubMed

    Rahantaniaina, Marie-Sylviane; Li, Shengchun; Chatel-Innocenti, Gilles; Tuzet, Andrée; Mhamdi, Amna; Vanacker, Hélène; Noctor, Graham

    2017-08-03

    Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H 2 O 2 detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function gstU8, gstU24, gstF8, prxIIE and prxIIF mutants as well as double gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H 2 O 2 metabolism.

  19. Decreased glutathione levels and impaired antioxidant enzyme activities in drug-naive first-episode schizophrenic patients

    PubMed Central

    2011-01-01

    Background The aim of this study was to determine glutathione levels and antioxidant enzyme activities in the drug-naive first-episode patients with schizophrenia in comparison with healthy control subjects. Methods It was a case-controlled study carried on twenty-three patients (20 men and 3 women, mean age = 29.3 ± 7.5 years) recruited in their first-episode of schizophrenia and 40 healthy control subjects (36 men and 9 women, mean age = 29.6 ± 6.2 years). In patients, the blood samples were obtained prior to the initiation of neuroleptic treatments. Glutathione levels: total glutathione (GSHt), reduced glutathione (GSHr) and oxidized glutathione (GSSG) and antioxidant enzyme activities: superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) were determined by spectrophotometry. Results GSHt and reduced GSHr were significantly lower in patients than in controls, whereas GSSG was significantly higher in patients. GPx activity was significantly higher in patients compared to control subjects. CAT activity was significantly lower in patients, whereas the SOD activity was comparable to that of controls. Conclusion This is a report of decreased plasma levels of GSHt and GSHr, and impaired antioxidant enzyme activities in drug-naive first-episode patients with schizophrenia. The GSH deficit seems to be implicated in psychosis, and may be an important indirect biomarker of oxidative stress in schizophrenia early in the course of illness. Finally, our results provide support for further studies of the possible role of antioxidants as neuroprotective therapeutic strategies for schizophrenia from early stages. PMID:21810251

  20. Enhancement of the depigmenting effect of hydroquinone and 4-hydroxyanisole by all-trans-retinoic acid (tretinoin): the impairment of glutathione-dependent cytoprotection?

    PubMed

    Kasraee, Behrooz; Handjani, Farhad; Aslani, Fatemeh S

    2003-01-01

    Many of the well-known depigmenting agents such as hydroquinone and 4-hydroxyanisole are, in fact, melanocytotoxic chemicals which are oxidized in melanocytes to produce highly toxic compounds such as quinones. These cytotoxic compounds are responsible for the destruction of pigment cells, which results in skin depigmentation. However, cells are capable of protecting themselves against cytotoxic agents by intracellular glutathione (GSH). This protection takes place under the enzymatic action of the detoxification enzyme glutathione S-transferase (GST), which is responsible for the conjugation of toxic species to GSH. The depigmenting effect of hydroquinone is shown to be potentiated by buthionine sulfoximine (BSO) and cystamine as the result of the reduction of intracellular levels of GSH by these two agents. Additionally, BSO and cystamine are shown to inhibit the activity of GST. The combination of all-trans-retinoic acid (tretinoin, TRA) with hydroquinone or 4-hydroxyanisole is also known to produce synergetic skin depigmentation. TRA serves as a potent inhibitor of mammalian GSTs and is known to make cells more susceptible to the cytotoxic effect of chemicals by inhibiting the activity of this enzyme. This agent is also shown to reduce the level of intracellular GSH in certain cells. We have proposed that the mechanism of action of TRA to synergistically enhance the melanocytotoxic effect of chemicals involves the inhibition of GST and the impairment of glutathione-dependent cytoprotection against melanocytotoxic agents. Copyright 2003 S. Karger AG, Basel

  1. Glutathione in cyanobacteria

    NASA Technical Reports Server (NTRS)

    Bermudes, D.

    1985-01-01

    The effects of light and O2 on glutathione production were determined. Results of light and dark studies under normal and reduced oxygen tensions were compared to determine the effect of reduction in oxygen tension on glutathione levels. The growth rate of Anacystis nidulans and concurrent production of glutathione is presented. The generation of time of Anacystis nidulans was approximately 12 hours. Results of light and dark incubation of Aphanothece halophytica dominated planktonic microbial community from Pond 4 and Anacystis nidulans under high and low oxygen tension is also presented. It appears that light grown Anacystis nidulans cells have equal amounts of glutathione while dark grown cells produce more glutathione in the presence of increased O2.

  2. A Dispersion-Dominated Chromogenic Strategy for Colorimetric Sensing of Glutathione at the Nanomolar Level Using Gold Nanoparticles.

    PubMed

    Xianyu, Yunlei; Xie, Yangzhouyun; Wang, Nuoxin; Wang, Zhuo; Jiang, Xingyu

    2015-11-04

    A dispersion-dominated chromogenic strategy for glutathione sensing is developed. Glutathione prevents the aggregation of arginine-modified gold nanoparticles via mercury-thiol interaction, which allows for glutathione sensing at the nanomolar level (10.9 × 10(-9) m) with facile operation and naked-eye readout. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Correction of glutathione deficiency in the lower respiratory tract of HIV seropositive individuals by glutathione aerosol treatment.

    PubMed

    Holroyd, K J; Buhl, R; Borok, Z; Roum, J H; Bokser, A D; Grimes, G J; Czerski, D; Cantin, A M; Crystal, R G

    1993-10-01

    Concentrations of glutathione, a ubiquitous tripeptide with immune enhancing and antioxidant properties, are decreased in the blood and lung epithelial lining fluid of human immunodeficiency virus (HIV) seropositive individuals. Since the lung is the most common site of infection in those who progress to AIDS it is rational to consider whether it is possible to safely augment glutathione levels in the epithelial lining fluid of HIV seropositive individuals, thus potentially improving local host defence. Purified reduced glutathione was delivered by aerosol to HIV seropositive individuals (n = 14) and the glutathione levels in lung epithelial lining fluid were compared before and at one, two, and three hours after aerosol administration. Before treatment total glutathione concentrations in the epithelial lining fluid were approximately 60% of controls. After three days of twice daily doses each of 600 mg reduced glutathione, total glutathione levels in the epithelial lining fluid increased and remained in the normal range for at least three hours after treatment. Strikingly, even though > 95% of the glutathione in the aerosol was in its reduced form, the percentage of oxidised glutathione in epithelial lining fluid increased from 5% before treatment to about 40% three hours after treatment, probably reflecting the use of glutathione as an antioxidant in vivo. No adverse effects were observed. It is feasible and safe to use aerosolised reduced glutathione to augment the deficient glutathione levels of the lower respiratory tract of HIV seropositive individuals. It is rational to evaluate further the efficacy of this tripeptide in improving host defence in HIV seropositive individuals.

  4. Correction of glutathione deficiency in the lower respiratory tract of HIV seropositive individuals by glutathione aerosol treatment.

    PubMed Central

    Holroyd, K. J.; Buhl, R.; Borok, Z.; Roum, J. H.; Bokser, A. D.; Grimes, G. J.; Czerski, D.; Cantin, A. M.; Crystal, R. G.

    1993-01-01

    BACKGROUND--Concentrations of glutathione, a ubiquitous tripeptide with immune enhancing and antioxidant properties, are decreased in the blood and lung epithelial lining fluid of human immunodeficiency virus (HIV) seropositive individuals. Since the lung is the most common site of infection in those who progress to AIDS it is rational to consider whether it is possible to safely augment glutathione levels in the epithelial lining fluid of HIV seropositive individuals, thus potentially improving local host defence. METHODS--Purified reduced glutathione was delivered by aerosol to HIV seropositive individuals (n = 14) and the glutathione levels in lung epithelial lining fluid were compared before and at one, two, and three hours after aerosol administration. RESULTS--Before treatment total glutathione concentrations in the epithelial lining fluid were approximately 60% of controls. After three days of twice daily doses each of 600 mg reduced glutathione, total glutathione levels in the epithelial lining fluid increased and remained in the normal range for at least three hours after treatment. Strikingly, even though > 95% of the glutathione in the aerosol was in its reduced form, the percentage of oxidised glutathione in epithelial lining fluid increased from 5% before treatment to about 40% three hours after treatment, probably reflecting the use of glutathione as an antioxidant in vivo. No adverse effects were observed. CONCLUSIONS--It is feasible and safe to use aerosolised reduced glutathione to augment the deficient glutathione levels of the lower respiratory tract of HIV seropositive individuals. It is rational to evaluate further the efficacy of this tripeptide in improving host defence in HIV seropositive individuals. PMID:8256245

  5. Blocking Lactate Export by Inhibiting the Myc Target MCT1 Disables Glycolysis and Glutathione Synthesis

    PubMed Central

    Doherty, Joanne R.; Yang, Chunying; Scott, Kristen E. N.; Cameron, Michael D.; Fallahi, Mohammad; Li, Weimin; Hall, Mark A.; Amelio, Antonio L.; Mishra, Jitendra K.; Li, Fangzheng; Tortosa, Mariola; Genau, Heide Marika; Rounbehler, Robert J.; Lu, Yunqi; Dang, Chi. V.; Kumar, K. Ganesh; Butler, Andrew A.; Bannister, Thomas D.; Hooper, Andrea T.; Unsal-Kacmaz, Keziban; Roush, William R.; Cleveland, John L.

    2014-01-01

    Myc oncoproteins induce genes driving aerobic glycolysis, including lactate dehydrogenase-A that generates lactate. Here we report that Myc controls transcription of the lactate transporter SLC16A1/MCT1, and that elevated MCT1 levels are manifest in premalignant and neoplastic Eμ-Myc transgenic B cells and in human malignancies with MYC or MYCN involvement. Notably, disrupting MCT1 function leads to an accumulation of intracellular lactate that rapidly disables tumor cell growth and glycolysis, provoking marked alterations in glycolytic intermediates, and reductions in glucose transport, and in levels of ATP, NADPH and glutathione. Reductions in glutathione then lead to increases in hydrogen peroxide, mitochondrial damage and, ultimately, cell death. Finally, forcing glycolysis by metformin treatment augments this response and the efficacy of MCT1 inhibitors, suggesting an attractive combination therapy for MYC/MCT1-expressing malignancies. PMID:24285728

  6. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

    PubMed

    Mazzari, Andre L D A; Milton, Flora; Frangos, Samantha; Carvalho, Ana C B; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum.

  7. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

    PubMed Central

    Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  8. Glutathione transferases, regulators of cellular metabolism and physiology.

    PubMed

    Board, Philip G; Menon, Deepthi

    2013-05-01

    The cytosolic glutathione transferases (GSTs) comprise a super family of proteins that can be categorized into multiple classes with a mixture of highly specific and overlapping functions. The review covers the genetics, structure and function of the human cytosolic GSTs with particular attention to their emerging roles in cellular metabolism. All the catalytically active GSTs contribute to the glutathione conjugation or glutathione dependant-biotransformation of xenobiotics and many catalyze glutathione peroxidase or thiol transferase reactions. GSTs also catalyze glutathione dependent isomerization reactions required for the synthesis of several prostaglandins and steroid hormones and the catabolism of tyrosine. An increasing body of work has implicated several GSTs in the regulation of cell signaling pathways mediated by stress-activated kinases like Jun N-terminal kinase. In addition, some members of the cytosolic GST family have been shown to form ion channels in intracellular membranes and to modulate ryanodine receptor Ca(2+) channels in skeletal and cardiac muscle. In addition to their well established roles in the conjugation and biotransformation of xenobiotics, GSTs have emerged as significant regulators of pathways determining cell proliferation and survival and as regulators of ryanodine receptors that are essential for muscle function. This article is part of a Special Issue entitled Cellular functions of glutathione. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. A Synthetic Chalcone as a Potent Inducer of Glutathione Biosynthesis

    PubMed Central

    Kachadourian, Remy; Day, Brian J.; Pugazhenti, Subbiah; Franklin, Christopher C.; Genoux-Bastide, Estelle; Mahaffey, Gregory; Gauthier, Charlotte; Di Pietro, Attilio; Boumendjel, Ahcène

    2014-01-01

    Chalcones continue to attract considerable interest due to their anti-inflammatory and antiangiogenic properties. We recently reported the ability of 2′,5′-dihydroxychalcone (2′,5′-DHC) to induce both breast cancer resistance protein-mediated export of glutathione (GSH) and c-Jun N-terminal kinase-mediated increased intracellular GSH levels. Herein, we report a structure–activity relationship study of a series of 30 synthetic chalcone derivatives with hydroxyl, methoxyl, and halogen (F and Cl) substituents and their ability to increase intracellular GSH levels. This effect was drastically improved with one or two electrowithdrawing groups on phenyl ring B and up to three methoxyl and/or hydroxyl groups on phenyl ring A. The optimal structure, 2-chloro-4′,6′-dimethoxy-2′-hydroxychalcone, induced both a potent NF-E2-related factor 2-mediated transcriptional response and an increased formation of glutamate cysteine ligase holoenzyme, as shown using a human breast cancer cell line stably expressing a luciferase reporter gene driven by antioxidant response elements. PMID:22239485

  10. Glutathione, glutathione-related enzymes, and oxidative stress in individuals with subacute occupational exposure to lead.

    PubMed

    Dobrakowski, Michał; Pawlas, Natalia; Hudziec, Edyta; Kozłowska, Agnieszka; Mikołajczyk, Agnieszka; Birkner, Ewa; Kasperczyk, Sławomir

    2016-07-01

    The aim of the study was to investigate the influence of subacute exposure to lead on the glutathione-related antioxidant defense and oxidative stress parameters in 36 males occupationally exposed to lead for 40±3.2days. Blood lead level in the examined population increased significantly by 359% due to lead exposure. Simultaneously, erythrocyte glutathione level decreased by 16%, whereas the activity of glutathione-6-phosphate dehydrogenase in erythrocytes and leukocytes decreased by 28% and 10%, respectively. Similarly, the activity of glutathione-S-transferase in erythrocytes decreased by 45%. However, the activity of glutathione reductase in erythrocytes and leukocytes increased by 26% and 6%, respectively, whereas the total oxidant status value in leukocytes increased by 37%. Subacute exposure to lead results in glutathione pool depletion and accumulation of lipid peroxidation products; however, it does not cause DNA damage. Besides, subacute exposure to lead modifies the activity of glutathione-related enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Alterations in brain glutathione homeostasis induced by the nerve gas soman.

    PubMed

    Klaidman, Lori K; Adams, James D; Cross, Robert; Pazdernik, Thomas L; Samson, Fred

    2003-01-01

    Public awareness of the dangers of chemical and biological warfare has been heightened in recent times. In particular, chemical nerve agents such as soman and its analogs have been developed and used in war as well as recent incidents, such as in Iraq and Japan. Soman, a rapid acting acetylcholinesterase inhibitor, produces a status epilepticus that leads to extensive neuropathology in vulnerable brain regions (eg, piriform cortex and hippocampus). This study was undertaken to determine whether oxidative mechanisms are involved in brain pathology during soman toxicity. Intracellular thiols such as glutathione (GSH) and protein sulfhydryls (PrSH) are among the most critical antioxidants used to combat oxidative stress. Here we report that during the seizure phase (1 h post soman exposure), PrSH levels in piriform cortex and hippocampus were decreased without changes in glutathione (GSH) levels. However, by 24 h post soman exposure (pathology phase), GSH levels were decreased by nearly 50% in the piriform cortex with a corresponding decrease in PrSH groups. The shift to a more oxidized thiol status indicates that oxygen free radicals likely participate in the neuropathology associated with soman-induced seizures.

  12. 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

  13. Glutathione metabolism as a determinant of therapeutic efficacy: a review.

    PubMed

    Arrick, B A; Nathan, C F

    1984-10-01

    Glutathione, as the chief nonprotein intracellular sulfhydryl, affects the efficacy and interactions of a variety of antineoplastic interventions, mainly through nucleophilic thioether formation or oxidation-reduction reactions. Thus, glutathione plays a role in the detoxification and repair of cellular injury by such diverse agents as mechlorethamine, melphalan, cyclophosphamide, nitrosoureas, 6-thiopurine, 4'-(9-acridinylamino)methanesulfon-m-anisidide, the quinone antibiotics (including Adriamycin, daunorubicin, and mitomycin C), the sesquiterpene lactones (such as vernolepin), and other sulfhydryl-reactive diterpenes (like jatrophone). Glutathione may play a similar role in host and tumor cell responses to radiation, hyperthermia, and the reactive reduction products of oxygen secreted by inflammatory cells. Further, glutathione participates in the formation of toxic metabolites of such chemotherapeutics as azathioprine and bleomycin and may affect the cellular uptake of other agents, such as methotrexate. It seems likely that alterations in glutathione metabolism of tumor or host as a result of one therapeutic intervention may affect the outcome of concurrent treatments. Knowledge of these interactions may be useful in designing combination therapy for neoplastic disease.

  14. The Assessments of the Intracellular Antioxidant Protection of the Organism after LLLT Irradiation

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

    Freitinger-Skalicka, Zuzana; Navratil, Leos; Zolzer, Friedo

    2009-06-19

    The antioxidants are chemical compounds that can bind to free oxygen radicals preventing these radicals from damaging healthy cells. Low levels of antioxidants, or inhibition of the antioxidant enzymes causes oxidative stress and may damage or kill cells. The purpose of this project was to establish the changes at intracellular antioxidant protection of the organism after LLLT irradiation. We used female mice of the strain CD1. The mice were exposed in the abdomen region to laser light. From the blood was assessment the Glutathione peroxidase, Reduced Glutathione and Plasma Antioxidant Capacity. The results obtained in the present study demonstrated thatmore » in vivo irradiation of the mice with low level lasers did not cause any statistically significant changes in superoxide dismutase and Glutathione peroxidase but we found changes in Reduced Glutathione and Plasma Antioxidant Capacity after exposing the mice to the LLLT during the 30 minutes after irradiation, as well on the 4th day. Do not replace the word ''abstract,'' but do replace the rest of this text. If you must insert a hard line break, please use Shift+Enter rather than just tapping your ''Enter'' key. You may want to print this page and refer to it as a style sample before you begin working on your paper.« less

  15. Effects Of Endothelin-1 On Intracellular Tetrahydrobiopterin Levels In Vascular Tissue.

    PubMed

    Cerrato, Ruha; Crabtree, Mark; Antoniades, Charalambos; Kublickiene, Karolina; Schiffrin, Ernesto L; Channon, Keith M; Böhm, Felix

    2018-03-23

    Tetrahydrobiopterin (BH4) is the essential cofactor of endothelial nitric oxide synthase (eNOS) and intracellular levels of BH4 is regulated by oxidative stress. The aim of this paper was to describe the influence of exogenous endothelin-1 on intracellular BH4 and its oxidation products dihydrobiopterin (BH2) and biopterin (B) in a wide range of vascular tissue. Segments of internal mammary artery (IMA) and human saphenous vein (SV) from 41 patients undergoing elective surgery were incubated in ET-1 (0.1 μM). Aorta and lung from transgenic mice overexpressing ET-1 in the endothelium (ET-TG) were analysed with regards to intracellular biopterin levels. Human umbilical vein endothelial cells (HUVEC) were incubated in ET-1 (0.1 μM) and intracellular biopterin levels were analysed. From 6 healthy women undergoing caesarean section, subcutaneous fat was harvested and the resistance arteries in these biopsies were tested for ET-mediated endothelial dysfunction. In HUVEC, exogenous ET-1 (0.1 μM) did not significantly change intracellular BH4, 1.54 ± 1.7 vs 1.68 ± 1.8 pmol/mg protein; p = .8. In IMA and SV, exogenous ET-1(0.1 μM) did not change intracellular BH4 n = 10, p = .4. In aorta from wild type vs ET-TG mice there was no significant difference in intracellular BH4 between the groups: 1.3 ± 0.49 vs 1.23 ± 0.3 pmol/mg protein; p = .6. In resistance arteries (n = 6) BH4 together with DTE (an antioxidant) was not able to prevent ET-mediated endothelial dysfunction. ET-1 did not significantly alter intracellular tetrahydrobiopterin levels in IMA, SV, HUVEC or aorta from ET-TG mice. These findings are important for future research in ET-1 mediated superoxide production and endothelial dysfunction.

  16. Low levels of glutathione are sufficient for survival of keratinocytes after UV irradiation and for healing of mouse skin wounds.

    PubMed

    Telorack, Michèle; Abplanalp, Jeannette; Werner, Sabine

    2016-08-01

    Reduced levels of the cellular antioxidant glutathione are associated with premature skin aging, cancer and impaired wound healing, but the in vivo functions of glutathione in the skin remain largely unknown. Therefore, we analyzed mice lacking the modifier subunit of the glutamate cysteine ligase (Gclm), the enzyme that catalyzes the rate-limiting step of glutathione biosynthesis. Glutathione levels in the skin of these mice were reduced by 70 %. However, neither skin development and homeostasis, nor UVA- or UVB-induced apoptosis in the epidermis were affected. Histomorphometric analysis of excisional wounds did not reveal wound healing abnormalities in young Gclm-deficient mice, while the area of hyperproliferative epithelium as well as keratinocyte proliferation were affected in aged mice. These findings suggest that low levels of glutathione are sufficient for wound repair in young mice, but become rate-limiting upon aging.

  17. 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

    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

  18. Passive regeneration of glutathione: glutathione reductase regulation in the freeze-tolerant North American wood frog, Rana sylvatica.

    PubMed

    Dawson, Neal J; Storey, Kenneth B

    2017-09-01

    Wood frogs inhabit a broad range across North America, extending from the southern tip of the Appalachian Mountains to the northern boreal forest. Remarkably, they can survive the winter in a frozen state, where as much as 70% of their body water is converted into ice. Whilst in the frozen state, their hearts cease to pump blood, causing their cells to experience ischemia, which can dramatically increase the production of reactive oxygen species within the cell. To overcome this, wood frogs have elevated levels of glutathione, a primary antioxidant. We examined the regulation of glutathione reductase, the enzyme involved in recycling glutathione, in both the frozen and unfrozen (control) state. Glutathione reductase activity from both the control and frozen state showed a dramatic reduction in substrate specificity ( K m ) for oxidized glutathione (50%) when measured in the presence of glucose (300 mmol l -1 ) and a increase (157%) when measured in the presence of levels of urea (75 mmol l -1 ) encountered in the frozen state. However, when we tested the synergistic effect of urea and glucose simultaneously, we observed a substantial reduction in the K m for oxidized glutathione (43%) to a value similar to that with glucose alone. In fact, we found no observable differences in the kinetic and structural properties of glutathione reductase between the two states. Therefore, a significant increase in the affinity for oxidized glutathione in the presence of endogenous levels of glucose suggests that increased glutathione recycling may occur as a result of passive regulation of glutathione reductase by rising levels of glucose during freezing. © 2017. Published by The Company of Biologists Ltd.

  19. Study the effect of Vitamin K on intracellular NAD level in yeast by fluorescence spectrum

    NASA Astrophysics Data System (ADS)

    Yahong, Chen; Ruxiu, Cai; Ke, Zhang

    2007-05-01

    The intracellular NAD level plays a pivotal role in numerous biological processes such as rhythm, senescence, cancer and death. The study of the intracellular NAD level has been one of the "hotspots" in biomedical research. We investigated the effect of Vitamin K on intracellular NAD level in yeast by fluorescence spectrum in this paper. Plasma membrane redox system of yeast was found to be greatly promoted by the addition of Vitamin K 3 or Vitamin K 1. Ferricyanide reduction catalyzed by Vitamin K was accompanied by the decrease in intracellular NADH concentration and the increase in intracellular NAD level of yeast cells.

  20. Multiscale modelling approach combining a kinetic model of glutathione metabolism with PBPK models of paracetamol and the potential glutathione-depletion biomarkers ophthalmic acid and 5-oxoproline in humans and rats.

    PubMed

    Geenen, Suzanne; Yates, James W T; Kenna, J Gerry; Bois, Frederic Y; Wilson, Ian D; Westerhoff, Hans V

    2013-06-01

    A key role of the antioxidant glutathione is detoxification of chemically reactive electrophilic drug metabolites within the liver. Therefore glutathione depletion can have severe toxic consequences. Ophthalmic acid and 5-oxoproline are metabolites involved in glutathione metabolism, which can be measured readily in the blood and urine and have been proposed as candidate biomarkers of hepatic glutathione content. However, currently it is unclear whether their concentrations in plasma exhibit a robust correlation with hepatic glutathione content. To explore this important question, we have developed a novel approach which combines a physiologically based pharmacokinetic (PBPK) model of metabolism and disposition of paracetamol (acetaminophen) with a previously developed mathematical systems model of hepatic glutathione homeostasis. Paracetamol is metabolised to reactive intermediates which deplete glutathione and cause toxicity when given at high doses. Our model correctly predicted that hepatic glutathione depletion following paracetamol administration resulted in elevated concentrations of 5-oxoproline and ophthalmic acid in blood and of 5-oxoproline in urine. However, we also found from the model that concentrations of both of the compounds were likely to be influenced by prolonged administration of paracetamol and by the concentrations of intracellular metabolites such as methionine. We conclude that care must be taken when extrapolating from concentrations of these biomarkers to hepatic glutathione status.

  1. [Effects of dissolved oxygen and pH on Candida utilis batch fermentation of glutathione].

    PubMed

    Wei, Gong-Yuan; Li, Yin; Du, Guo-Cheng; Chen, Jian

    2003-11-01

    The effects of dissolved oxygen (DO) and pH on glutathione batch fermentation by Candida utilis WSH-02-08 in a 7 liters stirred fermentor were investigated. It was shown that DO concentration is an important factor in glutathione production. With the initial glucose concentration of 30 g/L and a 5 L/min air flow rate, and the agitation rate less than 250 r/min, the DO concentration was not sufficient to satisfy the oxygen requirement during the fermentation. With an agitation rate of more than 300 r/min, the cell growth and glutathione production were enhanced significantly, with the dry cell mass and glutathione production were 20% and 25% higher than that at 200 r/min. When C. utilis WSH 02-08 was cultivated in a batch process without pH control, cell growth and glutathione production were inhibited, likely due to a dramatic decrease in the pH. Intracellular glutathione leakages were observed when the pH was 1.5 or less. To assess the effect of pH on glutathione production, six batch processes controlled at pH 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5 were conducted. The yield was highest at pH 5.5, when the dry cell mass and yield were 27% and 95% respectively higher than fermentation without pH control. The maximal intracellular glutathione content (2.15 %) was also achieved at the pH. To improve our understandings on the effect of pH on the batch glutathione production, a modified Logistic equation and Luedeking-Piret equation were used to simulate cell growth and glutathione production, respectively, under different pH. Based on the parameters obtained by the nonlinear estimation, kinetic analysis was performed to elucidate the effect of pH on the batch glutathione production. The process controlled at pH 5.5 was proven to be the best due to the higher value of K(I) (substrate inhibitory constant in the Logistic equation), lower value of a and higher value of beta (slope and intercept in the Luedeking-Piret equation, respectively).

  2. 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

  3. 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.

  4. Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity

    PubMed Central

    Hernandez-Fernaud, Juan R.; Ruengeler, Elena; Casazza, Andrea; Neilson, Lisa J.; Pulleine, Ellie; Santi, Alice; Ismail, Shehab; Lilla, Sergio; Dhayade, Sandeep; MacPherson, Iain R.; McNeish, Iain; Ennis, Darren; Ali, Hala; Kugeratski, Fernanda G.; Al Khamici, Heba; van den Biggelaar, Maartje; van den Berghe, Peter V.E.; Cloix, Catherine; McDonald, Laura; Millan, David; Hoyle, Aoisha; Kuchnio, Anna; Carmeliet, Peter; Valenzuela, Stella M.; Blyth, Karen; Yin, Huabing; Mazzone, Massimiliano; Norman, Jim C.; Zanivan, Sara

    2017-01-01

    The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion. PMID:28198360

  5. 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.

  6. Shikonin protects dopaminergic cell line PC12 against 6-hydroxydopamine-mediated neurotoxicity via both glutathione-dependent and independent pathways and by inhibiting apoptosis.

    PubMed

    Esmaeilzadeh, Emran; Gardaneh, Mossa; Gharib, Ehsan; Sabouni, Farzaneh

    2013-08-01

    We have investigated the mechanism of shikonin function on protection of dopaminergic neurons against 6-OHDA-induced neurotoxicity. Treatment of rat pheochromocytoma cell line PC12 by serial dilutions of shikonin determined 10 μM of the compound as its optimum concentration for protection saving nearly 70 % of the cells against toxicity. Reverse transcription-PCR analysis of shikonin-treated cells showed threefold increase in mRNA levels of glutathione peroxidase-1 (GPX-1) as a representative component of the intracellular anti-oxidant defense system. To elucidate shikonin-GPX1 relationships and maximize protection, we transduced PC12 cells using recombinant lentivirus vectors that harbored GPX-1 coding sequence. This change upregulated GPX-1 expression, increased peroxidase activity and made neuronal cells resistant to 6-OHDA-mediated toxicity. More importantly, addition of shikonin to GPX1-overexpressing PC12 cells augmented GPX-1 protein content by eightfold leading to fivefold increase of enzymatic activity, 91 % cell survival against neurotoxicity and concomitant increases in intracellular glutathione (GSH) levels. Depletion of intracellular GSH rendered all cell groups highly susceptible to toxicity; however, shikonin was capable of partially saving them. Subsequently, GSH-independent superoxide dismutase mRNA was found upregulated by shikonin. As signs of apoptosis inhibition, the compound upregulated Bcl-2, downregulated Bax, and prevented cell nuclei from undergoing morphological changes typical of apoptosis. Also, a co-staining method demonstrated GPX-1 overexpression significantly increases the percent of live cells that is maximized by shikonin treatment. Our data indicate that shikonin as an antioxidant compound protects dopaminergic neurons against 6-OHDA toxicity and enhances their survival via both glutathione-dependent and direct anti-apoptotic pathways.

  7. 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

  8. 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

  9. Comparison of glutathione levels measured using optimized monochlorobimane assay with those from ortho-phthalaldehyde assay in intact cells.

    PubMed

    Čapek, Jan; Hauschke, Martina; Brůčková, Lenka; Roušar, Tomáš

    2017-11-01

    Fluorometric glutathione assays have been generally preferred for their high specificity and sensitivity. An additional advantage offered by fluorescent bimane dyes is their ability to penetrate inside the cell. Their ability to react with glutathione within intact cells is frequently useful in flow cytometry and microscopy. Hence, the aims of our study were to use monochlorobimane for optimizing a spectrofluorometric glutathione assay in cells and then to compare that assay with the frequently used ortho-phthalaldehyde assay. We used glutathione-depleting agents (e.g., cisplatin and diethylmalonate) to induce cell impairment. For glutathione assessment, monochlorobimane (40μM) was added to cells and fluorescence was detected at 394/490nm. In addition to the regularly used calculation of glutathione levels from fluorescence change after 60min, we used an optimized calculation from the linear part of the fluorescence curve after 10min of measurement. We found that 10min treatment of cells with monochlorobimane is sufficient for evaluating cellular glutathione concentration and provides results entirely comparable with those from the standard ortho-phthalaldehyde assay. In contrast, the results obtained by the standardly used evaluation after 60min of monochlorobimane treatment provided higher glutathione values. We conclude that measuring glutathione using monochlorobimane with the here-described optimized evaluation of fluorescence signal could be a simple and useful method for routine and rapid assessment of glutathione within intact cells in large numbers of samples. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. In Vivo Monitoring of pH, Redox Status, and Glutathione Using L-Band EPR for Assessment of Therapeutic Effectiveness in Solid Tumors

    PubMed Central

    Bobko, Andrey A.; Eubank, Timothy D.; Voorhees, Jeffrey L.; Efimova, Olga V.; Kirilyuk, Igor A.; Petryakov, Sergey; Trofimiov, Dmitrii G.; Marsh, Clay B.; Zweier, Jay L.; Grigor’ev, Igor A.; Samouilov, Alexandre; Khramtsov, Valery V.

    2011-01-01

    Approach for in vivo real-time assessment of tumor tissue extracellular pH (pHe), redox, and intracellular glutathione based on L-band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony-stimulating factor. It was observed that tumor pHe is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony-stimulating factor decreased the value of pHe by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony-stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pHe mapping was performed using recently proposed variable frequency proton–electron double-resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pHe and a difference of about 0.4 pH units between average pHe values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pHe, extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors. Magn Reson Med 000:000–000, 2011. PMID:22113626

  11. Glutathione enzyme and selenoprotein polymorphisms associate with mercury biomarker levels in Michigan dental professionals

    PubMed Central

    Goodrich, Jaclyn M.; Wang, Yi; Gillespie, Brenda; Werner, Robert; Franzblau, Alfred; Basu, Niladri

    2012-01-01

    Mercury is a potent toxicant of concern to both the general public and occupationally exposed workers (e.g., dentists). Recent studies suggest that several genes mediating the toxicokinetics of mercury are polymorphic in humans and may influence inter-individual variability in mercury accumulation. This work hypothesizes that polymorphisms in key glutathione synthesizing enzyme, glutathione s-transferase, and selenoprotein genes underlie inter-individual differences in mercury body burden as assessed by analytical mercury measurement in urine and hair, biomarkers of elemental mercury and methylmercury, respectively. Urine and hair samples were collected from a population of dental professionals (n=515), and total mercury content was measured. Average urine (1.06±1.24 ug/L) and hair mercury levels (0.49±0.63 ug/g) were similar to national U.S. population averages. Taqman assays were used to genotype DNA from buccal swab samples at 15 polymorphic sites in genes implicated in mercury metabolism. Linear regression modeling assessed the ability of polymorphisms to modify the relationship between mercury biomarker levels and exposure sources (e.g., amalgams, fish consumption). Five polymorphisms were significantly associated with urine mercury levels (GSTT1 deletion), hair mercury levels (GSTP1-105, GSTP1-114, GSS 5’), or both (SEPP1 3’UTR). Overall, this study suggests that polymorphisms in selenoproteins and glutathione-related genes may influence elimination of mercury in the urine and hair or mercury retention following exposures to elemental mercury (via dental amalgams) and methylmercury (via fish consumption). PMID:21967774

  12. Relationship between Intracellular Magnesium Level, Lung Function, and Level of Asthma Control in Children with Chronic Bronchial Asthma.

    PubMed

    Sein, Htwe Htwe; Whye Lian, Cheah; Juan Loong, Kok; Sl Ng, Josephine; Rahardjai, Andy; Sultan, Mohamed Ameenudeen

    2014-01-01

    This study aimed to determine the intracellular (red blood cell (RBC)) magnesium levels in children with chronic bronchial asthma and to determine the relationship between the magnesium level and peak expiratory flow rate (PEFR), type of asthma treatment, and level of asthma control. A cross-sectional study was conducted at the Paediatric Clinic, Sarawak General Hospital. A total of 100 children, aged 6-12 years with chronic bronchial asthma, were recruited according to the study criteria. Venous blood samples were obtained to measure the intracellular (RBC) magnesium level using the GBC Avanta Flame Atomic Absorption Spectrophotometer. Mean age was 8.57 (SD 1.18) years, and 63% of the participants were male. Mean duration of asthma was 62.2 (SD 32.3) months. A normal intracellular magnesium level was found in 95% of the participants, with a mean of 2.27 (SD 0.33) mmol/L. Two-thirds of the participants had a normal peak flow expiratory rate (> 80% of predicted value). About 85% were using both reliever and controller. Almost half of the participants (49%) had chronic asthma that was well-controlled. No significant relationship was found between magnesium level and age (r = -0.089, P = 0.379), gender (t = 0.64, P = 0.52), duration of asthma (r = -0.03, P = 0.74), PEFR (t = 0.41, P = 0.68), current level of asthma control (t = 0.02, P = 0.97), and current treatment (t = 0.414, P = 0.680). There was no significant intracellular magnesium deficiency in children with chronic bronchial asthma. There was no significant relationship between therapeutic medications used for treatment of children with chronic asthma and intracellular magnesium levels.

  13. Low glutathione peroxidase activity levels in patients with vitiligo.

    PubMed

    Zedan, Hatem; Abdel-Motaleb, Amira Ali; Kassem, Nahed Mahmoud Ali; Hafeez, Heba Ahmed Abdel; Hussein, Mahmoud Rezk Abdelwhahed

    2015-01-01

    Vitiligo is an idiopathic skin disease characterized by white areas on the skin due to loss of the functional melanocytes, with possible involvement of oxidative stress. Glutathione peroxidase (GPx) is an antioxidant enzyme that protects cells against oxidative damage. To examine serum GPx levels in patients with vitiligo and to relate the findings to the clinical features. The study group included 60 patients with vitiligo and 30 matching healthy controls. GPx activity was evaluated using enzyme-linked immunosorbent assay. We found a significant decrease in serum GPx activity level in the patients with vitiligo compared to the healthy controls (0.29 ± 0.14 versus 0.47 ± 0.13, p < .001). The levels were significantly low in skin phenotypes III and IV (p < .001). Higher levels were also observed with increasing age (≥ 14 years), prolonged disease duration (≥ 3 years), and generalized and extensive vitiligo (< 50%). However, these variations were statistically insignificant. Low levels of serum GPx activity, indicative of a disturbed oxidant-antioxidant system, may contribute to the development of vitiligo. © 2014 Canadian Dermatology Association.

  14. Human gastric signet ring carcinoma (KATO-III) cell apoptosis induced by Vitex agnus-castus fruit extract through intracellular oxidative stress.

    PubMed

    Ohyama, Kunio; Akaike, Takenori; Imai, Masahiko; Toyoda, Hiroo; Hirobe, Chieko; Bessho, Toshio

    2005-07-01

    We have previously reported that an ethanol extract of the dried ripe fruit of Vitex agnus-castus (Vitex) displays cytotoxic activity against certain kinds of human cancer cell line resulting in the induction of apoptosis. In this paper, we investigate the molecular mechanism of apoptosis induced by Vitex using a human gastric signet ring carcinoma cell line, KATO-III. DNA fragmentation was observed in Vitex-treated KATO-III cells in a time- and dose-dependent manner. DNA fragmentation was accompanied by the following phenomena: elevation in the level of hemeoxygenase-1 protein and thioredoxin reductase mRNA; repression of Mn-superoxide dismutase and catalase mRNAs; release of cytochrome c from mitochondria into the cytosol; activation of caspases-8, -9 and -3; decrease in the level of Bcl-2, Bcl-XL and Bid protein; increase in the level of Bad protein. The intracellular oxidized state, measured using 2',7'-dichlorofluorescin diacetate, increased after Vitex treatment. While the amount of intracellular GSH decreased significantly after treatment with Vitex, the level of GSSG was unaffected. Furthermore, no significant perturbation in the amount of proteins/mRNAs related to glutathione metabolism could be detected. These apoptotic alterations induced by exposure to Vitex were blocked by the presence of an anti-oxidative reagent, N-acetyl-l-cysteine, or the addition of exogenous GSH. Our results demonstrate that intracellular oxidative stress and mitochondrial membrane damage is responsible for Vitex-induced apoptosis, which may be mediated by a diminution of reduced type glutathione within the cell.

  15. Relationships among Cortical Glutathione Levels, Brain Amyloidosis, and Memory in Healthy Older Adults Investigated In Vivo with 1H-MRS and Pittsburgh Compound-B PET.

    PubMed

    Chiang, G C; Mao, X; Kang, G; Chang, E; Pandya, S; Vallabhajosula, S; Isaacson, R; Ravdin, L D; Shungu, D C

    2017-06-01

    Oxidative stress has been implicated as an important pathologic mechanism in the development of Alzheimer disease. The purpose of this study was to assess whether glutathione levels, detected noninvasively with proton MR spectroscopy, are associated with brain amyloidosis and memory in a community-dwelling cohort of healthy older adults. Fifteen cognitively healthy subjects were prospectively enrolled in this study. All subjects underwent 1 H-MR spectroscopy of glutathione, a positron-emission tomography scan with an amyloid tracer, and neuropsychological testing by using the Repeatable Battery for the Assessment of Neuropsychological Status. Associations among glutathione levels, brain amyloidosis, and memory were assessed by using multivariate regression models. Lower glutathione levels were associated with greater brain amyloidosis in the temporal ( P = .03) and parietal ( P = .05) regions, adjusted for apolipoprotein E ε4 carrier status. There were no significant associations between glutathione levels and cognitive scores. This study found an association between cortical glutathione levels and brain amyloidosis in healthy older adults, suggesting a potential role for 1 H-MR spectroscopy measures of glutathione as a noninvasive biomarker of early Alzheimer disease pathogenesis. © 2017 by American Journal of Neuroradiology.

  16. Methylmercury alters glutathione homeostasis by inhibiting glutaredoxin 1 and enhancing glutathione biosynthesis in cultured human astrocytoma cells.

    PubMed

    Robitaille, Stephan; Mailloux, Ryan J; Chan, Hing Man

    2016-08-10

    Methylmercury (MeHg) is a neurotoxin that binds strongly to thiol residues on protein and low molecular weight molecules like reduced glutathione (GSH). The mechanism of its effects on GSH homeostasis particularly at environmentally relevant low doses is not fully known. We hypothesized that exposure to MeHg would lead to a depletion of reduced glutathione (GSH) and an accumulation of glutathione disulfide (GSSG) leading to alterations in S-glutathionylation of proteins. Our results showed exposure to low concentrations of MeHg (1μM) did not significantly alter GSH levels but increased GSSG levels by ∼12-fold. This effect was associated with a significant increase in total cellular glutathione content and a decrease in GSH/GSSG. Immunoblot analyses revealed that proteins involved in glutathione synthesis were upregulated accounting for the increase in cellular glutathione. This was associated an increase in cellular Nrf2 protein levels which is required to induce the expression of antioxidant genes in response to cellular stress. Intriguingly, we noted that a key enzyme involved in reversing protein S-glutathionylation and maintaining glutathione homeostasis, glutaredoxin-1 (Grx1), was inhibited by ∼50%. MeHg treatment also increased the S-glutathionylation of a high molecular weight protein. This observation is consistent with the inhibition of Grx1 and elevated H2O2 production however; contrary to our original hypothesis we found few S-glutathionylated proteins in the astrocytoma cells. Collectively, MeHg affects multiple arms of glutathione homeostasis ranging from pool management to protein S-glutathionylation and Grx1 activity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  17. Glutathione synthetase deficiency: a family report.

    PubMed Central

    Pejaver, R K; Watson, A H

    1994-01-01

    Glutathione synthetase deficiency is a rare inborn error of metabolism. Low levels of and at times unstable molecules of glutathione synthetase leads to glutathione deficiency affecting various systems of the body. The inheritance is thought to be of autosomal recessive variety. We diagnosed the condition in a neonate and proceeded to investigate the family. The results are discussed below. PMID:8158601

  18. Glutamine starvation enhances PCV2 replication via the phosphorylation of p38 MAPK, as promoted by reducing glutathione levels.

    PubMed

    Chen, Xingxiang; Shi, Xiuli; Gan, Fang; Huang, Da; Huang, Kehe

    2015-03-18

    Glutamine has a positive effect on ameliorating reproductive failure caused by porcine circovirus type 2 (PCV2). However, the mechanism by which glutamine affects PCV2 replication remains unclear. This study was conducted to investigate the effects of glutamine on PCV2 replication and its underlying mechanisms in vitro. The results show that glutamine promoted PK-15 cell viability. Surprisingly, glutamine starvation significantly increased PCV2 replication. The promotion of PCV2 replication by glutamine starvation disappeared after fresh media with 4 mM glutamine was added. Likewise, promotion of PCV2 was observed after adding buthionine sulfoximine (BSO). Glutamine starvation or BSO treatment increased the level of p38 MAPK phosphorylation and PCV2 replication in PK-15 cells. Meanwhile, p38 MAPK phosphorylation and PCV2 replication significantly decreased in p38-knockdown PK-15 cells. Promotion of PCV2 replication caused by glutamine starvation could be blocked in p38-knockdown PK-15 cells. Therefore, glutamine starvation increased PCV2 replication by promoting p38 MAPK activation, which was associated with the down regulation of intracellular glutathione levels. Our findings may contribute toward interpreting the possible pathogenic mechanism of PCV2 and provide a theoretical reference for application of glutamine in controlling porcine circovirus-associated diseases.

  19. 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

  20. Anethole dithiolethione prevents oxidative damage in glutathione-depleted astrocytes.

    PubMed

    Drukarch, B; Schepens, E; Stoof, J C; Langeveld, C H

    1997-06-25

    Astrocytes protect neurons against reactive oxygen species such as hydrogen peroxide, a capacity which reportedly is abolished following loss of the antioxidant glutathione. Anethole dithiolethione, a sulfur-containing compound which is used in humans, is known to increase cellular glutathione levels and thought thereby to protect against oxidative damage. In the present study we found that anethole dithiolethione increased the glutathione content of cultured rat striatal astrocytes. This effect was abolished by coincubation with the glutathione synthesis inhibitor buthionine sulfoximine. Nevertheless, in the presence of buthionine sulfoximine, despite the lack of an increase in the lowered glutathione level, anethole dithiolethione fully protected the astrocytes against the enhanced toxicity of hydrogen peroxide. Thus, apparently other mechanisms than stimulation of glutathione synthesis are involved in the compound's protective action in astrocytes. Considering the occurrence of lowered glutathione levels in neurodegenerative syndromes, we conclude that further evaluation of the therapeutic potential of anethole dithiolethione is warranted.

  1. Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient γ -ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation

    PubMed Central

    Jobe, Timothy O.; Sung, Dong-Yul; Akmakjian, Garo; Pham, Allis; Komives, Elizabeth A.; Mendoza-Cózatl, David G.; Schroeder, Julian I.

    2015-01-01

    Summary Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. The SULTR1;2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M2 seedlings were screened for an abnormal luciferase response during exposure to cadmium. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium (SRC) mutants; (ii) constitutive response to cadmium (CRC) mutants; or (iii) non-response and reduced response to cadmium (NRC) mutants. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione

  2. Glutathione enzyme and selenoprotein polymorphisms associate with mercury biomarker levels in Michigan dental professionals

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

    Goodrich, Jaclyn M.; Wang, Yi; Gillespie, Brenda

    Mercury is a potent toxicant of concern to both the general public and occupationally exposed workers (e.g., dentists). Recent studies suggest that several genes mediating the toxicokinetics of mercury are polymorphic in humans and may influence inter-individual variability in mercury accumulation. This work hypothesizes that polymorphisms in key glutathione synthesizing enzyme, glutathione s-transferase, and selenoprotein genes underlie inter-individual differences in mercury body burden as assessed by analytical mercury measurement in urine and hair, biomarkers of elemental mercury and methylmercury, respectively. Urine and hair samples were collected from a population of dental professionals (n = 515), and total mercury content wasmore » measured. Average urine (1.06 {+-} 1.24 ug/L) and hair mercury levels (0.49 {+-} 0.63 ug/g) were similar to national U.S. population averages. Taqman assays were used to genotype DNA from buccal swab samples at 15 polymorphic sites in genes implicated in mercury metabolism. Linear regression modeling assessed the ability of polymorphisms to modify the relationship between mercury biomarker levels and exposure sources (e.g., amalgams, fish consumption). Five polymorphisms were significantly associated with urine mercury levels (GSTT1 deletion), hair mercury levels (GSTP1-105, GSTP1-114, GSS 5 Prime ), or both (SEPP1 3 Prime UTR). Overall, this study suggests that polymorphisms in selenoproteins and glutathione-related genes may influence elimination of mercury in the urine and hair or mercury retention following exposures to elemental mercury (via dental amalgams) and methylmercury (via fish consumption). -- Highlights: Black-Right-Pointing-Pointer We explore the influence of 15 polymorphisms on urine and hair Hg levels. Black-Right-Pointing-Pointer Urine and hair Hg levels in dental professionals were similar to the US population. Black-Right-Pointing-Pointer GSTT1 and SEPP1 polymorphisms associated with urine Hg levels. Black

  3. Determination of intracellular glutathione and cysteine using HPLC with a monolithic column after derivatization with monobromobimane.

    PubMed

    Conlan, Xavier A; Stupka, Nicole; McDermott, Geoffrey P; Francis, Paul S; Barnett, Neil W

    2010-05-01

    An optimized high-performance liquid chromatography (HPLC) method is used to show that, as myoblasts differentiate into multinucleated muscle fibers, there is a shift to a more oxidized cell redox state. The HPLC method incorporated derivatization with monobromobimane for the determination of the reduced (GSH) and oxidized (GSSG) forms of glutathione and the reduced (Cys) and oxidized (CysSS) forms of cysteine. The derivatization was optimized to improve the sensitivity of the approach; the limits of detection for glutathione and cysteine were 3 x 10(-8) and 5 x 10(-8) M, respectively. 2009 John Wiley & Sons, Ltd.

  4. Mushrooms: A rich source of the antioxidants ergothioneine and glutathione.

    PubMed

    Kalaras, Michael D; Richie, John P; Calcagnotto, Ana; Beelman, Robert B

    2017-10-15

    While mushrooms are the highest dietary source for the unique sulfur-containing antioxidant ergothioneine, little is known regarding levels of the major biological antioxidant glutathione. Thus, our objectives were to determine and compare levels of glutathione, as well as ergothioneine, in different species of mushrooms. Glutathione levels varied >20-fold (0.11-2.41mg/gdw) with some varieties having higher levels than reported for other foods. Ergothioneine levels also varied widely (0.15-7.27mg/gdw) and were highly correlated with those of glutathione (r=0.62, P<0.001). Both antioxidants were more concentrated in pileus than stipe tissues in selected mushrooms species. Agaricus bisporus harvested during the third cropping flush contained higher levels of ergothioneine and glutathione compared to the first flush, possibly as a response to increased oxidative stress. This study demonstrated that certain mushroom species are high in glutathione and ergothioneine and should be considered an excellent dietary source of these important antioxidants. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Glutathione maintenance is crucial for survival of melanocytes after exposure to rhododendrol.

    PubMed

    Kondo, Masatoshi; Kawabata, Keigo; Sato, Kohji; Yamaguchi, Sayuri; Hachiya, Akira; Takahashi, Yoshito; Inoue, Shintaro

    2016-09-01

    Rhododendrol is a phenolic compound that shows a tyrosinase-dependent toxicity for melanocytes and occasionally induces a vitiligo-like skin depigmentation. The post-tyrosinase mechanisms determining melanocyte death or survival, however, are far from clear. Here, we find that rhododendrol treatment leads to a reduction in the levels of cellular glutathione but also induces a cellular antioxidant response that eventually increases glutathione levels. We further find that rhododendrol toxicity is enhanced when glutathione levels are experimentally reduced and alleviated when glutathione levels are increased. Hence, it appears that the size of the preexisting glutathione pool along with the capacity to supply glutathione via the antioxidant response determines whether melanocytes survive or die after rhododendrol exposure. It is conceivable, therefore, that rhododendrol-induced leukoderma depends on the capacity to maintain appropriate glutathione levels and that enhancement of glutathione levels may preserve a patient's melanocytes and potentially help in repigmentation. © 2016 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.

  6. JS-K, a glutathione/glutathione S-transferase-activated nitric oxide releasing prodrug inhibits androgen receptor and WNT-signaling in prostate cancer cells.

    PubMed

    Laschak, Martin; Spindler, Klaus-Dieter; Schrader, Andres J; Hessenauer, Andrea; Streicher, Wolfgang; Schrader, Mark; Cronauer, Marcus V

    2012-03-30

    Nitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). In consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC). Prostate cancer (PCa) cells were treated with JS-K, a diazeniumdiolate derivate capable of generating large amounts of intracellular NO following activation by glutathione S-transferase. Generation of NO was determined indirectly by the detection of nitrate in tissue culture medium or by immunodetection of nitrotyrosine in the cytoplasm. Effects of JS-K on intracellular AR-levels were determined by western blotting. AR-dimerization was analyzed by mammalian two hybrid assay, nuclear translocation of the AR was visualized in PCa cells transfected with a green fluorescent AR-Eos fusion protein using fluorescence microscopy. Modulation of AR- and WNT-signalling by JS-K was investigated using reporter gene assays. Tumor cell proliferation following JS-K treatment was measured by MTT-Assay. The NO-releasing compound JS-K was shown to inhibit AR-mediated reporter gene activity in 22Rv1 CRPC cells. Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization. In contrast to previously tested NO-donors, JS-K was able to reduce the intracellular concentration of functional AR. This could be attributed to the generation of extremely high intracellular levels of the free radical NO as demonstrated indirectly by high levels of nitrotyrosine in JS-K treated cells. Moreover, JS-K diminished WNT-signaling in AR-positive 22Rv1 cells. In line with these observations, castration resistant 22Rv1 cells were found to be more susceptible to the growth inhibitory effects of JS-K than the androgen dependent LNCaP which do not exhibit an active WNT-signaling pathway. Our results

  7. JS-K, a glutathione/glutathione S-transferase-activated nitric oxide releasing prodrug inhibits androgen receptor and WNT-signaling in prostate cancer cells

    PubMed Central

    2012-01-01

    Background Nitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). In consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC). Methods Prostate cancer (PCa) cells were treated with JS-K, a diazeniumdiolate derivate capable of generating large amounts of intracellular NO following activation by glutathione S-transferase. Generation of NO was determined indirectly by the detection of nitrate in tissue culture medium or by immunodetection of nitrotyrosine in the cytoplasm. Effects of JS-K on intracellular AR-levels were determined by western blotting. AR-dimerization was analyzed by mammalian two hybrid assay, nuclear translocation of the AR was visualized in PCa cells transfected with a green fluorescent AR-Eos fusion protein using fluorescence microscopy. Modulation of AR- and WNT-signalling by JS-K was investigated using reporter gene assays. Tumor cell proliferation following JS-K treatment was measured by MTT-Assay. Results The NO-releasing compound JS-K was shown to inhibit AR-mediated reporter gene activity in 22Rv1 CRPC cells. Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization. In contrast to previously tested NO-donors, JS-K was able to reduce the intracellular concentration of functional AR. This could be attributed to the generation of extremely high intracellular levels of the free radical NO as demonstrated indirectly by high levels of nitrotyrosine in JS-K treated cells. Moreover, JS-K diminished WNT-signaling in AR-positive 22Rv1 cells. In line with these observations, castration resistant 22Rv1 cells were found to be more susceptible to the growth inhibitory effects of JS-K than the androgen dependent LNCaP which do not exhibit an active WNT

  8. Stimulation of hepatic glutathione formation by administration of L-2-oxothiazolidine-4-carboxylate, a 5-oxo-L-prolinase substrate.

    PubMed Central

    Williamson, J M; Meister, A

    1981-01-01

    5-Oxo-L-prolinase, the enzyme that catalyzes the conversion of 5-oxo-L-proline to L-glutamate coupled to the cleavage of ATP to ADP and Pi, also acts on L-2-oxothiazolidine-4-carboxylate (an analog of 5-oxoproline in which the 4-methylene moiety is replaced by sulfur) and ATP to yield cysteine and ADP. The enzyme, which exhibits an affinity for the analog similar to that for the natural substrate, is inhibited by the analog in vitro and in vivo. L-2-oxothiazolidine-4-carboxylate thus serves as a potent inhibitor of the gamma-glutamyl cycle at the step of 5-oxoprolinase. Administration of L-2-oxothiazolidine-4-carboxylate to mice that had been depleted of hepatic glutathione led to restoration of normal hepatic glutathione levels. Since L-2-oxothiazolidine-4-carboxylate is an excellent substrate of the enzyme, it may serve as an intracellular delivery system for cysteine and thus has potential as a therapeutic agent for conditions in which there is depletion of hepatic glutathione. PMID:6940159

  9. Plastid-Localized Glutathione Reductase2–Regulated Glutathione Redox Status Is Essential for Arabidopsis Root Apical Meristem Maintenance[C][W

    PubMed Central

    Yu, Xin; Pasternak, Taras; Eiblmeier, Monika; Ditengou, Franck; Kochersperger, Philip; Sun, Jiaqiang; Wang, Hui; Rennenberg, Heinz; Teale, William; Paponov, Ivan; Zhou, Wenkun; Li, Chuanyou; Li, Xugang; Palme, Klaus

    2013-01-01

    Glutathione is involved in thiol redox signaling and acts as a major redox buffer against reactive oxygen species, helping to maintain a reducing environment in vivo. Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide (GSSG) into reduced glutathione (GSH). The Arabidopsis thaliana genome encodes two GRs: GR1 and GR2. Whereas the cytosolic/peroxisomal GR1 is not crucial for plant development, we show here that the plastid-localized GR2 is essential for root growth and root apical meristem (RAM) maintenance. We identify a GR2 mutant, miao, that displays strong inhibition of root growth and severe defects in the RAM, with GR activity being reduced to ∼50%. miao accumulates high levels of GSSG and exhibits increased glutathione oxidation. The exogenous application of GSH or the thiol-reducing agent DTT can rescue the root phenotype of miao, demonstrating that the RAM defects in miao are triggered by glutathione oxidation. Our in silico analysis of public microarray data shows that auxin and glutathione redox signaling generally act independently at the transcriptional level. We propose that glutathione redox status is essential for RAM maintenance through both auxin/PLETHORA (PLT)-dependent and auxin/PLT-independent redox signaling pathways. PMID:24249834

  10. Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes.

    PubMed

    Deponte, Marcel

    2013-05-01

    Glutathione-dependent catalysis is a metabolic adaptation to chemical challenges encountered by all life forms. In the course of evolution, nature optimized numerous mechanisms to use glutathione as the most versatile nucleophile for the conversion of a plethora of sulfur-, oxygen- or carbon-containing electrophilic substances. This comprehensive review summarizes fundamental principles of glutathione catalysis and compares the structures and mechanisms of glutathione-dependent enzymes, including glutathione reductase, glutaredoxins, glutathione peroxidases, peroxiredoxins, glyoxalases 1 and 2, glutathione transferases and MAPEG. Moreover, open mechanistic questions, evolutionary aspects and the physiological relevance of glutathione catalysis are discussed for each enzyme family. It is surprising how little is known about many glutathione-dependent enzymes, how often reaction geometries and acid-base catalysts are neglected, and how many mechanistic puzzles remain unsolved despite almost a century of research. On the one hand, several enzyme families with non-related protein folds recognize the glutathione moiety of their substrates. On the other hand, the thioredoxin fold is often used for glutathione catalysis. Ancient as well as recent structural changes of this fold did not only significantly alter the reaction mechanism, but also resulted in completely different protein functions. Glutathione-dependent enzymes are excellent study objects for structure-function relationships and molecular evolution. Notably, in times of systems biology, the outcome of models on glutathione metabolism and redox regulation is more than questionable as long as fundamental enzyme properties are neither studied nor understood. Furthermore, several of the presented mechanisms could have implications for drug development. This article is part of a Special Issue entitled Cellular functions of glutathione. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Biochemical heterogeneity in glutathione synthetase deficiency.

    PubMed Central

    Spielberg, S P; Garrick, M D; Corash, L M; Butler, J D; Tietze, F; Rogers, L; Schulman, J D

    1978-01-01

    Two different clinical syndromes are associated with glutathione synthetase deficiency, one presenting with hemolytic anemia and 5-oxoprolinuria, the other with isolated hemolysis. We have differentiated these disorders on an enzymatic basis. In 5-oxoprolinuria, all cell types examined have grossly deficient enzyme activity and glutathione content. In contrast, in the nonoxoprolinuric variant, erythrocytes have decreased enzyme activity and glutathione content, whereas nucleated cells maintain substantial levels of both. The enzyme in this disorder is unstable in vitro and has shortened survival in intact erythrocytes. Nucleated cells appear able to maintain sufficient enzyme activity and concentrations of glutathione to suppress overproduction of 5-oxoproline. PMID:659603

  12. Emerging regulatory paradigms in glutathione metabolism

    PubMed Central

    Liu, Yilin; Hyde, Annastasia S.; Simpson, Melanie A.; Barycki, Joseph J.

    2015-01-01

    One of the hallmarks of cancer is the ability to generate and withstand unusual levels of oxidative stress. In part, this property of tumor cells is conferred by elevation of the cellular redox buffer glutathione. Though enzymes of the glutathione synthesis and salvage pathways have been characterized for several decades, we still lack a comprehensive understanding of their independent and coordinate regulatory mechanisms. Recent studies have further revealed that overall central metabolic pathways are frequently altered in various tumor types, resulting in significant increases in biosynthetic capacity, and feeding into glutathione synthesis. In this review, we will discuss the enzymes and pathways affecting glutathione flux in cancer, and summarize current models for regulating cellular glutathione through both de novo synthesis and efficient salvage. In addition, we examine the integration of glutathione metabolism with other altered fates of intermediary metabolites, and highlight remaining questions about molecular details of the accepted regulatory modes. PMID:24974179

  13. LPS alters pattern of sickness behavior but does not affect glutathione level in aged male rats.

    PubMed

    Wrotek, Sylwia; Jędrzejewski, Tomasz; Nowakowska, Anna; Kozak, Wiesław

    2016-08-01

    Behavioral symptoms of sickness, such as fever and motor activity are a coordinated set of changes that develop during infection. The aim of study was to compare the sickness behaviour (SB) in healthy old and young rats treated with pyrogenic dose of endotoxin and to check their glutathione level. Before experimentation male Wistar rats were selected according to standard body mass, motor activity, and white blood cells count. Intraperitoneal injection of lipopolysaccharide (LPS) from E. coli was used to provoke SB. The level of liver glutathione, interleukin (IL) -6, deep body temperature (Tb) and motor activity were measured. Glutathione level in old and young rats did not differ significantly. In both young and old rats LPS administration provoked fever (the mean value of Tb was 38.06 ± 0.01 °C in old rats, and 38.19 ± 0.06 °C in young rats). LPS injection affected night-time activity in both groups (12 h averages were 1.56 ± 0.40 counts in old LPS-treated rats vs 2.74 ± 0.53 counts in not-treated old rats and 3.44 ± 0.60 counts for young LPS-treated vs 4.28 ± 0.57 counts for young not-treated rats). The injection of LPS provoked an elevation of plasma IL-6 concentration (from values below the lowest detectable standard in not-treated groups of animals to 6322.82 ± 537.00 pg/mL in old LPS-treated rats and 7415.62 ± 451.88 pg/mL in young LPS-treated rats). Based on these data, we conclude that good health of aged rats prevents decrease in the glutathione level. Old rats are still able to develop SB in response to pyrogenic dose of LPS, although its components have changed pattern compared to young animals.

  14. Expression of the Laccase Gene from a White Rot Fungus in Pichia pastoris Can Enhance the Resistance of This Yeast to H2O2-Mediated Oxidative Stress by Stimulating the Glutathione-Based Antioxidative System

    PubMed Central

    Fan, Fangfang; Zhuo, Rui; Ma, Fuying; Gong, Yangmin; Wan, Xia; Jiang, Mulan

    2012-01-01

    Laccase is a copper-containing polyphenol oxidase that has great potential in industrial and biotechnological applications. Previous research has suggested that fungal laccase may be involved in the defense against oxidative stress, but there is little direct evidence supporting this hypothesis, and the mechanism by which laccase protects cells from oxidative stress also remains unclear. Here, we report that the expression of the laccase gene from white rot fungus in Pichia pastoris can significantly enhance the resistance of yeast to H2O2-mediated oxidative stress. The expression of laccase in yeast was found to confer a strong ability to scavenge intracellular H2O2 and to protect cells from lipid oxidative damage. The mechanism by which laccase gene expression increases resistance to oxidative stress was then investigated further. We found that laccase gene expression in Pichia pastoris could increase the level of glutathione-based antioxidative activity, including the intracellular glutathione levels and the enzymatic activity of glutathione peroxidase, glutathione reductase, and γ-glutamylcysteine synthetase. The transcription of the laccase gene in Pichia pastoris was found to be enhanced by the oxidative stress caused by exogenous H2O2. The stimulation of laccase gene expression in response to exogenous H2O2 stress further contributed to the transcriptional induction of the genes involved in the glutathione-dependent antioxidative system, including PpYAP1, PpGPX1, PpPMP20, PpGLR1, and PpGSH1. Taken together, these results suggest that the expression of the laccase gene in Pichia pastoris can enhance the resistance of yeast to H2O2-mediated oxidative stress by stimulating the glutathione-based antioxidative system to protect the cell from oxidative damage. PMID:22706050

  15. ROS production, intracellular HSP70 levels and their relationship in human neutrophils: effects of age.

    PubMed

    Kovalenko, Elena I; Boyko, Anna A; Semenkov, Victor F; Lutsenko, Gennady V; Grechikhina, Maria V; Kanevskiy, Leonid M; Azhikina, Tatyana L; Telford, William G; Sapozhnikov, Alexander M

    2014-12-15

    ROS production and intracellular HSP70 levels were measured in human neutrophils for three age groups: young (20-59 years), elders (60-89 years) and nonagenarians (90 years and older). Elders showed higher levels of spontaneous intracellular ROS content compared with young and nonagenarian groups, which had similar intracellular ROS levels. Zymosan-induced (non-spontaneous) extracellular ROS levels were also similar for young and nonagenarians but were lower in elders. However, spontaneous extracellular ROS production increased continuously with age. Correlation analysis revealed positive relationships between HSP70 levels and zymosan-stimulated ROS production in the elder group. This was consistent with a promoting role for HSP70 in ROS-associated neutrophils response to pathogens. No positive correlation between ROS production and intracellular HSP70 levels was found for groups of young people and nonagenarians. In contrast, significant negative correlations of some ROS and HSP70 characteriscics were found for neutrophils from young people and nonagenarians. The observed difference in ROS and HSP70 correlations in elders and nonagenarians might be associated with an increased risk of mortality in older individuals less than 90 years old.

  16. Glutathione and multidrug resistance protein transporter mediate a self-propelled disposal of bismuth in human cells

    PubMed Central

    Hong, Yifan; Lai, Yau-Tsz; Chan, Godfrey Chi-Fung; Sun, Hongzhe

    2015-01-01

    Glutathione and multidrug resistance protein (MRP) play an important role on the metabolism of a variety of drugs. Bismuth drugs have been used to treat gastrointestinal disorder and Helicobacter pylori infection for decades without exerting acute toxicity. They were found to interact with a wide variety of biomolecules, but the major metabolic pathway remains unknown. For the first time (to our knowledge), we systematically and quantitatively studied the metabolism of bismuth in human cells. Our data demonstrated that over 90% of bismuth was passively absorbed, conjugated to glutathione, and transported into vesicles by MRP transporter. Mathematical modeling of the system reveals an interesting phenomenon. Passively absorbed bismuth consumes intracellular glutathione, which therefore activates de novo biosynthesis of glutathione. Reciprocally, sequestration by glutathione facilitates the passive uptake of bismuth and thus completes a self-sustaining positive feedback circle. This mechanism robustly removes bismuth from both intra- and extracellular space, protecting critical systems of human body from acute toxicity. It elucidates the selectivity of bismuth drugs between human and pathogens that lack of glutathione, such as Helicobacter pylori, opening new horizons for further drug development. PMID:25737551

  17. 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.

  18. Diverse Effects of Glutathione and UPF Peptides on Antioxidant Defense System in Human Erythroleukemia Cells K562.

    PubMed

    Kairane, Ceslava; Mahlapuu, Riina; Ehrlich, Kersti; Kilk, Kalle; Zilmer, Mihkel; Soomets, Ursel

    2012-01-01

    The main goal of the present paper was to examine the influence of the replacement of γ-Glu moiety to α-Glu in glutathione and in its antioxidative tetrapeptidic analogue UPF1 (Tyr(Me)-γ-Glu-Cys-Gly), resulting in α-GSH and UPF17 (Tyr(Me)-Glu-Cys-Gly), on the antioxidative defense system in K562 cells. UPF1 and GSH increased while UPF17 and α-GSH decreased the activity of CuZnSOD in K562 cells, at peptide concentration of 10 μM by 42% and 38% or 35% and 24%, respectively. After three-hour incubation, UPF1 increased and UPF17 decreased the intracellular level of total GSH. Additionally, it was shown that UPF1 is not degraded by γ-glutamyltranspeptidase, which performs glutathione breakdown. These results indicate that effective antioxidative character of peptides does not depend only on the reactivity of the thiol group, but also of the other functional groups, and on the spatial structure of peptides.

  19. Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient γ-ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation.

    PubMed

    Jobe, Timothy O; Sung, Dong-Yul; Akmakjian, Garo; Pham, Allis; Komives, Elizabeth A; Mendoza-Cózatl, David G; Schroeder, Julian I

    2012-06-01

    Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. The SULTR1;2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M(2) seedlings were screened for an abnormal luciferase response during exposure to cadmium. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium (SRC) mutants; (ii) constitutive response to cadmium (CRC) mutants; or (iii) non-response and reduced response to cadmium (NRC) mutants. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione depletion

  20. Fast and Selective Two-Stage Ratiometric Fluorescent Probes for Imaging of Glutathione in Living Cells.

    PubMed

    Gong, Deyan; Han, Shi-Chong; Iqbal, Anam; Qian, Jing; Cao, Ting; Liu, Wei; Liu, Weisheng; Qin, Wenwu; Guo, Huichen

    2017-12-19

    Two fluorescent, m-nitrophenol-substituted difluoroboron dipyrromethene dyes have been designed by nucleophilic substitution reaction of 3,5-dichloro-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY). Nonsymmetric and symmetric probes, that is. BODIPY 1 (with one nitrophenol group at the position 3) and BODIPY 2 (with two nitrophenol groups at the positions 3 and 5) were applied to ratiometric fluorescent glutathione detection. The detection is based on the two-step nucleophilic aromatic substitution of the nitrophenol groups of the probes by glutathione in buffer solution containing CTAB. In the first stage, probe 1 showed ratiometric fluorescent color change from green (λ em = 530 nm) to yellow (λ em = 561 nm) because of monosubstitution with glutathione (I 561nm /I 530nm ). Addition of excess glutathione caused the second stage of ratiometric fluorescent color change from yellow to reddish orange (λ em = 596 nm, I 596nm /I 561nm ) due to disubstitution with glutathione. Therefore, different concentration ranges of glutathione (from less to excess) could be rapidly detected by the two-stage ratiometric fluorescent probe 1 in 5 min. While, probe 2 shows single-stage ratiometric fluorescent detection to GSH (from green to reddish orange, I 596nm /I 535nm ). Probes 1 and 2 exhibit excellent properties with sensitive, specific colorimetric response and ratiometric fluorescent response to glutathione over other sulfur nucleophiles. Application to cellular ratiometric fluorescence imaging indicated that the probes were highly responsive to intracellular glutathione.

  1. Spectrofluorimetric assay method for glutathione and glutathione transferase using monobromobimane.

    PubMed

    Yakubu, S I; Yakasai, I A; Musa, A

    2011-06-01

    The primary role of glutathione transferase is to defend an organism from toxicities through catalyzing the reaction of glutathione (GSH) with potentially toxic compounds or metabolites to their chemically and biologically inert conjugates. The objective of the study was to develop a simple and sensitive spectrofluorimetric assay method for glutathione transferase using monobromobimane (MBB), a non fluorescent compound with electrophilic site. MBB slowly reacted with glutathione to form fluorescent glutathione conjugate and that the reaction was catalysed by glutathione transferase. Both non-enzymatic and enzymatic reaction products of MBB, in presence of GSH in phosphate buffer (pH 6.5), were measured by following increase of fluorescence at wavelength of 475nm. For validation of the assay method, the kinetic parameters such as the apparent Michaelis-Mente constants and maximum rates of conjugate formation as well as the specific activity of rat hepatic glutathione transferase were determined. The method was found to be sensitive, thus, applied to measure glutathione contents of crude preparation of rat hepatic cytosol fraction.

  2. 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.

  3. Intracellular metabolite levels shape sulfur isotope fractionation during microbial sulfate respiration

    PubMed Central

    Wing, Boswell A.; Halevy, Itay

    2014-01-01

    We present a quantitative model for sulfur isotope fractionation accompanying bacterial and archaeal dissimilatory sulfate respiration. By incorporating independently available biochemical data, the model can reproduce a large number of recent experimental fractionation measurements with only three free parameters: (i) the sulfur isotope selectivity of sulfate uptake into the cytoplasm, (ii) the ratio of reduced to oxidized electron carriers supporting the respiration pathway, and (iii) the ratio of in vitro to in vivo levels of respiratory enzyme activity. Fractionation is influenced by all steps in the dissimilatory pathway, which means that environmental sulfate and sulfide levels control sulfur isotope fractionation through the proximate influence of intracellular metabolites. Although sulfur isotope fractionation is a phenotypic trait that appears to be strain specific, we show that it converges on near-thermodynamic behavior, even at micromolar sulfate levels, as long as intracellular sulfate reduction rates are low enough (<<1 fmol H2S⋅cell−1⋅d−1). PMID:25362045

  4. Subcellular Distribution of Glutathione Precursors in Arabidopsis thaliana

    PubMed Central

    Koffler, Barbara Eva; Maier, Romana; Zechmann, Bernd

    2011-01-01

    Abstract Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) – the first enzyme of glutathione synthesis – causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells. PMID:22050910

  5. Effect of glutathione on phytochelatin synthesis in tomato cells.

    PubMed

    Mendum, M L; Gupta, S C; Goldsbrough, P B

    1990-06-01

    Growth of cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, in the presence of cadmium is inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. Cell growth and phytochelatin synthesis are restored to cells treated with buthionine sulfoximine by the addition of glutathione to the medium. Glutathione stimulates the accumulation of phytochelatins in cadmium treated cells, indicating that availability of glutathione can limit synthesis of these peptides. Exogenous glutathione causes a disproportionate increase in the level of smaller phytochelatins, notably [gamma-Glu-Cys](2)-Gly. In the presence of buthionine sulfoximine and glutathione, phytochelatins that are produced upon exposure to cadmium incorporate little [(35)S]cysteine, indicating that these peptides are probably not synthesized by sequential addition of cysteine and glutamate to glutathione.

  6. Cell internalizable and intracellularly degradable cationic polyurethane micelles as a potential platform for efficient imaging and drug delivery.

    PubMed

    Ding, Mingming; Zeng, Xin; He, Xueling; Li, Jiehua; Tan, Hong; Fu, Qiang

    2014-08-11

    A cell internalizable and intracellularly degradable micellar system, assembled from multiblock polyurethanes bearing cell-penetrating gemini quaternary ammonium pendent groups in the side chain and redox-responsive disulfide linkages throughout the backbone, was developed for potential magnetic resonance imaging (MRI) and drug delivery. The nanocarrier is featured as a typical "cleavable core-internalizable shell-protective corona" architecture, which exhibits small size, positive surface charge, high loading capacity, and reduction-triggered destabilization. Furthermore, it can rapidly enter tumor cells and release its cargo in response to an intracellular level of glutathione, resulting in enhanced drug efficacy in vitro. The magnetic micelles loaded with superparamagnetic iron oxide (SPIO) nanoparticles demonstrate excellent MRI contrast enhancement, with T2 relaxivity found to be affected by the morphology of SPIO-clustering inside the micelle core. The multifunctional carrier with good cytocompatibility and nontoxic degradation products can serve as a promising theranostic candidate for efficient intracellular delivery of anticancer drugs and real-time monitoring of therapeutic effect.

  7. 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.

  8. Effect of cyanamide on toxicity and glutathione depletion in rat hepatocyte cultures: differences between two dichloropropanol isomers.

    PubMed

    Hammond, A H; Fry, J R

    1999-09-30

    The effect of aldehyde dehydrogenase inhibition by cyanamide pre-treatment in vitro on dichloropropanol-dependent toxicity and glutathione depletion was investigated in 24 h rat hepatocyte cultures. Cyanamide pre-treatment had no effect on nitrophenol hydroxylase, 7-methoxy-, 7-ethoxy- or 7-benzyloxyresorufin O-dealkylase activities in 24 h cultures from untreated rats, and had no effect on intracellular glutathione content in cultures from untreated rats, or in cultures from isoniazid-treated rats in which cytochrome P4502E1 (CYP2E1) is increased. In cultures from untreated animals the primary alcohol, 2,3-dichloropropanol, was not toxic and did not significantly deplete glutathione. Cyanamide pre-treatment however, potentiated both toxicity and glutathione depletion. Induction of CYP2E1 also potentiated the toxicity of 2,3-dichloropropanol, and in these cultures cyanamide pre-treatment significantly increased both toxicity and glutathione depletion. Cyanamide did not alter the toxicity or glutathione depletion due to the secondary alcohol, 1,3-dichloropropanol, irrespective of CYP2E1 induction. These results indicate that the primary alcohol isomer is metabolised to an aldehyde intermediate which depletes glutathione. Under basal conditions this metabolite appears to be effectively detoxified, but increased CYP2E1 activity and/or decreased aldehyde dehydrogenase activity promotes accumulation of metabolite, and therefore increases glutathione depletion and toxicity.

  9. Glutathione Degradation.

    PubMed

    Bachhawat, Anand Kumar; Kaur, Amandeep

    2017-11-20

    Glutathione degradation has for long been thought to occur only on noncytosolic pools. This is because there has been only one enzyme known to degrade glutathione (γ-glutamyl transpeptidase) and this localizes to either the plasma membrane (mammals, bacteria) or the vacuolar membrane (yeast, plants) and acts on extracellular or vacuolar pools. The last few years have seen the discovery of several new enzymes of glutathione degradation that function in the cytosol, throwing new light on glutathione degradation. Recent Advances: The new enzymes that have been identified in the last few years that can initiate glutathione degradation include the Dug enzyme found in yeast and fungi, the ChaC1 enzyme found among higher eukaryotes, the ChaC2 enzyme found from bacteria to man, and the RipAY enzyme found in some bacteria. These enzymes play roles ranging from housekeeping functions to stress responses and are involved in processes such as embryonic neural development and pathogenesis. In addition to delineating the pathways of glutathione degradation in detail, a critical issue is to find how these new enzymes impact cellular physiology and homeostasis. Glutathione degradation plays a far greater role in cellular physiology than previously envisaged. The differential regulation and differential specificities of various enzymes, each acting on distinct pools, can lead to different consequences to the cell. It is likely that the coming years will see these downstream effects being unraveled in greater detail and will lead to a better understanding and appreciation of glutathione degradation. Antioxid. Redox Signal. 27, 1200-1216.

  10. Bioavailable Concentrations of Delphinidin and Its Metabolite, Gallic Acid, Induce Antioxidant Protection Associated with Increased Intracellular Glutathione in Cultured Endothelial Cells

    PubMed Central

    Goszcz, Katarzyna; Deakin, Sherine J.; Duthie, Garry G.; Stewart, Derek

    2017-01-01

    Despite limited bioavailability and rapid degradation, dietary anthocyanins are antioxidants with cardiovascular benefits. This study tested the hypothesis that the antioxidant protection conferred by the anthocyanin, delphinidin, is mediated by modulation of endogenous antioxidant defences, driven by its degradation product, gallic acid. Delphinidin was found to degrade rapidly (t1/2 ~ 30 min), generating gallic acid as a major degradation product. Both delphinidin and gallic acid generated oxygen-centred radicals at high (100 μM) concentrations in vitro. In a cultured human umbilical vein endothelial cell model of oxidative stress, the antioxidant protective effects of both delphinidin and gallic acid displayed a hormesic profile; 100 μM concentrations of both were cytotoxic, but relatively low concentrations (100 nM–1 μM) protected the cells and were associated with increased intracellular glutathione. We conclude that delphinidin is intrinsically unstable and unlikely to confer any direct antioxidant activity in vivo yet it offered antioxidant protection to cells at low concentrations. This paradox might be explained by the ability of the degradation product, gallic acid, to confer benefit. The findings are important in understanding the mode of protection conferred by anthocyanins and reinforce the necessity to conduct in vitro experiments at biologically relevant concentrations. PMID:29081896

  11. Reactions of benzene oxide with thiols including glutathione.

    PubMed

    Henderson, Alistair P; Barnes, Martine L; Bleasdale, Christine; Cameron, Richard; Clegg, William; Heath, Sarah L; Lindstrom, Andrew B; Rappaport, Stephen M; Waidyanatha, Suramya; Watson, William P; Golding, Bernard T

    2005-02-01

    S-Phenylmercapturic acid is a minor metabolite of benzene used as a biomarker for human benzene exposures. The reaction of intracellular glutathione with benzene oxide-oxepin, the initial metabolite of benzene, is presumed to give 1-(S-glutathionyl)-cyclohexa-3,5-dien-2-ol, which undergoes dehydration to S-phenylglutathione, the precursor of S-phenylmercapturic acid. To validate the proposed route to S-phenylglutathione, reactions of benzene oxide-oxepin with glutathione and other sulfur nucleophiles have been studied. The reaction of benzene oxide with an excess of aqueous sodium sulfide, followed by acetylation, gave bis-(6-trans-5-acetoxycyclohexa-1,3-dienyl)sulfide, the structure of which was proved by X-ray crystallography. Reactions of benzene oxide-oxepin in a 95:5 (v/v) mixture of phosphate buffer in D2O with (CD3)2SO were monitored by 1H NMR spectroscopy. In the absence of glutathione, the half-life of benzene oxide-oxepin was ca. 34 min at 25 degrees C and pD 7.0. The half-life was not affected in the range of 2-15 mM glutathione in the presence and absence of a commercial sample of human glutathione S-transferase (at pH 7.0, 8.0, 8.5, or 10.0). The adduct 1-(S-glutathionyl)-cyclohexa-3,5-diene-2-ol was identified in these reaction mixtures, especially at higher pH, by mass spectrometry and by its acid-catalyzed decomposition to S-phenylglutathione. Incubation of benzene oxide with N-acetyl-L-cysteine at 37 degrees C and pH 10.0 and subsequent mass spectrometric analysis of the mixture showed formation of pre-S-phenylmercapturic acid and the dehydration product, S-phenylmercapturic acid. The data validate the premise that benzene oxide-oxepin can be captured by glutathione to give (1R,2R)- and/or (1S,2S)-1-(S-glutathionyl)-cyclohexa-3,5-dien-2-ol, which dehydrate to S-phenylglutathione. The capture is a relatively inefficient process at pH 7 that is accelerated at higher pH. These studies account for the observation that the metabolism of benzene is

  12. [Effect of UV-radiation on the level of ascorbic acid, SH-groups, and activity of glutathione reductase in the eye lens].

    PubMed

    Byshneva, L N; Senchuk, V V

    2002-01-01

    The effect of UV radiation in vitro on the level of ascorbate, SH-groups and glutathione reductase activity in the soluble fraction of bovine eye lens was studied. UV-Irradiation increased NADPH-oxidoreductase activity, the level of ascorbate oxidation and decreased the content of SH-groups and activity of glutathione reductase. Significant activation of the NADPH-oxidoreductase activity in the presence of ascorbate and Cu2+ was observed after UV-irradiation. It is suggested that ascorbate may play an important role in the UV-induced lens pathology.

  13. Decreased Glutathione S-transferase Level and Neonatal Hyperbilirubinemia Associated with Glucose-6-phosphate Dehydrogenase Deficiency: A Perspective Review.

    PubMed

    Al-Abdi, Sameer Yaseen

    2017-02-01

    Classically, genetically decreased bilirubin conjugation and/or hemolysis account for the mechanisms contributing to neonatal hyperbilirubinemia associated with glucose-6-phosphate dehydrogenase (G6PD) deficiency. However, these mechanisms are not involved in most cases of this hyperbilirubinemia. Additional plausible mechanisms for G6PD deficiency-associated hyperbilirubinemia need to be considered. Glutathione S-transferases (GST) activity depends on a steady quantity of reduced form of glutathione (GSH). If GSH is oxidized, it is reduced back by glutathione reductase, which requires the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). The main source of NADPH is the pentose phosphate pathway, in which G6PD is the first enzyme. Rat kidney GSH, rat liver GST, and human red blood cell GST levels have been found to positively correlate with G6PD levels in their respective tissues. As G6PD is expressed in hepatocytes, it is expected that GST levels would be significantly decreased in hepatocytes of G6PD-deficient neonates. As hepatic GST binds bilirubin and prevents their reflux into circulation, hypothesis that decreased GST levels in hepatocytes is an additional mechanism contributing to G6PD deficiency-associated hyperbilirubinemia seems plausible. Evidence for and against this hypothesis are discussed in this article hoping to stimulate further research on the role of GST in G6PD deficiency-associated hyperbilirubinemia. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  14. Glutathione synthesis is essential for pollen germination in vitro

    PubMed Central

    2011-01-01

    Background The antioxidant glutathione fulfills many important roles during plant development, growth and defense in the sporophyte, however the role of this important molecule in the gametophyte generation is largely unclear. Bioinformatic data indicate that critical control enzymes are negligibly transcribed in pollen and sperm cells. Therefore, we decided to investigate the role of glutathione synthesis for pollen germination in vitro in Arabidopsis thaliana accession Col-0 and in the glutathione deficient mutant pad2-1 and link it with glutathione status on the subcellular level. Results The depletion of glutathione by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, reduced pollen germination rates to 2-5% compared to 71% germination in wildtype controls. The application of reduced glutathione (GSH), together with BSO, restored pollen germination and glutathione contents to control values, demonstrating that inhibition of glutathione synthesis is responsible for the decrease of pollen germination in vitro. The addition of indole-3-acetic acid (IAA) to media containing BSO restored pollen germination to control values, which demonstrated that glutathione depletion in pollen grains triggered disturbances in auxin metabolism which led to inhibition of pollen germination. Conclusions This study demonstrates that glutathione synthesis is essential for pollen germination in vitro and that glutathione depletion and auxin metabolism are linked in pollen germination and early elongation of the pollen tube, as IAA addition rescues glutathione deficient pollen. PMID:21439079

  15. Nrf2 activation ameliorates cytotoxic effects of arsenic trioxide in acute promyelocytic leukemia cells through increased glutathione levels and arsenic efflux from cells

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

    Nishimoto, Shoichi; Suzuki, Toshihiro; Koike, Shin

    Carnosic acid (CA), a phenolic diterpene isolated from Rosmarinus officinalis, has been shown to activate nuclear transcription factor E2-related factor 2 (Nrf2), which plays a central role in cytoprotective responses to oxidative and electrophilic stress. Recently, the Nrf2-Kelch ECH associating protein 1 (Keap1) pathway has been associated with cancer drug resistance attributable to modulation of the expression and activation of antioxidant and detoxification enzymes. However, the exact mechanisms by which Nrf2 activation results in chemoresistance are insufficiently understood to date. This study investigated the mechanisms by which the cytotoxic effects of arsenic trioxide (ATO), an anticancer drug, were decreased inmore » acute promyelocytic leukemia cells treated with CA, a typical activator of Nrf2 used to stimulate the Nrf2/Keap1 system. Our findings suggest that arsenic is non-enzymatically incorporated into NB4 cells and forms complexes that are dependent on intracellular glutathione (GSH) concentrations. In addition, the arsenic complexes are recognized as substrates by multidrug resistance proteins and subsequently excreted from the cells. Therefore, Nrf2-associated activation of the GSH biosynthetic pathway, followed by increased levels of intracellular GSH, are key mechanisms underlying accelerated arsenic efflux and attenuation of the cytotoxic effects of ATO. - Highlights: • Nrf2 activation attenuates the effect of arsenic trioxide to acute promyelocytic leukemia cells. • The sensitivity of arsenic trioxide to NB4 cells was dependent on efflux rate of arsenic. • Activation of the GSH biosynthesis is essential in Nrf2-regulated responses for arsenic efflux.« less

  16. Glutathione-mediated release of Bodipy® from PEG cofunctionalized gold nanoparticles

    PubMed Central

    Kumar, Dhiraj; Meenan, Brian J; Dixon, Dorian

    2012-01-01

    Gold nanoparticles synthesized via sodium citrate reduction of chloroauric acid (HAuCl4) were functionalized with either various concentrations of thiol-terminated Bodipy® FL L-cystine (0.5, 1.0, 1.5, and 2.0 μg/mL) or Bodipy-poly(ethylene glycol) at concentrations of 0.5–18.75, 1.0–12.50, and 1.5–6.25 μg/mL to form a mixed monolayer of BODIPY-PEG. Thiol-terminated Bodipy, a fluorescing molecule, was used as the model drug, while PEG is widely used in drug-delivery applications to shield nanoparticles from unwanted immune responses. Understanding the influence of PEG-capping on payload release is critical because it is the most widely used type of nanoparticle functionalization in drug delivery studies. It has been previously reported that glutathione can trigger release of thiol-bound payloads from gold nanoparticles. Bodipy release from Bodipy capped and from Bodipy-PEG functionalized gold nanoparticles was studied at typical intracellular glutathione levels. It was observed that the addition of PEG capping inhibits the initial burst release observed in gold nanoparticles functionalized only with Bodipy and inhibits nanoparticle aggregation. Efficient and controlled payload release was observed in gold nanoparticles cofunctionalized with only a limited amount of PEG, thus enabling the coattachment of large amounts of drug, targeting groups or other payloads. PMID:22915847

  17. Dynamic compartment specific changes in glutathione and ascorbate levels in Arabidopsis plants exposed to different light intensities

    PubMed Central

    2013-01-01

    Background Excess light conditions induce the generation of reactive oxygen species (ROS) directly in the chloroplasts but also cause an accumulation and production of ROS in peroxisomes, cytosol and vacuoles. Antioxidants such as ascorbate and glutathione occur in all cell compartments where they detoxify ROS. In this study compartment specific changes in antioxidant levels and related enzymes were monitored among Arabidopsis wildtype plants and ascorbate and glutathione deficient mutants (vtc2-1 and pad2-1, respectively) exposed to different light intensities (50, 150 which was considered as control condition, 300, 700 and 1,500 μmol m-2 s-1) for 4 h and 14 d. Results The results revealed that wildtype plants reacted to short term exposure to excess light conditions with the accumulation of ascorbate and glutathione in chloroplasts, peroxisomes and the cytosol and an increased activity of catalase in the leaves. Long term exposure led to an accumulation of ascorbate and glutathione mainly in chloroplasts. In wildtype plants an accumulation of ascorbate and hydrogen peroxide (H2O2) could be observed in vacuoles when exposed to high light conditions. The pad2-1 mutant reacted to long term excess light exposure with an accumulation of ascorbate in peroxisomes whereas the vtc2-1 mutant reacted with an accumulation of glutathione in the chloroplasts (relative to the wildtype) and nuclei during long term high light conditions indicating an important role of these antioxidants in these cell compartments for the protection of the mutants against high light stress. Conclusion The results obtained in this study demonstrate that the accumulation of ascorbate and glutathione in chloroplasts, peroxisomes and the cytosol is an important reaction of plants to short term high light stress. The accumulation of ascorbate and H2O2 along the tonoplast and in vacuoles during these conditions indicates an important route for H2O2 detoxification under these conditions. PMID

  18. Plasma interleukin-6 levels, glutathione peroxidase and isoprostane in obese women before and after weight loss. Association with cardiovascular risk factors.

    PubMed

    Bougoulia, Maria; Triantos, Athanassios; Koliakos, George

    2006-01-01

    To evaluate the levels of Interleukin-6 (IL-6), glutathione peroxidase and isoprostane in obese women and their association with markers of cardiovascular risk factors before and after weight loss. 36 healthy obese women of reproductive age (group A: age (mean+/-SD) 35.4+/-9.2 years, Body Mass Index (BMI) 38.5+/-7 kg/m2) and 30 healthy, normal weight women (group B: age mean+/-SD 34.9+/-7.4 y., BMI 24+/-1.1 kg/m2) were included in the study. Glucose tolerance was normal in all participating women. Il-6, glutathione peroxidase and isoprostane, C-Reactive Protein (CRP), insulin, fasting plasma glucose, HOMA-IR as well as the lipid profile were evaluated. Body weight, BMI, Waist to Hip ratio (W/H) ratio, Waist Circumference (WC), %free fat mass and the %fat mass were also measured. A hypo-caloric diet was prescribed for the obese women and all participants were re-examined after six months. In obese women after weight loss, anthropometric obesity markers (BMI, W/H ratio), %fat, lipid profile, insulin levels and inflammation indices such as IL-6 and CRP, the oxidative stress index isoprostane, as well as glutathione peroxidase were significantly ameliorated. The levels of serum glutathione peroxidase activity were negatively correlated with IL-6 levels and were significantly increased after weight reduction. In obese women there was an association between IL-6 levels and the values of %fat, %free fat mass, insulin and HOMA-IR before and after weight loss. Weight loss is related to reduction of oxidative stress and inflammation; this beneficial effect could possibly be translated into reduction of cardiovascular risk in obese individuals.

  19. Effects of sub-lethal exposure of rats to the herbicide glyphosate in drinking water: glutathione transferase enzyme activities, levels of reduced glutathione and lipid peroxidation in liver, kidneys and small intestine.

    PubMed

    Larsen, K; Najle, R; Lifschitz, A; Virkel, G

    2012-11-01

    Glyphosate (GLP), the active ingredient of many weed killing formulations, is a broad spectrum herbicide compound. Wistar rats were exposed during 30 or 90 days to the highest level (0.7 mg/L) of GLP allowed in water for human consumption (US EPA, 2011) and a 10-fold higher concentration (7 mg/L). The low levels of exposure to the herbicide did not produce histomorphological changes. The production of TBARS was similar or tended to be lower compared to control animals not exposed to the herbicide. In rats exposed to GLP, increased levels of reduced glutathione (GSH) and enhanced glutathione peroxidase (GPx) activity may act as a protective mechanism against possible detrimental effects of the herbicide. Overall, this work showed certain biochemical modifications, even at 3-20-fold lower doses of GLP than the oral reference dose of 2mg/kg/day (US EPA, 1993). The toxicological significance of these findings remains to be clarified. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Imaging in real-time with FRET the redox response of tumorigenic cells to glutathione perturbations in a microscale flow†

    PubMed Central

    Lin, Chunchen; Kolossov, Vladimir L.; Tsvid, Gene; Trump, Lisa; Henry, Jennifer Jo; Henderson, Jerrod L.; Rund, Laurie A.; Kenis, Paul J.A.; Schook, Lawrence B.; Gaskins, H. Rex; Timp, Gregory

    2012-01-01

    Despite the potential benefits of selective redox-modulating strategies for cancer therapy, an efficacious methodology for testing therapies remains elusive because of the difficulty in measuring intracellular redox potentials over time. In this report, we have incorporated a new FRET-based biosensor to follow in real time redox-sensitive processes in cells transformed to be tumorigenic and cultured in a microfluidic channel. A microfluidic network was used to control micro-scale flow near the cells and at the same time deliver drugs exogenously. Subsequently, the response of a redox homeostasis circuit was tested, namely reduced glutathione (GSH)/oxidized glutathione(GSSG), to diamide, a thiol oxidant, and two drugs used for cancer therapies: BSO (l-buthionine-[SR]-sulfoximine) and BCNU (carmustine). The main outcome from these experiments is a comparison of the temporal depletion and recovery of GSH in single living cells in real-time. These data demonstrate that mammalian cells are capable of restoring a reduced intracellular redox environment in minutes after an acute oxidative insult is removed. This recovery is significantly delayed by (i) the inhibition of GSH biosynthesis by BSO; (ii) the inactivation of glutathione reductase by BCNU; and (iii) in tumorigenic cells relative to an isogenic non-tumorigenic control cell line. PMID:21183971

  1. Blocking lactate export by inhibiting the Myc target MCT1 Disables glycolysis and glutathione synthesis.

    PubMed

    Doherty, Joanne R; Yang, Chunying; Scott, Kristen E N; Cameron, Michael D; Fallahi, Mohammad; Li, Weimin; Hall, Mark A; Amelio, Antonio L; Mishra, Jitendra K; Li, Fangzheng; Tortosa, Mariola; Genau, Heide Marika; Rounbehler, Robert J; Lu, Yunqi; Dang, Chi V; Kumar, K Ganesh; Butler, Andrew A; Bannister, Thomas D; Hooper, Andrea T; Unsal-Kacmaz, Keziban; Roush, William R; Cleveland, John L

    2014-02-01

    Myc oncoproteins induce genes driving aerobic glycolysis, including lactate dehydrogenase-A that generates lactate. Here, we report that Myc controls transcription of the lactate transporter SLC16A1/MCT1 and that elevated MCT1 levels are manifest in premalignant and neoplastic Eμ-Myc transgenic B cells and in human malignancies with MYC or MYCN involvement. Notably, disrupting MCT1 function leads to an accumulation of intracellular lactate that rapidly disables tumor cell growth and glycolysis, provoking marked alterations in glycolytic intermediates, reductions in glucose transport, and in levels of ATP, NADPH, and ultimately, glutathione (GSH). Reductions in GSH then lead to increases in hydrogen peroxide, mitochondrial damage, and ultimately, cell death. Finally, forcing glycolysis by metformin treatment augments this response and the efficacy of MCT1 inhibitors, suggesting an attractive combination therapy for MYC/MCT1-expressing malignancies.

  2. Increased Zn/Glutathione Levels and Higher Superoxide Dismutase-1 Activity as Biomarkers of Oxidative Stress in Women with Long-Term Dental Amalgam Fillings: Correlation between Mercury/Aluminium Levels (in Hair) and Antioxidant Systems in Plasma

    PubMed Central

    Cabaña-Muñoz, María Eugenia; Parmigiani-Izquierdo, José María; Bravo-González, Luis Alberto; Kyung, Hee-Moon; Merino, José Joaquín

    2015-01-01

    Background The induction of oxidative stress by Hg can affect antioxidant enzymes. However, epidemiological studies have failed to establish clear association between dental fillings presence and health problems. Objectives To determine whether heavy metals (in hair), antioxidant enzymes (SOD-1) and glutathione levels could be affected by the chronic presence of heavy metals in women who had dental amalgam fillings. Materials and Methods 55 hair samples (42 females with amalgam fillings and 13 female control subjects) were obtained. All subjects (mean age 44 years) who had dental amalgam filling for more than 10 years (average 15 years). Certain metals were quantified by ICP-MS (Mass Spectrophotometry) in hair (μg/g: Al, Hg, Ba, Ag, Sb, As, Be, Bi, Cd, Pb, Pt, Tl, Th, U, Ni, Sn, Ti) and SOD-1 and Glutathione (reduced form) levels in plasma. Data were compared with controls without amalgams, and analyzed to identify any significant relation between metals and the total number of amalgam fillings, comparing those with four or less (n = 27) with those with more than four (n = 15). As no significant differences were detected, the two groups were pooled (Amlgam; n = 42). Findings Hg, Ag, Al and Ba were higher in the amalgam group but without significant differences for most of the heavy metals analyzed. Increased SOD-1 activity and glutathione levels (reduced form) were observed in the amalgam group. Aluminum (Al) correlated with glutathione levels while Hg levels correlated with SOD-1. The observed Al/glutathione and Hg/SOD-1 correlation could be adaptive responses against the chronic presence of mercury. Conclusions Hg, Ag, Al and Ba levels increased in women who had dental amalgam fillings for long periods. Al correlated with glutathione, and Hg with SOD-1. SOD-1 may be a possible biomarker for assessing chronic Hg toxicity. PMID:26076368

  3. Antiprotozoal glutathione derivatives with flagellar membrane binding activity against T. brucei rhodesiense.

    PubMed

    Daunes, Sylvie; Yardley, Vanessa; Croft, Simon L; D'Silva, Claudius

    2017-02-15

    A new series of N-substituted S-(2,4-dinitrophenyl)glutathione dibutyl diesters were synthesized to improve in vitro anti-protozoal activity against the pathogenic parasites Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania donovani. The results obtained indicate that N-substituents enhance the inhibitory properties of glutathione diesters whilst showing reduced toxicity against KB cells as in the cases of compounds 5, 9, 10, 16, 18 and 19. We suggest that the interaction of N-substituted S-(2,4-dinitrophenyl) glutathione dibutyl diesters with T. b. brucei occurs mainly by weak hydrophobic interactions such as London and van der Waals forces. A QSAR study indicated that the inhibitory activity of the peptide is associated negatively with the average number of C atoms, N C and positively to S ZX, the ZX shadow a geometric descriptor related to molecular size and orientation of the compound. HPLC-UV studies in conjunction with optical microscopy indicate that the observed selectivity of inhibition of these compounds against bloodstream form T. b. brucei parasites in comparison to L. donovani under the same conditions is due to intracellular uptake via endocytosis in the flagellar pocket. Copyright © 2016. Published by Elsevier Ltd.

  4. Flavonoid-induced glutathione depletion: Potential implications for cancer treatment☆

    PubMed Central

    Kachadourian, Remy; Day, Brian J.

    2014-01-01

    The ability of a number of flavonoids to induce glutathione (GSH) depletion was measured in lung (A549), myeloid (HL-60), and prostate (PC-3) human tumor cells. The hydroxychalcone (2′-HC) and the dihydroxychalcones (2′,2-, 2′,3-, 2′,4-, and 2′,5′-DHC) were the most effective in A549 and HL-60 cells, depleting more than 50% of intracellular GSH within 4 h of exposure at 25 µM. In contrast, the flavones chrysin and apigenin were the most effective in PC-3 cells, depleting 50–70% of intracellular GSH within 24 h of exposure at 25 µM. In general, these flavonoids were more effective than three classical substrates of multidrug resistance protein 1 (MK-571, indomethacin, and verapamil). Prototypic flavonoids (2′,5′-DHC and chrysin) were subsequently tested for their abilities to potentiate the toxicities of prooxidants (etoposide, rotenone, 2-methoxyestradiol, and curcumin). In A549 cells, 2′,5′-DHC potentiated the cytotoxicities of rotenone, 2-methoxyestradiol, and curcumin, but not etoposide. In HL-60 and PC-3 cells, chrysin potentiated the cytotoxicity of curcumin, cytotoxicity that was attenuated by the catalytic antioxidant manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP). Assessments of mitochondrial GSH levels mitochondrial membrane potential and cytochrome c release showed that the potentiation effects induced by 2′,5′-DHC and chrysin involve mitochondrial dysfunction. PMID:16781454

  5. The effects of glutathione depletion on thermotolerance and heat stress protein synthesis.

    PubMed Central

    Russo, A.; Mitchell, J. B.; McPherson, S.

    1984-01-01

    The effects of cellular glutathione depletion by buthionine sulfoximine on the development of thermotolerance and synthesis of heat stress protein was studied. Cellular glutathione levels were found to increase rapidly following an acute heat treatment of either 12 min at 45.5 degrees C or 1 h at 43 degrees C and remain elevated for prolonged periods. Glutathione depletion and prevention of glutathione synthesis by buthionine sulfoximine resulted in inhibition of the development of thermotolerance and a decrease in total protein as well as specific heat stress proteins. While the degree of inhibition of thermotolerance was similar for both glutathione depletion protocols, inhibition in heat stress protein synthesis was greater when glutathione was depleted to low levels prior to heating. The possible role of glutathione and the cellular redox state to thermotolerance and synthesis of heat stress protein is discussed. Images Figure 2 PMID:6733022

  6. Glutathione peroxidases of the potato cyst nematode Globodera Rostochiensis.

    PubMed

    Jones, J T; Reavy, B; Smant, G; Prior, A E

    2004-01-07

    We report the cloning and characterisation of full-length DNAs complementary to RNA (cDNAs) encoding two glutathione peroxidases (GpXs) from a plant parasitic nematode, the potato cyst nematode (PCN) Globodera rostochiensis. One protein has a functional signal peptide that targets the protein for secretion from animal cells while the other is predicted to be intracellular. Both genes are expressed in all parasite stages tested. The mRNA encoding the intracellular GpX is present throughout the nematode second stage juvenile and is particularly abundant in metabolically active tissues including the genital primordia. The mRNA encoding the secreted GpX is restricted to the hypodermis, the outermost cellular layer of the nematode, a location from which it is likely to be secreted to the parasite surface. Biochemical studies confirmed the secreted protein as a functional GpX and showed that, like secreted GpXs of other parasitic nematodes, it does not metabolise hydrogen peroxide but has a preference for larger hydroperoxide substrates. The intracellular protein is likely to have a role in metabolism of active oxygen species derived from internal body metabolism while the secreted protein may protect the parasite from host defences. Other functional roles for this protein are discussed.

  7. The peptide transporter PepT2 mediates the uptake of the glutathione precursor CysGly in astroglia-rich primary cultures.

    PubMed

    Dringen, R; Hamprecht, B; Bröer, S

    1998-07-01

    The intracellular content of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was used as an indicator for the ability of these cultures to utilize cysteinylglycine (CysGly) for glutathione synthesis. After a 24-h starvation period in the absence of glucose and amino acids, CysGly was able to substitute for cysteine plus glycine in the restoration of glutathione. Glutathione restoration from CysGly plus glutamate was only slightly affected by the dipeptides carnosine or serylglycine in a 200-fold excess. Captopril, a substrate of the peptide transporter PepT1, had almost no effect on glutathione restoration. In contrast, with increasing concentrations of alanylalanine or cefadroxil, known substrates of the peptide transporter PepT2, the amount of glutathione restored in the presence of CysGly and glutamate was strongly reduced. Cefadroxil in a 200-fold excess totally prevented the utilization of CysGly for glutathione restoration. The presence of mRNA for PepT2 in astroglia-rich primary cultures was demonstrated by application of RT-PCR. These results demonstrate that PepT2 is expressed in astroglia-rich primary cultures and that this transporter is highly likely to be responsible for the uptake of CysGly in these cultures.

  8. The glutathione cycle: Glutathione metabolism beyond the γ-glutamyl cycle.

    PubMed

    Bachhawat, Anand Kumar; Yadav, Shambhu

    2018-04-17

    Glutathione was discovered in 1888, over 125 years ago. Since then, our understanding of various functions and metabolism of this important molecule has grown over these years. But it is only now, in the last decade, that a somewhat complete picture of its metabolism has emerged. Glutathione metabolism has till now been largely depicted and understood by the γ-glutamyl cycle that was proposed in 1970. However, new findings and knowledge particularly on the transport and degradation of glutathione have revealed that many aspects of the γ-glutamyl cycle are incorrect. Despite this, an integrated critical analysis of the cycle has never been undertaken and this has led to the cycle and its errors perpetuating in the literature. This review takes a careful look at the γ-glutamyl cycle and its shortcomings and presents a "glutathione cycle" that captures the current understanding of glutathione metabolism. © 2018 IUBMB Life, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

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

    PubMed

    Singhal, Sharad S; Singh, Sharda P; Singhal, Preeti; Horne, David; Singhal, Jyotsana; Awasthi, Sanjay

    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 hydroperoxides 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. Copyright © 2015 Elsevier Inc. All rights

  10. An anthocyanin/polyphenolic-rich fruit juice reduces oxidative DNA damage and increases glutathione level in healthy probands.

    PubMed

    Weisel, Tamara; Baum, Matthias; Eisenbrand, Gerhard; Dietrich, Helmut; Will, Frank; Stockis, Jean-Pierre; Kulling, Sabine; Rüfer, Corinna; Johannes, Christian; Janzowski, Christine

    2006-04-01

    Oxidative cell damage is involved in the pathogenesis of atherosclerosis, cancer, diabetes and other diseases. Uptake of fruit juice with especially high content of antioxidant flavonoids/polyphenols, might reduce oxidative cell damage. Therefore, an intervention study was performed with a red mixed berry juice [trolox equivalent antioxidative capacity (TEAC): 19.1 mmol/L trolox] and a corresponding polyphenol-depleted juice (polyphenols largely removed, TEAC 2.4 mmol/L trolox), serving as control. After a 3-week run-in period, 18 male probands daily consumed 700 mL juice, and 9 consumed control juice, in a 4-week intervention, followed by a 3-week wash-out. Samples were collected weekly to analyze DNA damage (comet assay), lipid peroxidation (plasma malondialdehyde: HPLC/fluorescence; urinary isoprostanes: GC-MS), blood glutathione (photometrically), DNA-binding activity of nuclear factor-kappaB (ELISA) and plasma carotenoid/alpha-tocopherol levels (HPLC-DAD). During intervention with the fruit juice, a decrease of oxidative DNA damage (p<5x10(-4)) and an increase of reduced glutathione (p<5x10(-4)) and of glutathione status (p<0.05) were observed, which returned to the run-in levels in the subsequent wash-out phase. The other biomarkers were not significantly modulated by the juice supplement. Intervention with the control juice did not result in reduction of oxidative damage. In conclusion, the fruit juice clearly reduces oxidative cell damage in healthy probands.

  11. Melanoma-targeted delivery system (part 1): design, synthesis and evaluation of releasable disulfide drug by glutathione.

    PubMed

    El Aissi, Radhia; Chezal, Jean-Michel; Tarrit, Sébastien; Chavignon, Olivier; Moreau, Emmanuel

    2015-08-28

    Here we describe the design and synthesis of a prodrug developed for pigmented melanoma therapy, consisting of a Melanin-Targeting Probe (MTP) conjugated to 5-iodo-2'-deoxyuridine (IUdR) with a reduction-sensitive pre-determined breaking point. Compared with the non-cleavable conjugate (17b), prodrug (17a) bearing a self-immolative disulfide linker achieved complete release of IUdR within 20 min in the presence of reducing agents such as DTT or glutathione. Analytical results also showed that prodrug (17a) was more sensitive than parent non-cleavable conjugate (17b) for a concentration range of glutathione similar to that found in the intracellular compartment of tumours. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  12. Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells*

    PubMed Central

    Pei, Shanshan; Minhajuddin, Mohammad; Callahan, Kevin P.; Balys, Marlene; Ashton, John M.; Neering, Sarah J.; Lagadinou, Eleni D.; Corbett, Cheryl; Ye, Haobin; Liesveld, Jane L.; O'Dwyer, Kristen M.; Li, Zheng; Shi, Lei; Greninger, Patricia; Settleman, Jeffrey; Benes, Cyril; Hagen, Fred K.; Munger, Joshua; Crooks, Peter A.; Becker, Michael W.; Jordan, Craig T.

    2013-01-01

    The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells. PMID:24089526

  13. 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.

  14. Compensation for intracellular environment in expression levels of mammalian circadian clock genes

    PubMed Central

    Matsumura, Ritsuko; Okamoto, Akihiko; Node, Koichi; Akashi, Makoto

    2014-01-01

    The circadian clock is driven by transcriptional oscillation of clock genes in almost all body cells. To investigate the effect of cell type-specific intracellular environment on the circadian machinery, we examined gene expression profiles in five peripheral tissues. As expected, the phase relationship between expression rhythms of nine clock genes was similar in all tissues examined. We also compared relative expression levels of clock genes among tissues, and unexpectedly found that quantitative variation remained within an approximately three-fold range, which was substantially smaller than that of metabolic housekeeping genes. Interestingly, circadian gene expression was little affected even when fibroblasts were cultured with different concentrations of serum. Together, these findings support a hypothesis that expression levels of clock genes are quantitatively compensated for the intracellular environment, such as redox potential and metabolite composition. However, more comprehensive studies are required to reach definitive conclusions. PMID:24504324

  15. Mitochondrial Glutathione: Regulation and Functions.

    PubMed

    Calabrese, Gaetano; Morgan, Bruce; Riemer, Jan

    2017-11-20

    Mitochondrial glutathione fulfills crucial roles in a number of processes, including iron-sulfur cluster biosynthesis and peroxide detoxification. Recent Advances: Genetically encoded fluorescent probes for the glutathione redox potential (E GSH ) have permitted extensive new insights into the regulation of mitochondrial glutathione redox homeostasis. These probes have revealed that the glutathione pools of the mitochondrial matrix and intermembrane space (IMS) are highly reduced, similar to the cytosolic glutathione pool. The glutathione pool of the IMS is in equilibrium with the cytosolic glutathione pool due to the presence of porins that allow free passage of reduced glutathione (GSH) and oxidized glutathione (GSSG) across the outer mitochondrial membrane. In contrast, limited transport of glutathione across the inner mitochondrial membrane ensures that the matrix glutathione pool is kinetically isolated from the cytosol and IMS. In contrast to the situation in the cytosol, there appears to be extensive crosstalk between the mitochondrial glutathione and thioredoxin systems. Further, both systems appear to be intimately involved in the removal of reactive oxygen species, particularly hydrogen peroxide (H 2 O 2 ), produced in mitochondria. However, a detailed understanding of these interactions remains elusive. We postulate that the application of genetically encoded sensors for glutathione in combination with novel H 2 O 2 probes and conventional biochemical redox state assays will lead to fundamental new insights into mitochondrial redox regulation and reinvigorate research into the physiological relevance of mitochondrial redox changes. Antioxid. Redox Signal. 27, 1162-1177.

  16. Effect of glutathione on phytochelatin synthesis in tomato cells. [Lycopersicon esculentum

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

    Mendum, M.L.; Gupta, S.C.; Goldsbrough, P.B.

    Growth of cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, in the presence of cadmium is inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. Cell growth and phytochelatin synthesis are restored to cells treated with buthionine sulfoximine by the addition of glutathione to the medium. Glutathione stimulates the accumulation of phytochelatins in cadmium treated cells, indicating that availability of glutathione can limit synthesis of these peptides. Exogenous glutathione causes a disproportionate increase in the level of smaller phytochelatins, notably ({gamma}-Glu-Cys){sub 2}-Gly. In the presence of buthionine sulfoximine and glutathione, phytochelatins that are produced upon exposure to cadmiummore » incorporate little ({sup 35}S)cysteine, indicating that these peptides are probably not synthesized by sequential addition of cysteine and glutamate to glutathione.« less

  17. Chemotherapy triggers HIF-1–dependent glutathione synthesis and copper chelation that induces the breast cancer stem cell phenotype

    PubMed Central

    Lu, Haiquan; Samanta, Debangshu; Xiang, Lisha; Zhang, Huimin; Hu, Hongxia; Chen, Ivan; Bullen, John W.; Semenza, Gregg L.

    2015-01-01

    Triple negative breast cancer (TNBC) accounts for 10–15% of all breast cancer but is responsible for a disproportionate share of morbidity and mortality because of its aggressive characteristics and lack of targeted therapies. Chemotherapy induces enrichment of breast cancer stem cells (BCSCs), which are responsible for tumor recurrence and metastasis. Here, we demonstrate that chemotherapy induces the expression of the cystine transporter xCT and the regulatory subunit of glutamate-cysteine ligase (GCLM) in a hypoxia-inducible factor (HIF)-1–dependent manner, leading to increased intracellular glutathione levels, which inhibit mitogen-activated protein kinase kinase (MEK) activity through copper chelation. Loss of MEK-ERK signaling causes FoxO3 nuclear translocation and transcriptional activation of the gene encoding the pluripotency factor Nanog, which is required for enrichment of BCSCs. Inhibition of xCT, GCLM, FoxO3, or Nanog blocks chemotherapy-induced enrichment of BCSCs and impairs tumor initiation. These results suggest that, in combination with chemotherapy, targeting BCSCs by inhibiting HIF-1–regulated glutathione synthesis may improve outcome in TNBC. PMID:26229077

  18. 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.

  19. Levels of glutathione and 2,3-diphosphoglycerate in the red blood cells of Australian Aborigines.

    PubMed

    Agar, N S

    1980-01-01

    There were no significant differences in packed cell volume (PCV) and red cell 2,3-diphosphoglycerate (2,3-DPG) levels in Australian Aborigines and Caucasians. A highly significant negative correlation was found between PCV and 2,3-DPG in both Aborigines (r = 0.251; n = 231) and Caucasians (r = 0.435; n = 227). Levels of reduced glutathione (GSH) in the red blood cells of Aborigines were significantly lower (P < 0.001) compared to those of Caucasians. There was a significant negative correlation between PCV and GSH in both the groups; (Aborigines r = -0.637, n = 115; Caucasians r = 0.388, n = 111).

  20. Positive correlation between decreased cellular uptake, NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines.

    PubMed

    Scheulen, M E; Hoensch, H; Kappus, H; Seeber, S; Schmidt, C G

    1987-01-01

    From a wild type strain of Ehrlich ascites tumor (EATWT) sublines resistant to daunorubicin (EATDNM), etoposide (EATETO), and cisplatinum (EATCIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i.p. with EAT cells. Adriamycin concentrations causing 50% inhibition of 3H-thymidine (ICT) and 3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EATWT - EATCIS - EATDNM - EATETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICTc, ICUc), ICTc and ICUc still varied up to the 3.2 fold in EATCIS, EATDNM and EATETO in comparison to EATWT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p less than 0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICTc, and ICUc, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.

  1. 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.

  2. Self-Assembly of Multi-nanozymes to Mimic an Intracellular Antioxidant Defense System.

    PubMed

    Huang, Yanyan; Liu, Zhen; Liu, Chaoqun; Ju, Enguo; Zhang, Yan; Ren, Jinsong; Qu, Xiaogang

    2016-06-01

    In this work, for the first time, we constructed a novel multi-nanozymes cooperative platform to mimic intracellular antioxidant enzyme-based defense system. V2 O5 nanowire served as a glutathione peroxidase (GPx) mimic while MnO2 nanoparticle was used to mimic superoxide dismutase (SOD) and catalase (CAT). Dopamine was used as a linker to achieve the assembling of the nanomaterials. The obtained V2 O5 @pDA@MnO2 nanocomposite could serve as one multi-nanozyme model to mimic intracellular antioxidant enzyme-based defense procedure in which, for example SOD, CAT, and GPx co-participate. In addition, through assembling with dopamine, the hybrid nanocomposites provided synergistic antioxidative effect. Importantly, both in vitro and in vivo experiments demonstrated that our biocompatible system exhibited excellent intracellular reactive oxygen species (ROS) removal ability to protect cell components against oxidative stress, showing its potential application in inflammation therapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. The glutathione-dependent system of antioxidant defense is not modulated by temperature acclimation in muscle tissues from striped bass, Morone saxatilis.

    PubMed

    Grim, Jeffrey M; Simonik, Elizabeth A; Semones, Molly C; Kuhn, Donald E; Crockett, Elizabeth L

    2013-02-01

    Cold temperature generally induces an enhancement of oxidative capacities, a greater content of intracellular lipids, and a remodeling of lipids in biological membranes. These physiological responses may pose a heightened risk of lipid peroxidation (LPO), while warm temperature could result in greater risk of LPO since rates involving reactive oxygen species and LPO will be elevated. The current study examines responses of the glutathione system of antioxidant defense after temperature acclimation. We measured total glutathione (tGSH), and protein levels of GPx1, GPx4, and GST (cardiac and skeletal muscles), and enzymatic activity (skeletal muscle) of glutathione-dependent antioxidants (GPx, GPx4, and GST) in tissues from striped bass (Morone saxatilis) acclimated for six weeks to 7 °C or 25 °C. tGSH of cardiac muscle from cold-acclimated animals was 1.2-times higher than in warm-bodied counterparts, but unchanged with temperature acclimation in skeletal muscle. A second low molecular weight antioxidant, ascorbate was 1.4- and 1.5-times higher in cardiac and skeletal muscle, respectively in warm- than cold-acclimated animals. Despite 1.2-times higher oxidative capacities (as indicated by citrate synthase activity), in skeletal muscle from cold- versus warm-acclimated fish, levels and activities of antioxidant enzymes were similar between acclimation groups. Lipid peroxidation products (as indicated by TBARS), normalized to tissue wet weight, were more than 2-times higher in skeletal muscle from cold- than warm-acclimated animals, however, when normalized to phospholipid content there was no statistical difference between acclimation groups. Our results demonstrate that the physiological changes, associated with acclimation to low temperature in the eurythermal striped bass, are not accompanied by an enhanced antioxidant defense in the glutathione-dependent system. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. A Glutathione-independent Glyoxalase of the DJ-1 Superfamily Plays an Important Role in Managing Metabolically Generated Methylglyoxal in Candida albicans*

    PubMed Central

    Hasim, Sahar; Hussin, Nur Ahmad; Alomar, Fadhel; Bidasee, Keshore R.; Nickerson, Kenneth W.; Wilson, Mark A.

    2014-01-01

    Methylglyoxal is a cytotoxic reactive carbonyl compound produced by central metabolism. Dedicated glyoxalases convert methylglyoxal to d-lactate using multiple catalytic strategies. In this study, the DJ-1 superfamily member ORF 19.251/GLX3 from Candida albicans is shown to possess glyoxalase activity, making this the first demonstrated glutathione-independent glyoxalase in fungi. The crystal structure of Glx3p indicates that the protein is a monomer containing the catalytic triad Cys136-His137-Glu168. Purified Glx3p has an in vitro methylglyoxalase activity (Km = 5.5 mm and kcat = 7.8 s−1) that is significantly greater than that of more distantly related members of the DJ-1 superfamily. A close Glx3p homolog from Saccharomyces cerevisiae (YDR533C/Hsp31) also has glyoxalase activity, suggesting that fungal members of the Hsp31 clade of the DJ-1 superfamily are all probable glutathione-independent glyoxalases. A homozygous glx3 null mutant in C. albicans strain SC5314 displays greater sensitivity to millimolar levels of exogenous methylglyoxal, elevated levels of intracellular methylglyoxal, and carbon source-dependent growth defects, especially when grown on glycerol. These phenotypic defects are complemented by restoration of the wild-type GLX3 locus. The growth defect of Glx3-deficient cells in glycerol is also partially complemented by added inorganic phosphate, which is not observed for wild-type or glucose-grown cells. Therefore, C. albicans Glx3 and its fungal homologs are physiologically relevant glutathione-independent glyoxalases that are not redundant with the previously characterized glutathione-dependent GLO1/GLO2 system. In addition to its role in detoxifying glyoxals, Glx3 and its close homologs may have other important roles in stress response. PMID:24302734

  5. Stabilization of anthocyanins in blackberry juice by glutathione fortification.

    PubMed

    Stebbins, Nathan B; Howard, Luke R; Prior, Ronald L; Brownmiller, Cindi; Mauromoustakos, Andy

    2017-10-18

    Blackberry anthocyanins provide attractive color and antioxidant activity. However, anthocyanins degrade during juice processing and storage, so maintaining high anthocyanin concentrations in berry juices may lead to greater antioxidant and health benefits for the consumer. This study evaluated potential additives to stabilize anthocyanins during blackberry juice storage. The anthocyanin stabilizing agents used were: glutathione, galacturonic acid, diethylenetriaminepentaacetic acid and tannic acid, which were added at a level of 500 mg L -1 . Juice anthocyanin, flavonol, and ellagitannin content and percent polymeric color were measured over five weeks of accelerated storage at 30 °C. Glutathione had the greatest protective effect on total anthocyanins and polymeric color. Therefore a second study was performed with glutathione in combination with lipoic and ascorbic acids in an effort to use antioxidant recycling to achieve a synergistic effect. However, the antioxidant recycling system had no protective effect relative to glutathione alone. Glutathione appears to be a promising blackberry juice additive to protect against anthocyanin degradation during storage.

  6. Fragrance chemicals lyral and lilial decrease viability of HaCat cells' by increasing free radical production and lowering intracellular ATP level: protection by antioxidants.

    PubMed

    Usta, Julnar; Hachem, Yassmine; El-Rifai, Omar; Bou-Moughlabey, Yolla; Echtay, Karim; Griffiths, David; Nakkash-Chmaisse, Hania; Makki, Rajaa Fakhoury

    2013-02-01

    We investigate in this study the biochemical effects on cells in culture of two commonly used fragrance chemicals: lyral and lilial. Whereas both chemicals exerted a significant effect on primary keratinocyte(s), HaCat cells, no effect was obtained with any of HepG2, Hek293, Caco2, NIH3T3, and MCF7 cells. Lyral and lilial: (a) decreased the viability of HaCat cells with a 50% cell death at 100 and 60 nM respectively; (b) decreased significantly in a dose dependant manner the intracellular ATP level following 12-h of treatment; (c) inhibited complexes I and II of electron transport chain in liver sub-mitochondrial particles; and (d) increased reactive oxygen species generation that was reversed by N-acetyl cysteine and trolox and the natural antioxidant lipoic acid, without influencing the level of free and/or oxidized glutathione. Lipoic acid protected HaCat cells against the decrease in viability induced by either compound. Dehydrogenation of lyral and lilial produce α,β-unsaturated aldehydes, that reacts with lipoic acid requiring proteins resulting in their inhibition. We propose lyral and lilial as toxic to mitochondria that have a direct effect on electron transport chain, increase ROS production, derange mitochondrial membrane potential, and decrease cellular ATP level, leading thus to cell death. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Effect of altitude on brain intracellular pH and inorganic phosphate levels

    PubMed Central

    Shi, Xian-Feng; Carlson, Paul J.; Kim, Tae-Suk; Sung, Young-Hoon; Hellem, Tracy L.; Fiedler, Kristen K.; Kim, Seong-Eun; Glaeser, Breanna; Wang, Kristina; Zuo, Chun S.; Jeong, Eun-Kee; Renshaw, Perry F.; Kondo, Douglas G.

    2015-01-01

    Normal brain activity is associated with task-related pH changes. Although central nervous system syndromes associated with significant acidosis and alkalosis are well understood, the effects of less dramatic and chronic changes in brain pH are uncertain. One environmental factor known to alter brain pH is the extreme, acute change in altitude encountered by mountaineers. However, the effect of long-term exposure to moderate altitude has not been studied. The aim of this two-site study was to measure brain intracellular pH and phosphate-bearing metabolite levels at two altitudes in healthy volunteers, using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Increased brain pH and reduced inorganic phosphate (Pi) levels were found in healthy subjects who were long-term residents of Salt Lake City, UT (4720 ft/1438 m), compared with residents of Belmont, MA (20 ft/6 m). Brain intracellular pH at the altitude of 4720 ft was more alkaline than that observed near sea level. In addition, the ratio of inorganic phosphate to total phosphate signal also shifted toward lower values in the Salt Lake City region compared with the Belmont area. These results suggest that long-term residence at moderate altitude is associated with brain chemical changes. PMID:24768210

  8. 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

  9. 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.

  10. Endoplasmic Reticulum Transport of Glutathione by Sec61 Is Regulated by Ero1 and Bip.

    PubMed

    Ponsero, Alise J; Igbaria, Aeid; Darch, Maxwell A; Miled, Samia; Outten, Caryn E; Winther, Jakob R; Palais, Gael; D'Autréaux, Benoit; Delaunay-Moisan, Agnès; Toledano, Michel B

    2017-09-21

    In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative GSH and GSSG biosensors to monitor glutathione import into the ER of yeast cells. We found that glutathione enters the ER by facilitated diffusion through the Sec61 protein-conducting channel, while oxidized Bip (Kar2) inhibits transport. Increased ER glutathione import triggers H 2 O 2 -dependent Bip oxidation through Ero1 reductive activation, which inhibits glutathione import in a negative regulatory loop. During ER stress, transport is activated by UPR-dependent Ero1 induction, and cytosolic glutathione levels increase. Thus, the ER redox poise is tuned by reciprocal control of glutathione import and Ero1 activation. The ER protein-conducting channel is permeable to small molecules, provided the driving force of a concentration gradient. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Methyl vinyl ketone, a toxic ingredient in cigarette smoke extract, modifies glutathione in mouse melanoma cells.

    PubMed

    Horiyama, Shizuyo; Takahashi, Yuta; Hatai, Mayuko; Honda, Chie; Suwa, Kiyoko; Ichikawa, Atsushi; Yoshikawa, Noriko; Nakamura, Kazuki; Kunitomo, Masaru; Date, Sachiko; Masujima, Tsutomu; Takayama, Mitsuo

    2014-01-01

    Cigarette smoke contains many harmful chemicals, which contribute to the pathogenesis of smoking-related diseases such as chronic obstructive pulmonary disease, cancer and cardiovascular disease. The cytotoxicity of cigarette smoke is well documented, but the definitive mechanism behind its toxicity remains unknown. Ingredients in cigarette smoke are known to deplete intracellular glutathione (GSH), the most abundant cellular thiol antioxidant, and to cause oxidative stress. In the present study, we investigated the mechanism of cigarette smoke extract (CSE)-induced cytotoxicity in B16-BL6 mouse melanoma (B16-BL6) cells using liquid chromatography-tandem mass spectrometry. CSE and ingredients in cigarette smoke, methyl vinyl ketone (MVK) and crotonaldehyde (CA), reduced cell viability in a concentration-dependent manner. Also, CSE and the ingredients (m/z 70, each) irreversibly reacted with GSH (m/z 308) to form GSH adducts (m/z 378) in cells and considerably decreased cellular GSH levels at concentrations that do not cause cell death. Mass spectral data showed that the major product formed in cells exposed to CSE was the GSH-MVK adduct via Michael-addition and was not the GSH-CA adduct. These results indicate that MVK included in CSE reacts with GSH in cells to form the GSH-MVK adduct, and thus a possible reason for CSE-induced cytotoxicity is a decrease in intracellular GSH levels.

  12. Balneotherapy and platelet glutathione metabolism in type II diabetic patients

    NASA Astrophysics Data System (ADS)

    Ohtsuka, Yoshinori; Yabunaka, Noriyuki; Watanabe, Ichiro; Noro, Hiroshi; Agishi, Yuko

    1996-09-01

    Effects of balneotherapy on platelet glutathione metabolism were investigated in 12 type II (non-insulin-dependent) diabetic patients. Levels of the reduced form of glutathione (GSH) on admission were well correlated with those of fasting plasma glucose (FPG; r=0.692, P<0.02). After 4 weeks of balneotherapy, the mean level of GSH showed no changes; however, in well-controlled patients (FPG <150 mg/dl), the level increased ( P<0.01) and in poorly controlled patients (FPG >150 mg/dl), the value decreased ( P<0.05). There was a negative correlation between glutathione peroxidase (GPX) activities and the levels of FPG ( r=-0.430, P<0.05). After balneotherapy, the activity increased in 5 patients, decreased in 3 patients and showed no changes (alteration within ±3%) in all the other patients. From these findings in diabetic patients we concluded: (1) platelet GSH synthesis appeared to be induced in response to oxidative stress; (2) lowered GPX activities indicated that the antioxidative defense system was impaired; and (3) platelet glutathione metabolism was partially improved by 4 weeks balneotherapy, an effect thought to be dependent on the control status of plasma glucose levels. It is suggested that balneotherapy is beneficial for patients whose platelet antioxidative defense system is damaged, such as those with diabetes mellitus and coronary heart disease.

  13. Impact of glutathione metabolism on zinc homeostasis in Saccharomyces cerevisiae.

    PubMed

    Steiger, Matthias G; Patzschke, Anett; Holz, Caterina; Lang, Christine; Causon, Tim; Hann, Stephan; Mattanovich, Diethard; Sauer, Michael

    2017-06-01

    Zinc is a crucial mineral for all organisms as it is an essential cofactor for the proper function of a plethora of proteins and depletion of zinc causes oxidative stress. Glutathione is the major redox buffering agent in the cell and therefore important for mitigation of the adverse effects of oxidative stress. In mammalian cells, zinc deficiency is accompanied by a glutathione depletion. In the yeast Saccharomyces cerevisiae, the opposite effect is observed: under low zinc conditions, an elevated glutathione concentration is found. The main regulator to overcome zinc deficiency is Zap1p. However, we show that Zap1p is not involved in this glutathione accumulation phenotype. Furthermore, we found that in glutathione-accumulating strains also the metal ion-binding phytochelatin-2, which is an oligomer of glutathione, is accumulated. This increased phytochelatin concentration correlates with a lower free zinc level in the vacuole. These results suggest that phytochelatin is important for zinc buffering in S. cerevisiae and thus explains how zinc homeostasis is connected with glutathione metabolism. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Proteasome inhibitors alter levels of intracellular peptides in HEK293T and SH-SY5Y cells.

    PubMed

    Dasgupta, Sayani; Castro, Leandro M; Dulman, Russell; Yang, Ciyu; Schmidt, Marion; Ferro, Emer S; Fricker, Lloyd D

    2014-01-01

    The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell.

  15. Proteasome Inhibitors Alter Levels of Intracellular Peptides in HEK293T and SH-SY5Y Cells

    PubMed Central

    Dasgupta, Sayani; Castro, Leandro M.; Dulman, Russell; Yang, Ciyu; Schmidt, Marion; Ferro, Emer S.; Fricker, Lloyd D.

    2014-01-01

    The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell. PMID:25079948

  16. High Glutathione and Glutathione Peroxidase-2 Levels Mediate Cell-Type-Specific DNA Damage Protection in Human Induced Pluripotent Stem Cells

    PubMed Central

    Dannenmann, Benjamin; Lehle, Simon; Hildebrand, Dominic G.; Kübler, Ayline; Grondona, Paula; Schmid, Vera; Holzer, Katharina; Fröschl, Mirjam; Essmann, Frank; Rothfuss, Oliver; Schulze-Osthoff, Klaus

    2015-01-01

    Summary Pluripotent stem cells must strictly maintain genomic integrity to prevent transmission of mutations. In human induced pluripotent stem cells (iPSCs), we found that genome surveillance is achieved via two ways, namely, a hypersensitivity to apoptosis and a very low accumulation of DNA lesions. The low apoptosis threshold was mediated by constitutive p53 expression and a marked upregulation of proapoptotic p53 target genes of the BCL-2 family, ensuring the efficient iPSC removal upon genotoxic insults. Intriguingly, despite the elevated apoptosis sensitivity, both mitochondrial and nuclear DNA lesions induced by genotoxins were less frequent in iPSCs compared to fibroblasts. Gene profiling identified that mRNA expression of several antioxidant proteins was considerably upregulated in iPSCs. Knockdown of glutathione peroxidase-2 and depletion of glutathione impaired protection against DNA lesions. Thus, iPSCs ensure genomic integrity through enhanced apoptosis induction and increased antioxidant defense, contributing to protection against DNA damage. PMID:25937369

  17. Loss of Selenium-Binding Protein 1 Decreases Sensitivity to Clastogens and Intracellular Selenium Content in HeLa Cells.

    PubMed

    Zhao, Changhui; Zeng, Huawei; Wu, Ryan T Y; Cheng, Wen-Hsing

    2016-01-01

    Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesize that SBP1 sequesters cellular selenium and sensitizes cancer cells to DNA-damaging agents. To test this hypothesis, we knocked down SBP1 expression in HeLa cervical cancer cells by employing a short hairpin RNA (shRNA) approach. Reduced sensitivity to hydrogen peroxide, paraquat and camptothecin, reactive oxygen species content, and intracellular retention of selenium after selenomethionine treatment were observed in SBP1 shRNA HeLa cells. Results from Western analyses showed that treatment of HeLa cells with selenomethionine resulted in increased SBP1 protein expression in a dose-dependent manner. Knockdown of SBP1 rendered HeLa cells increased expression of glutathione peroxidase-1 but not glutathione peroxidase-4 protein levels and accelerated migration from a wound. Altogether, SBP1 retains supplemental selenium and sensitizes HeLa cancer cells to clastogens, suggesting a new cancer treatment strategy by sequestering selenium through SBP1.

  18. Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients.

    PubMed

    Wang, Xu-Zhen; Jin, Zhan-Kui; Tian, Xiao-Hui; Xue, Wu-Jun; Tian, Pu-Xun; Ding, Xiao-Ming; Zheng, Jin; Li, Yang; Jing, Xin; Luo, Zi-Zhen

    2014-01-01

    Peripheral blood CD4+ T cell adenosine triphosphate (ATP) release has been reported to be an adjunct tool to evaluate global cellular immune response in solid-organ transplant recipients. However, the correlation between the ATP level and rejection was controversial. The aim of this prospective clinical study was to explore the association between the intracellular ATP level and the occurrence, progression, and treatment of acute rejection (AR) episodes, determine the predicting value of intracellular ATP level for AR in kidney transplant (KT) recipients. In the period of October 2011 to October 2012, 140 KT recipients were recruited and followed for six months after transplantation. Patients were categorized into stable group and AR group according to their clinical course. Whole blood samples were collected pretransplantation, and at 7, 14, 21, and 28days, and at 2, 3, 4, 5 and 6months post-transplantation. Additional blood samples were obtained from AR patients on the day AR occurred, on the day before and 3 and 7days after intravenous anti-rejection therapy started, and on the day when AR reversed. The intracellular ATP in CD4+ T cells was detected by ImmuKnow Immune Cell Function Assay according to the manufacturer's instruction. The absolute number of CD4+ T cells and the trough levels of tacrolimus and cyclosporine were also measured. The ATP level detected on the day AR occurred (627.07±149.85ng/ml) was obviously higher than that of the stable group (320.48±149.11ng/ml, P<0.05). ATP value decreased to 265.35±84.33ng/m at the end of anti-rejection therapy, which was obviously lower than that measured on the day before the anti-rejection therapy started (665.87±162.85ng/ml, P<0.05). ROC analysis revealed that increased intracellular adenosine triphosphate level showed better sensitivity and specificity than those obtained using single time point detection (89.5% vs 85.0%;95.0% vs 88.9%). The best cutoff value was 172.55ng/ml. A positive correlation

  19. Optimization of glutathione production in batch and fed-batch cultures by the wild-type and recombinant strains of the methylotrophic yeast Hansenula polymorpha DL-1

    PubMed Central

    2011-01-01

    Background Tripeptide glutathione (gamma-glutamyl-L-cysteinyl-glycine) is the most abundant non-protein thiol that protects cells from metabolic and oxidative stresses and is widely used as medicine, food additives and in cosmetic industry. The methylotrophic yeast Hansenula polymorpha is regarded as a rich source of glutathione due to the role of this thiol in detoxifications of key intermediates of methanol metabolism. Cellular and extracellular glutathione production of H. polymorpha DL-1 in the wild type and recombinant strains which overexpress genes of glutathione biosynthesis (GSH2) and its precursor cysteine (MET4) was studied. Results Glutathione producing capacity of H. polymorpha DL-1 depending on parameters of cultivation (dissolved oxygen tension, pH, stirrer speed), carbon substrate (glucose, methanol) and type of overexpressed genes of glutathione and its precursor biosynthesis during batch and fed-batch fermentations were studied. Under optimized conditions of glucose fed-batch cultivation, the glutathione productivity of the engineered strains was increased from ~900 up to ~ 2300 mg of Total Intracellular Glutathione (TIG) or GSH+GSSGin, per liter of culture medium. Meantime, methanol fed-batch cultivation of one of the recombinant strains allowed achieving the extracellular glutathione productivity up to 250 mg of Total Extracellular Glutathione (TEG) or GSH+GSSGex, per liter of the culture medium. Conclusions H. polymorpha is an competitive glutathione producer as compared to other known yeast and bacteria strains (Saccharomyces cerevisiae, Candida utilis, Escherichia coli, Lactococcus lactis etc.) with good perspectives for further improvement especially for production of extracellular form of glutathione. PMID:21255454

  20. 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.

  1. Ebselen exerts antifungal activity by regulating glutathione (GSH) and reactive oxygen species (ROS) production in fungal cells.

    PubMed

    Thangamani, Shankar; Eldesouky, Hassan E; Mohammad, Haroon; Pascuzzi, Pete E; Avramova, Larisa; Hazbun, Tony R; Seleem, Mohamed N

    2017-01-01

    Ebselen, an organoselenium compound and a clinically safe molecule has been reported to possess potent antifungal activity, but its antifungal mechanism of action and in vivo antifungal activity remain unclear. The antifungal effect of ebselen was tested against Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, Cryptococcus neoformans, and C. gattii clinical isolates. Chemogenomic profiling and biochemical assays were employed to identify the antifungal target of ebselen. Ebselen's antifungal activity in vivo was investigated in a Caenorhabditis elegans animal model. Ebselen exhibits potent antifungal activity against both Candida spp. and Cryptococcus spp., at concentrations ranging from 0.5 to 2μg/ml. Ebselen rapidly eradicates a high fungal inoculum within 2h of treatment. Investigation of the drug's antifungal mechanism of action indicates that ebselen depletes intracellular glutathione (GSH) levels, leading to increased production of reactive oxygen species (ROS), and thereby disturbs the redox homeostasis in fungal cells. Examination of ebselen's in vivo antifungal activity in two Caenorhabditis elegans models of infection demonstrate that ebselen is superior to conventional antifungal drugs (fluconazole, flucytosine and amphotericin) in reducing Candida and Cryptococcus fungal load. Ebselen possesses potent antifungal activity against clinically relevant isolates of both Candida and Cryptococcus by regulating GSH and ROS production. The potent in vivo antifungal activity of ebselen supports further investigation for repurposing it for use as an antifungal agent. The present study shows that ebselen targets glutathione and also support that glutathione as a potential target for antifungal drug development. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. 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

  3. Optimization of Reduced Glutathione Production by a Lactobacillus plantarum Isolate Using Plackett-Burman and Box-Behnken Designs.

    PubMed

    Al-Madboly, Lamiaa A; Khedr, Eman G; Ali, Safaa M

    2017-01-01

    In this work, we aim to optimize the production of reduced glutathione (GSH) synthesized intracellularly by a food-grade microorganism through a statistical approach. Using a colorimetric method, 25 Lactobacillus plantarum isolates were screened in an attempt to find a GSH-producing strain. It was found that 36% of the tested isolates showed positive result. Isolate (L 7 ) was found to produce 152.61 μM glutathione per gram which was the highest amount produced intracellularly. Accordingly, the later isolate was selected for the optimization process using Plackett-Burman and Box-Behnken designs. Temperature, amino acids, and urea were found to be the most significant independent variables. Following data analysis, the composition of the optimized medium was De Man-Sharp-Rogosa broth as a basal medium supplemented with NaCl (5%), H 2 O 2 (0.05%), sodium dodecyl sulfate (0.05%), amino acids (0.0281%), and urea (0.192%). The pH of the medium was adjusted to 8 and incubated for 24 h at 40°C. The GSH amount was increased by 10-fold (851%) using the optimized medium. Hence, our optimization design estimated the biotechnological potential of L. plantarum (L 7 ) for the production of GSH in the industry.

  4. Buthionine sulfoximine, a glutathione depletor, attenuates endotoxic fever and reduces IL-1β and IL-6 level in rats.

    PubMed

    Wrotek, Sylwia; Domagalski, Krzysztof; Jędrzejewski, Tomasz; Dec, Eliza; Kozak, Wiesław

    2017-02-01

    The aim of our study was to investigate the effect of buthionine sulfoximine (BSO) - a glutathione depletor - on a course of endotoxic fever and IL-1β and IL-6 production. Male Wistar rats were subjected to intraperitoneal injection of lipopolysaccharide (LPS) from E. coli (50μg/kg, ip) to provoke fever. The level of spleen glutathione, plasma interleukin (IL)-1β, IL-6, and deep body temperature (Tb) were measured. The LPS administration provoked fever (the average Tb was 38.14±0.05°C in NaCl/LPS-treated rats vs 37.10±0.03°C in control, not-treated rats; p<0.001). We observed that LPS injection induced a decrease in spleen glutathione level (7.67±0.92nM/g vs 13.27±0.47nM/g in not-treated rats; p<0.001). Furthermore, the injection of LPS provoked an elevation of plasma IL-1β and IL-6 concentration (from values below the lowest detectable standard in not-treated animals to 199.99±34.89pg/mL and 7500±542.21pg/mL, respectively; p<0.001). Pretreatment with BSO enhanced glutathione decrease in LPS-treated rats (5.05±0.49nM/g), and significantly affected fever (maximal Tb was 37.81±0.07°C in BSO/LPS-treated rats vs 38.76±0.11°C in NaCl/LPS-treated rats). BSO 4h after LPS injection decreased IL-1β and IL-6 gene expression (about 1.5 fold, and 2 fold, respectively). In a consequence we observed a decrease in plasma IL-6 concentration (4h after LPS injection plasma IL-6 was 4167.17±956.54pg/mL in BSO/LPS-treated rats vs 7500±542.21pg/mL in NaCl/LPS-treated rats; p<0.001), and later IL-1β (7h after LPS injection the IL-1β concentration was not detected). Based on these data, we conclude that BSO, in addition to well-known application as an inhibitor of glutathione synthesis, is an antipyretic agent which reduces both IL-1β and IL-6 concentration. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Glutathione conjugation and contaminant transformation

    USGS Publications Warehouse

    Field, Jennifer A.; Thurman, E.M.

    1996-01-01

    The recent identification of a novel sulfonated metabolite of alachlor in groundwater and metolachlor in soil is likely the result of glutathione conjugation. Glutathione conjugation is an important biochemical reaction that leads, in the case of alachlor, to the formation of a rather difficult to detect, water-soluble, and therefore highly mobile, sulfonated metabolite. Research from weed science, toxicology, and biochemistry is discussed to support the hypothesis that glutathione conjugation is a potentially important detoxification pathway carried out by aquatic and terrestrial plants and soil microorganisms. A brief review of the biochemical basis for glutathione conjugation is presented. We recommend that multidisciplinary research focus on the occurrence and expression of glutathione and its attendant enzymes in plants and microorganisms, relationships between electrophilic substrate structure and enzyme activity, and the potential exploitation of plants and microorganisms that are competent in glutathione conjugation for phytoremediation and bioremediation.

  6. 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

  7. Yi-gan san restores behavioral alterations and a decrease of brain glutathione level in a mouse model of schizophrenia.

    PubMed

    Makinodan, Manabu; Yamauchi, Takahira; Tatsumi, Kouko; Okuda, Hiroaki; Noriyama, Yoshinobu; Sadamatsu, Miyuki; Kishimoto, Toshifumi; Wanaka, Akio

    2009-01-01

    The traditional Chinese herbal medicine yi-gan san has been used to cure neuropsychological disorders. Schizophrenia can be one of the target diseases of yi-gan san. We aimed at evaluating the possible use of yi-gan san in improving the schizophrenic symptoms of an animal model. Yi-gan san or distilled water was administered to mice born from pregnant mice injected with polyinosinic-polycytidilic acid or phosphate buffered saline. The former is a model of schizophrenia based on the epidemiological data that maternal infection leads to psychotic disorders including schizophrenia in the offspring. Prepulse inhibition and sensitivity to methamphetamine in open field tests were analyzed and the total glutathione content of whole brains was measured. Yi-gan san reversed the decrease in prepulse inhibition, hypersensitivity to methamphetamine and cognitive deficits found in the model mice to the level of control mice. Total glutathione content in whole brains was reduced in the model mice but was restored to normal levels by yi-gan san treatment. These results suggest that yi-gan san may have ameliorating effects on the pathological symptoms of schizophrenia.

  8. Withanolide A Prevents Neurodegeneration by Modulating Hippocampal Glutathione Biosynthesis during Hypoxia

    PubMed Central

    Baitharu, Iswar; Jain, Vishal; Deep, Satya Narayan; Shroff, Sabita; Sahu, Jayanta Kumar; Naik, Pradeep Kumar; Ilavazhagan, Govindasamy

    2014-01-01

    Withania somnifera root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer. Present study explores the ameliorative effect of withanolide A, a major component of withania root extract and its molecular mechanism against hypoxia induced memory impairment. Withanolide A was administered to male Sprague Dawley rats before a period of 21 days pre-exposure and during 07 days of exposure to a simulated altitude of 25,000 ft. Glutathione level and glutathione dependent free radicals scavenging enzyme system, ATP, NADPH level, γ-glutamylcysteinyl ligase (GCLC) activity and oxidative stress markers were assessed in the hippocampus. Expression of apoptotic marker caspase 3 in hippocampus was investigated by immunohistochemistry. Transcriptional alteration and expression of GCLC and Nuclear factor (erythroid-derived 2)–related factor 2 (Nrf2) were investigated by real time PCR and immunoblotting respectively. Exposure to hypobaric hypoxia decreased reduced glutathione (GSH) level and impaired reduced gluatathione dependent free radical scavenging system in hippocampus resulting in elevated oxidative stress. Supplementation of withanolide A during hypoxic exposure increased GSH level, augmented GSH dependent free radicals scavenging system and decreased the number of caspase and hoescht positive cells in hippocampus. While withanolide A reversed hypoxia mediated neurodegeneration, administration of buthionine sulfoximine along with withanolide A blunted its neuroprotective effects. Exogenous administration of corticosterone suppressed Nrf2 and GCLC expression whereas inhibition of corticosterone synthesis upregulated Nrf2 as well as GCLC. Thus present study infers that withanolide A reduces neurodegeneration by restoring hypoxia induced glutathione depletion in hippocampus. Further, Withanolide A increases glutathione biosynthesis in neuronal cells by upregulating GCLC level through Nrf2 pathway in a corticosterone dependenet manner

  9. Withanolide A prevents neurodegeneration by modulating hippocampal glutathione biosynthesis during hypoxia.

    PubMed

    Baitharu, Iswar; Jain, Vishal; Deep, Satya Narayan; Shroff, Sabita; Sahu, Jayanta Kumar; Naik, Pradeep Kumar; Ilavazhagan, Govindasamy

    2014-01-01

    Withania somnifera root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer. Present study explores the ameliorative effect of withanolide A, a major component of withania root extract and its molecular mechanism against hypoxia induced memory impairment. Withanolide A was administered to male Sprague Dawley rats before a period of 21 days pre-exposure and during 07 days of exposure to a simulated altitude of 25,000 ft. Glutathione level and glutathione dependent free radicals scavenging enzyme system, ATP, NADPH level, γ-glutamylcysteinyl ligase (GCLC) activity and oxidative stress markers were assessed in the hippocampus. Expression of apoptotic marker caspase 3 in hippocampus was investigated by immunohistochemistry. Transcriptional alteration and expression of GCLC and Nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) were investigated by real time PCR and immunoblotting respectively. Exposure to hypobaric hypoxia decreased reduced glutathione (GSH) level and impaired reduced gluatathione dependent free radical scavenging system in hippocampus resulting in elevated oxidative stress. Supplementation of withanolide A during hypoxic exposure increased GSH level, augmented GSH dependent free radicals scavenging system and decreased the number of caspase and hoescht positive cells in hippocampus. While withanolide A reversed hypoxia mediated neurodegeneration, administration of buthionine sulfoximine along with withanolide A blunted its neuroprotective effects. Exogenous administration of corticosterone suppressed Nrf2 and GCLC expression whereas inhibition of corticosterone synthesis upregulated Nrf2 as well as GCLC. Thus present study infers that withanolide A reduces neurodegeneration by restoring hypoxia induced glutathione depletion in hippocampus. Further, Withanolide A increases glutathione biosynthesis in neuronal cells by upregulating GCLC level through Nrf2 pathway in a corticosterone dependenet manner.

  10. In vitro antileishmanial activity of fisetin flavonoid via inhibition of glutathione biosynthesis and arginase activity in Leishmania infantum.

    PubMed

    Adinehbeigi, Keivan; Razi Jalali, Mohammad Hossein; Shahriari, Ali; Bahrami, Somayeh

    2017-06-01

    With the increasing emergence of drug resistant Leishmania sp. in recent years, combination therapy has been considered as a useful way to treat and control of Leishmaniasis. The present study was designed to evaluate the antileishmanial effects of the fisetin alone and combination of fisetin plus Meglumine antimoniate (Fi-MA) against Leishmania infantum. The IC50 values for fisetin were obtained 0.283 and 0.102 μM against promastigotes and amastigote forms, respectively. Meglumine antimoniate (MA, Glucantime) as control drug also revealed IC50 values of 0.247 and 0.105 μM for promastigotes and amastigotes of L. infantum, respectively. In order to determine the mode of action of fisetin and Meglumine antimoniate (MA, Glucantime), the activities of arginase (ARG), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured. Moreover, intracellular glutathione (GSH) and nitric oxide (NO) levels in L. infantum-infected macrophages and L. infantum promastigotes which were treated with IC50 concentrations of fisetin, MA and Fi-MA were investigated. Our results showed that MA decreased CAT and SOD activity and increased NO levels in L. infantum-infected macrophages. In promastigotes, MA inhibited parasite SOD activity and reduced parasite NO production. The decreased levels of most of the antioxidant enzymes, accompanying by the raised level of NO in treated macrophages with MA, were observed to regain their normal profiles due to Fi-MA treatment. Furthermore, fisetin could prevent the growth of promastigotes by inhibition of ARG activity and reduction of GSH levels and NO production. In conclusion, these findings showed that fisetin improves MA side effects.

  11. In vitro antileishmanial activity of fisetin flavonoid via inhibition of glutathione biosynthesis and arginase activity in Leishmania infantum

    PubMed Central

    Razi Jalali, Mohammad Hossein; Shahriari, Ali; Bahrami, Somayeh

    2017-01-01

    With the increasing emergence of drug resistant Leishmania sp. in recent years, combination therapy has been considered as a useful way to treat and control of Leishmaniasis. The present study was designed to evaluate the antileishmanial effects of the fisetin alone and combination of fisetin plus Meglumine antimoniate (Fi-MA) against Leishmania infantum. The IC50 values for fisetin were obtained 0.283 and 0.102 μM against promastigotes and amastigote forms, respectively. Meglumine antimoniate (MA, Glucantime) as control drug also revealed IC50 values of 0.247 and 0.105 μM for promastigotes and amastigotes of L. infantum, respectively. In order to determine the mode of action of fisetin and Meglumine antimoniate (MA, Glucantime), the activities of arginase (ARG), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured. Moreover, intracellular glutathione (GSH) and nitric oxide (NO) levels in L. infantum-infected macrophages and L. infantum promastigotes which were treated with IC50 concentrations of fisetin, MA and Fi-MA were investigated. Our results showed that MA decreased CAT and SOD activity and increased NO levels in L. infantum-infected macrophages. In promastigotes, MA inhibited parasite SOD activity and reduced parasite NO production. The decreased levels of most of the antioxidant enzymes, accompanying by the raised level of NO in treated macrophages with MA, were observed to regain their normal profiles due to Fi-MA treatment. Furthermore, fisetin could prevent the growth of promastigotes by inhibition of ARG activity and reduction of GSH levels and NO production. In conclusion, these findings showed that fisetin improves MA side effects. PMID:28385129

  12. Rapid accumulation of glutathione during light stress in Arabidopsis.

    PubMed

    Choudhury, Feroza K; Devireddy, Amith R; Azad, Rajeev K; Shulaev, Vladimir; Mittler, Ron

    2018-05-25

    Environmental stress conditions can drastically affect plant growth and productivity. In contrast to soil moisture or salinity that can gradually change over a period of days or weeks, changes in light intensity or temperature can occur very rapidly, sometimes over the course of minutes or seconds. We previously reported that in response to rapid changes in light intensity (0-60 sec), Arabidopsis thaliana plants mount a large-scale transcriptomic response that includes several different transcripts essential for light stress acclimation. Here, we expand our analysis of the rapid response of Arabidopsis to light stress using a metabolomics approach and identify 111 metabolites that significantly alter in their level during the first 90 sec of light stress exposure. We further show that the levels of free and total glutathione accumulate rapidly during light stress in Arabidopsis and that the accumulation of total glutathione during light stress is associated with an increase in nitric oxide (NO) levels. We further suggest that the increase in precursors for glutathione biosynthesis could be linked to alterations in photorespiration, and that phosphoenolpyruvate could represent a major energy and carbon source for rapid metabolic responses. Taken together, our analysis could be used as an initial road map for the identification of different pathways that could be used to augment the rapid response of plants to abiotic stress. In addition, it highlights the important role of glutathione in these responses.

  13. Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

    PubMed Central

    Volland, Stefanie; Lütz, Cornelius; Michalke, Bernhard; Lütz-Meindl, Ursula

    2012-01-01

    Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 μM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

  14. Effect of glutathione during bottle storage of sparkling wine.

    PubMed

    Webber, Vanessa; Dutra, Sandra Valduga; Spinelli, Fernanda Rodrigues; Carnieli, Gilberto João; Cardozo, Alejandro; Vanderlinde, Regina

    2017-02-01

    Reduced glutathione (GSH) is an efficient antioxidant on limiting browning, losing varietal aromas and off-flavor formation. Therefore, this study aims to evaluate the effect of GSH addition (10, 20 and 30mgL(-1)) after the disgorging of the sparkling wine during storage. The sparkling wines were analyzed at 1, 6, 12 and 18months of storage according to the color index, concentration of the free SO2, phenolic compounds, catechin, epicatechin, caffeic acid, coumaric acid, acetaldehyde, total and reduced glutathione. The results show that GSH concentration decreased to the level of the control sparkling wine during the first 6months, and the total glutathione gradually declined up to 12months. The GSH reduces browning and acetaldehyde formation for up to 12months. However, the presence of glutathione had low or no influence on the concentration of free SO2, total phenolics, catechin, epicatechin, caffeic and coumaric acids. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Human glutathione transferases catalyzing the bioactivation of anticancer thiopurine prodrugs.

    PubMed

    Eklund, Birgitta I; Gunnarsdottir, Sjofn; Elfarra, Adnan A; Mannervik, Bengt

    2007-06-01

    cis-6-(2-Acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG) are thiopurine prodrugs provisionally inactivated by an alpha,beta-unsaturated substituent on the sulfur of the parental thiopurines 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). The active thiopurines are liberated intracellularly by glutathione (GSH) in reactions catalyzed by glutathione transferases (GSTs) (EC 2.5.1.18). Catalytic activities of 13 human GSTs representing seven distinct classes of soluble GSTs have been determined. The bioactivation of cAVTP and tAVTG occurs via a transient addition of GSH to the activated double bond of the S-substituent of the prodrug, followed by elimination of the thiopurine. The first of these consecutive reactions is rate-limiting for thiopurine release, but GST-activation of this first addition is shifting the rate limitation to the subsequent elimination. Highly active GSTs reveal the transient intermediate, which is detectable by UV spectroscopy and HPLC analysis. LC/MS analysis of the reaction products demonstrates that the primary GSH conjugate, 4-glutathionylbuten-2-one, can react with a second GSH molecule to form the 4-(bis-glutathionyl)butan-2-one. GST M1-1 and GST A4-4 were the most efficient enzymes with tAVTG, and GST M1-1 and GST M2-2 had highest activity with cAVTP. The highly efficient GST M1-1 is polymorphic and is absent in approximately half of the human population. GST P1-1, which is overexpressed in many cancer cells, had no detectable activity with cAVTP and only minor activity with tAVTG. Other GST-activated prodrugs have targeted GST P1-1-expressing cancer cells. Tumors expressing high levels of GST M1-1 or GST A4-4 can be predicted to be particularly vulnerable to chemotherapy with cAVTP or tAVTG.

  16. [Effect of inducible nitric oxide on intracellular homeostasis of hepatocytes].

    PubMed

    Tang, Xi-Feng; Zhou, Dong-Yao; Kang, Ge-Fei

    2002-02-01

    To investigate the effects of inducible nitric oxide (NO) and exogenous NO on the intracellular homeostasis of the hepatocytes. Endogenous NO was induced by combined action of lipopolysaccharide (LPS) and cytokines in cultured rat hepatocytes, and exogenous NO was supplied by sodium nitroprusside (SNP) to stimulate the hepatocytes. The changes in intracellular malondialdehyde (MDA), reduced glutathione(GSH) and free calcium ([Ca2+]i) were observed. substantial increase by 7.97 times in intracellular MDA level and a decrease by 57.9% in GSH occurred in the hepatocytes after the cells had been incubated with LPS and cytokines for 24 h, which were reversed by 43.5% and 98.4% respectively by treatment with N(G)-monomethyl-L-arginine (NMMA), a competitive nitric oxide synthase (NOS) inhibitor. Verapamil significantly reduced both endogenous NO production and oxidative stress, while the effect of A23187 was not conspicuous. Incubation with chlorpromazine and Vitamine E (VitE), however, did not result in decreased release of NO by LPS- and cytokines-induced hepatocytes. After SNP exposure of the hepatocytes, the oxidative status was reversibly enhanced in a time-dependent manner. Short exposure to SNP led to a concentration-dependent inhibition of the rapid and transient increase in free calcium induced by K(+) depolarization and hepatopoietin-coupled calcium mobilization. Inducible NO may initiate and play a key role in the latter stages of metabolic and functional stress responses of hepatocytes against endotoxin and cytokines, when the reduction occurs in the capacity of NO to independently mediate lipid peroxidation and counteract oxidation. The inhibitory effect of NO on [Ca2+]i mobilization may be an important autoregulatory mechanism by means of negative feedback on protein kinase C-associated NOS induction.

  17. 1-Methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons

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

    Drechsel, Derek A.; Liang, L.-P.; Patel, Manisha

    2007-05-01

    Decreased glutathione levels associated with increased oxidative stress are a hallmark of numerous neurodegenerative diseases, including Parkinson's disease. GSH is an important molecule that serves as an anti-oxidant and is also a major determinant of cellular redox environment. Previous studies have demonstrated that neurotoxins can cause changes in reduced and oxidized GSH levels; however, information regarding steady state levels remains unexplored. The goal of this study was to characterize changes in cellular GSH levels and its regulatory enzymes in a dopaminergic cell line (N27) following treatment with the Parkinsonian toxin, 1-methyl-4-phenylpyridinium (MPP{sup +}). Cellular GSH levels were initially significantly decreasedmore » 12 h after treatment, but subsequently recovered to values greater than controls by 24 h. However, oxidized glutathione (GSSG) levels were increased 24 h following treatment, concomitant with a decrease in GSH/GSSG ratio prior to cell death. In accordance with these changes, ROS levels were also increased, confirming the presence of oxidative stress. Decreased enzymatic activities of glutathione reductase and glutamate-cysteine ligase by 20-25% were observed at early time points and partly account for changes in GSH levels after MPP{sup +} exposure. Additionally, glutathione peroxidase activity was increased 24 h following treatment. MPP{sup +} treatment was not associated with increased efflux of glutathione to the medium. These data further elucidate the mechanisms underlying GSH depletion in response to the Parkinsonian toxin, MPP{sup +}.« less

  18. Dysregulation of Glutathione Homeostasis in Neurodegenerative Diseases

    PubMed Central

    Johnson, William M.; Wilson-Delfosse, Amy L.; Mieyal, John. J.

    2012-01-01

    Dysregulation of glutathione homeostasis and alterations in glutathione-dependent enzyme activities are increasingly implicated in the induction and progression of neurodegenerative diseases, including Alzheimer’s, Parkinson’s and Huntington’s diseases, amyotrophic lateral sclerosis, and Friedreich’s ataxia. In this review background is provided on the steady-state synthesis, regulation, and transport of glutathione, with primary focus on the brain. A brief overview is presented on the distinct but vital roles of glutathione in cellular maintenance and survival, and on the functions of key glutathione-dependent enzymes. Major contributors to initiation and progression of neurodegenerative diseases are considered, including oxidative stress, protein misfolding, and protein aggregation. In each case examples of key regulatory mechanisms are identified that are sensitive to changes in glutathione redox status and/or in the activities of glutathione-dependent enzymes. Mechanisms of dysregulation of glutathione and/or glutathione-dependent enzymes are discussed that are implicated in pathogenesis of each neurodegenerative disease. Limitations in information or interpretation are identified, and possible avenues for further research are described with an aim to elucidating novel targets for therapeutic interventions. The pros and cons of administration of N-acetylcysteine or glutathione as therapeutic agents for neurodegenerative diseases, as well as the potential utility of serum glutathione as a biomarker, are critically evaluated. PMID:23201762

  19. Investigation of electroacupuncture and manual acupuncture on carnitine and glutathione in muscle.

    PubMed

    Toda, Shizuo

    2011-01-01

    Electroacupuncture (EA) and manual acupuncture (MA) have therapeutic effects on muscle fatigue in muscle disease. The deficiencies of carnitine and glutathione induce muscle fatigue. This report investigated the effects of EA and MA on carnitine and glutathione in muscle. After the mice of EA group were fixed in the animal cage, right Zusanli (ST36) and Jiexi (ST41) were acupunctured and stimulated with uniform reinforcing and reducing method by twirling the acupuncture needle for 15 min. And then, the needle handles were connected to an electric stimulator for stimulating the acupoint with dense-sparse waves. After the mice of MA group were fixed in an animal cage, right ST36 and ST41 were acupunctured and allowed for 15 min. The mice of normal control group were not acupunctured and stimulated for 15 min. The mice of all groups were killed for collecting muscle tissue 1 h after the final treatment. Carnitine and glutathione in homogenate of muscle tissue were determined with carnitine (Kainos Laboratories Co., Tokyo, Japan) and glutathione assay kit (Dojin Chemicals Co., Kumamoto, Japan). Carnitine level in muscle tissue of MA group was significantly higher than those of EA group and normal control group. Carnitine level in muscle tissue of EA group was not significantly different from that of normal control group. Glutathione levels in muscle tissue of EA group and MA group were significantly higher than that of normal control group. This report presented that carnitine in muscle is increased by MA, and not increased by EA, and that glutathione in muscle is increased by EA and MA.

  20. The effect of prolonged oral contraceptive steroid use on erythrocyte glutathione peroxidase activity.

    PubMed

    Capel, I D; Jenner, M; Williams, D C; Donaldson, D; Nath, A

    1981-08-01

    A clinical study was undertaken to determine whether oral contraceptives (OCs) affect the activity of the enzyme glutathione peroxidase. OC users recruited for the study were volunteers attending the Redhill Family Planning Clinic in England. Their demographic characteristics were noted. Pre- and postmenopausal comparative subjects were also used. The laboratory procedures involved in the study are described. Findings are tabulated. The average erythrocyte glutathione peroxidase levels of women using OCs for more than 7 months were significantly higher than those of the pre- and postmenopausal subjects. These levels increased progressively with duration of OC use. These levels did not fluctuate with the menstrual cycle in either OC or non-OC users. Levels of erythrocyte selenium and plasma pyridoxal were not significantly altered by OC use. Riboflavin status, however, as estimated by glutathione reductase activity was substantially lower in OC users and was lowest in women who had used OCs for the longest amount of time. Riboflavin status was found to be directly correlated with erythrocyte glutathione peroxidase levels. These findings may be important because selenium is currently believed to offer protective benefits against carcinogenesis, especially breast cancer. All the OCs studied produced the same effects.

  1. 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.

  2. Insulin-Like Growth Factor Binding Proteins Increase Intracellular Calcium Levels in Two Different Cell Lines

    PubMed Central

    Seurin, Danielle; Lombet, Alain; Babajko, Sylvie; Godeau, François; Ricort, Jean-Marc

    2013-01-01

    Background Insulin-like growth factor binding proteins (IGFBPs) are six related secreted proteins that share IGF-dependent and -independent functions. If the former functions begin to be well described, the latter are somewhat more difficult to investigate and to characterize. At the cellular level, IGFBPs were shown to modulate numerous processes including cell growth, differentiation and apoptosis. However, the molecular mechanisms implicated remain largely unknown. We previously demonstrated that IGFBP-3, but not IGFBP-1 or IGFBP-5, increase intracellular calcium concentration in MCF-7 cells (Ricort J-M et al. (2002) FEBS lett 527: 293–297). Methodology/Principal Findings We perform a global analysis in which we studied, by two different approaches, the binding of each IGFBP isoform (i.e., IGFBP-1 to -6) to the surface of two different cellular models, MCF-7 breast adenocarcinoma cells and C2 myoblast proliferative cells, as well as the IGFBP-induced increase of intracellular calcium concentration. Using both confocal fluorescence microscopy and flow cytometry analysis, we showed that all IGFBPs bind to MCF-7 cell surface. By contrast, only four IGFBPs can bind to C2 cell surface since neither IGFBP-2 nor IGFBP-4 were detected. Among the six IGFBPs tested, only IGFBP-1 did not increased intracellular calcium concentration whatever the cellular model studied. By contrast, IGFBP-2, -3, -4 and -6, in MCF-7 cells, and IGFBP-3, -5 and -6, in C2 proliferative cells, induce a rapid and transient increase in intracellular free calcium concentration. Moreover, IGFBP-2 and -3 (in MCF-7 cells) and IGFBP-5 (in C2 cells) increase intracellular free calcium concentration by a pertussis toxin sensitive signaling pathway. Conclusions Our results demonstrate that IGFBPs are able to bind to cell surface and increase intracellular calcium concentration. By characterizing the IGFBPs-induced cell responses and intracellular couplings, we highlight the cellular specificity and

  3. Alternative oxidase impacts ganoderic acid biosynthesis by regulating intracellular ROS levels in Ganoderma lucidum.

    PubMed

    Shi, Deng-Ke; Zhu, Jing; Sun, Ze-Hua; Zhang, Guang; Liu, Rui; Zhang, Tian-Jun; Wang, Sheng-Li; Ren, Ang; Zhao, Ming-Wen

    2017-10-01

    The alternative oxidase (AOX), which forms a branch of the mitochondrial respiratory electron transport pathway, functions to sustain electron flux and alleviate reactive oxygen species (ROS) production. In this article, a homologous AOX gene was identified in Ganoderma lucidum. The coding sequence of the AOX gene in G. lucidum contains 1038 nucleotides and encodes a protein of 39.48 kDa. RNA interference (RNAi) was used to study the function of AOX in G. lucidum, and two silenced strains (AOXi6 and AOXi21) were obtained, showing significant decreases of approximately 60 and 50 %, respectively, in alternative pathway respiratory efficiency compared to WT. The content of ganoderic acid (GA) in the mutant strains AOXi6 and AOXi21 showed significant increases of approximately 42 and 44 %, respectively, compared to WT. Elevated contents of intermediate metabolites in GA biosynthesis and elevated transcription levels of corresponding genes were also observed in the mutant strains AOXi6 and AOXi21. In addition, the intracellular ROS content in strains AOXi6 and AOXi21 was significantly increased, by approximately 1.75- and 1.93-fold, respectively, compared with WT. Furthermore, adding N-acetyl-l-cysteine (NAC), a ROS scavenger, significantly depressed the intracellular ROS content and GA accumulation in AOX-silenced strains. These results indicate that AOX affects GA biosynthesis by regulating intracellular ROS levels. Our research revealed the important role of AOX in the secondary metabolism of G. lucidum.

  4. Silencing Prx1 and/or Prx5 sensitizes human esophageal cancer cells to ionizing radiation and increases apoptosis via intracellular ROS accumulation

    PubMed Central

    Gao, Mai-cang; Jia, Xiao-di; Wu, Qi-fei; Cheng, Yan; Chen, Fen-rong; Zhang, Jun

    2011-01-01

    Aim: To investigate whether down-regulation of peroxiredoxin 1 (Prx1) and/or peroxiredoxin 5 (Prx5) sensitizes human esophageal cancer cells to ionizing radiation (IR). Methods: Human esophageal carcinoma cell lines Eca-109 and TE-1 were used. Prx mRNA expression profiles in Eca-109 and TE-1 cells were determined using RT-PCR. Two highly expressed isoforms of Prxs, Prx1 and Prx5, were silenced by RNA interference (RNAi). Following IR, intracellular reactive oxygen species (ROS) and apoptosis were measured using flow cytometry, the activities of catalase, superoxide dismutase and glutathione peroxidase were measured, and the radiosensitizing effect of RNAi was observed. Tumor xenograft model was also used to examine the radiosensitizing effect of RNAi in vivo. Results: Down-regulation of Prx1 and/or Prx5 by RNAi does not alter the activities of catalase, superoxide dismutase and glutathione peroxidase, but made human tumor cells more sensitive to IR-induced apoptosis both in vitro and in vivo. When the two isoforms were decreased simultaneously, intracellular ROS and apoptosis significantly increased after IR. Conclusion: Silencing Prx1 and/or Prx5 by RNAi sensitizes human Eca-109 and TE-1 cells to IR, and the intracellular ROS accumulation may contribute to the radiosensitizing effect of the RNAi. PMID:21468086

  5. Relationship between oxidizable fatty acid content and level of antioxidant glutathione peroxidases in marine fish

    PubMed Central

    Grim, Jeffrey M.; Hyndman, Kelly A.; Kriska, Tamas; Girotti, Albert W.; Crockett, Elizabeth L.

    2011-01-01

    SUMMARY Biological membranes can be protected from lipid peroxidation by antioxidant enzymes including catalase (CAT) and selenium-dependent glutathione peroxidases 1 and 4 (GPx1 and GPx4). Unlike GPx1, GPx4 can directly detoxify lipid hydroperoxides in membranes without prior action of phospholipase A2. We hypothesized that (1) GPx4 is enhanced in species that contain elevated levels of highly oxidizable polyunsaturated fatty acids (PUFA) and (2) activities of antioxidant enzymes are prioritized to meet species-specific oxidative stresses. In this study we examined (i) activities of the oxidative enzyme citrate synthase (CS) and antioxidant (CAT, GPx1 and GPx4) enzymes, (ii) GPx4 protein expression, and (iii) phospholipid composition in livers of five species of marine fish (Myxine glutinosa, Petromyzon marinus, Squalus acanthias, Fundulus heteroclitus and Myoxocephalus octodecemspinosus) that contain a range of PUFA. GPx4 activity was, on average, 5.8 times higher in F. heteroclitus and S. acanthias than in the other three marine fish species sampled. Similarly, activities of CAT and GPx1 were highest in S. acanthias and F. heteroclitus, respectively. GPx4 activity for all species correlates with membrane unsaturation, as well as oxidative activity as indicated by CS. These data support our hypothesis that GPx4 level in marine fish is a function, at least in part, of high PUFA content in these animals. GPx1 activity was also correlated with membrane unsaturation, indicating that marine species partition resources among glutathione-dependent defenses for protection from the initial oxidative insult (e.g. H2O2) and to repair damaged lipids within biological membranes. PMID:22031739

  6. Downregulation of Glutathione Biosynthesis Contributes to Oxidative Stress and Liver Dysfunction in Acute Kidney Injury

    PubMed Central

    Siow, Yaw L.; Isaak, Cara K.

    2016-01-01

    Ischemia-reperfusion is a common cause for acute kidney injury and can lead to distant organ dysfunction. Glutathione is a major endogenous antioxidant and its depletion directly correlates to ischemia-reperfusion injury. The liver has high capacity for producing glutathione and is a key organ in modulating local and systemic redox balance. In the present study, we investigated the mechanism by which kidney ischemia-reperfusion led to glutathione depletion and oxidative stress. The left kidney of Sprague-Dawley rats was subjected to 45 min ischemia followed by 6 h reperfusion. Ischemia-reperfusion impaired kidney and liver function. This was accompanied by a decrease in glutathione levels in the liver and plasma and increased hepatic lipid peroxidation and plasma homocysteine levels. Ischemia-reperfusion caused a significant decrease in mRNA and protein levels of hepatic glutamate-cysteine ligase mediated through the inhibition of transcription factor Nrf2. Ischemia-reperfusion inhibited hepatic expression of cystathionine γ-lyase, an enzyme responsible for producing cysteine (an essential precursor for glutathione synthesis) through the transsulfuration pathway. These results suggest that inhibition of glutamate-cysteine ligase expression and downregulation of the transsulfuration pathway lead to reduced hepatic glutathione biosynthesis and elevation of plasma homocysteine levels, which, in turn, may contribute to oxidative stress and distant organ injury during renal ischemia-reperfusion. PMID:27872680

  7. Docosahexaenoic acid prevents paraquat-induced reactive oxygen species production in dopaminergic neurons via enhancement of glutathione homeostasis

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

    Lee, Hyoung Jun; Han, Jeongsu; Jang, Yunseon

    Highlights: • DHA prevents PQ-induced dopaminergic neuronal loss via decreasing of excessive ROS. • DHA increases GR and GCLm derivate GSH pool by enhancement of Nrf2 expression. • Protective mechanism is removal of PQ-induced ROS via DHA-dependent GSH pool. • DHA may be a good preventive strategy for Parkinson’s disease (PD) therapy. - Abstract: Omega-3 polyunsaturated fatty acid levels are reduced in the substantia nigra area in Parkinson’s disease patients and animal models, implicating docosahexaenoic acid (DHA) as a potential treatment for preventing Parkinson’s disease and suggesting the need for investigations into how DHA might protect against neurotoxin-induced dopaminergic neuronmore » loss. The herbicide paraquat (PQ) induces dopaminergic neuron loss through the excessive production of reactive oxygen species (ROS). We found that treatment of dopaminergic SN4741 cells with PQ reduced cell viability in a dose-dependent manner, but pretreatment with DHA ameliorated the toxic effect of PQ. To determine the toxic mechanism of PQ, we measured intracellular ROS content in different organelles with specific dyes. As expected, all types of ROS were increased by PQ treatment, but DHA pretreatment selectively decreased cytosolic hydrogen peroxide content. Furthermore, DHA treatment-induced increases in glutathione reductase and glutamate cysteine ligase modifier subunit (GCLm) mRNA expression were positively correlated with glutathione (GSH) content. Consistent with this increase in GCLm mRNA levels, Western blot analysis revealed that DHA pretreatment increased nuclear factor-erythroid 2 related factor 2 (Nrf2) protein levels. These findings indicate that DHA prevents PQ-induced neuronal cell loss by enhancing Nrf2-regulated GSH homeostasis.« less

  8. Investigation of Electroacupuncture and Manual Acupuncture on Carnitine and Glutathione in Muscle

    PubMed Central

    Toda, Shizuo

    2011-01-01

    Electroacupuncture (EA) and manual acupuncture (MA) have therapeutic effects on muscle fatigue in muscle disease. The deficiencies of carnitine and glutathione induce muscle fatigue. This report investigated the effects of EA and MA on carnitine and glutathione in muscle. After the mice of EA group were fixed in the animal cage, right Zusanli (ST36) and Jiexi (ST41) were acupunctured and stimulated with uniform reinforcing and reducing method by twirling the acupuncture needle for 15 min. And then, the needle handles were connected to an electric stimulator for stimulating the acupoint with dense-sparse waves. After the mice of MA group were fixed in an animal cage, right ST36 and ST41 were acupunctured and allowed for 15 min. The mice of normal control group were not acupunctured and stimulated for 15 min. The mice of all groups were killed for collecting muscle tissue 1 h after the final treatment. Carnitine and glutathione in homogenate of muscle tissue were determined with carnitine (Kainos Laboratories Co., Tokyo, Japan) and glutathione assay kit (Dojin Chemicals Co., Kumamoto, Japan). Carnitine level in muscle tissue of MA group was significantly higher than those of EA group and normal control group. Carnitine level in muscle tissue of EA group was not significantly different from that of normal control group. Glutathione levels in muscle tissue of EA group and MA group were significantly higher than that of normal control group. This report presented that carnitine in muscle is increased by MA, and not increased by EA, and that glutathione in muscle is increased by EA and MA. PMID:19592478

  9. The influence of royal jelly and human interferon-alpha (HuIFN-αN3) on proliferation, glutathione level and lipid peroxidation in human colorectal adenocarcinoma cells in vitro.

    PubMed

    Filipič, Bratko; Gradišnik, Lidija; Rihar, Klemen; Šooš, Eugen; Pereyra, Adriana; Potokar, Jana

    2015-12-01

    Among royal jelly's (RJ) various biological activities, its possible antitumour activity deserves particular attention. The purpose of this study was to investigate the influence of RJ, its bioactive component 10-hydroxy-2-decenoic acid (10- HDA), and human interferon-alpha (HuIFN-αN3) on the proliferation of human colorectal adenocarcinoma cells (CaCo- 2), and ascertain their effect on intracellular glutathione (GSH) level and lipid peroxidation. We studied the antiproliferative (AP) activity of RJ [(0.1 g/10 mL phosphate buffer saline (PBS)], HuIFN-αN3 (1000 I.U. mL⁻¹), 10-HDA at 100.0 μmol L⁻¹, and their different combinations, in the ratio 1:1, 1:2, and 2:1 on CaCo-2 cells. The GSH level was measured by glutathione assay. The lipid peroxidation was measured by malondialdehyde (MDA) assay. Single RJ had a low AP activity: 2.0 (0.5 mg mL⁻¹). HuIFN-αN3 had an AP activity of 2.5 (208.33 I.U. mL⁻¹), while 10-HDA had an AP activity of 1.5 (37.5 μmol mL⁻¹). The highest AP activity of 3.8 was obtained when RJ and HuIFN-αN3 were applied at the ratio 2:1. In that combination the level of GSH was 24.9±2.4 nmol g⁻³ of proteins (vs. 70.2±3.2 nmol g⁻³ in the control) and the level of MDA was 72.3±3.1 nmol g⁻³ (vs. 23.6±9.1 nmol g⁻³ in the control). It is generally assumed that 10-HDA, an important constituent of RJ, together with HuIFN-αN3, is responsible for the inhibition of CaCo-2 cells proliferation in vitro. In our study, however, RJ and HuIFN-αN3 applied at 2:1 decreased the level of GSH the most and significantly increased lipid peroxidation via MDA in CaCo-2 cells. Future studies should show whether these GSH- and MDA-related activities of RJ, HuIFN-αN3, 10-HDA, and their combinations may decrease the tumorigenicity index and tumorigenic potential of various tumour cells in vitro.

  10. Oxidative stress protection and glutathione metabolism in response to hydrogen peroxide and menadione in riboflavinogenic fungus Ashbya gossypii.

    PubMed

    Kavitha, S; Chandra, T S

    2014-11-01

    Ashbya gossypii is a plant pathogen and a natural overproducer of riboflavin and is used for industrial riboflavin production. A few literature reports depict a link between riboflavin overproduction and stress in this fungus. However, the stress protection mechanisms and glutathione metabolism are not much explored in A. gossypii. In the present study, an increase in the activity of catalase and superoxide dismutase was observed in response to hydrogen peroxide and menadione. The lipid peroxide and membrane lipid peroxide levels were increased by H2O2 and menadione, indicating oxidative damage. The glutathione metabolism was altered with a significant increase in oxidized glutathione (GSSG), glutathione peroxidase (GPX), glutathione S transferase (GST), and glutathione reductase (GR) and a decrease in reduced glutathione (GSH) levels in the presence of H2O2 and menadione. Expression of the genes involved in stress mechanism was analyzed in response to the stressors by semiquantitative RT-PCR. The messenger RNA (mRNA) levels of CTT1, SOD1, GSH1, YAP1, and RIB3 were increased by H2O2 and menadione, indicating the effect of stress at the transcriptional level. A preliminary bioinformatics study for the presence of stress response elements (STRE)/Yap response elements (YRE) depicted that the glutathione metabolic genes, stress genes, and the RIB genes hosted either STRE/YRE, which may enable induction of these genes during stress.

  11. Intracellular delivery and antitumor effects of a redox-responsive polymeric paclitaxel conjugate based on hyaluronic acid.

    PubMed

    Yin, Shaoping; Huai, Jue; Chen, Xi; Yang, Yong; Zhang, Xinxin; Gan, Yong; Wang, Guangji; Gu, Xiaochen; Li, Juan

    2015-10-01

    Polymer-drug conjugates have demonstrated application potentials in optimizing chemotherapeutics. In this study a new bioconjugate, HA-ss-PTX, was designed and synthesized with cooperative dual characteristics of active tumor targeting and selective intracellular drug release. Paclitaxel (PTX) was covalently attached to hyaluronic acid (HA) with various sizes (MW 9.5, 35, 770 kDa); a cross-linker containing disulfide bond was also used to shield drug leakage in blood circulation and to achieve rapid drug release in tumor cells in response to glutathione. Incorporation of HA to the conjugate enhanced the capabilities of drug loading, intracellular endocytosis and tumor targeting of micelles in comparison to mPEG. HA molecular weight showed significant effect on properties and antitumor efficacy of the synthesized conjugates. Intracellular uptake of HA-ss-PTX toward MCF-7 cells was mediated by CD44-caveolae-mediated endocytosis. Compared to Taxol and mPEG-ss-PTX, HA9.5-ss-PTX demonstrated improved tumor growth inhibition in vivo with a TIR of 83.27 ± 5.20%. It was concluded that HA9.5-ss-PTX achieved rapid intracellular release of PTX and enhanced its therapeutic efficacy, thus providing a platform for specific drug targeting and controlled intracellular release in chemotherapeutics. Polymer-drug conjugates, promising nanomedicines, still face some technical challenges including a lack of specific targeting and rapid intracellular drug release at the target site. In this manuscript we designed and constructed a novel bioconjugate HA-ss-PTX, which possessed coordinated dual characteristics of active tumor targeting and selective intracellular drug release. Redox-responsive disulfide bond was introduced to the conjugate to shield drug leakage in blood circulation and to achieve rapid drug release at tumor site in response to reductant like glutathione. Paclitaxel was selected as a model drug to be covalently attached to hyaluronic acid (HA) with various sizes to

  12. 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.

  13. [The activity of glutathione antioxidant system at melaksen and valdoxan action under experimental hyperthyroidism in rats].

    PubMed

    Gorbenko, M V; Popova, T N; Shul'gin, K K; Popov, S S

    2013-01-01

    Investigation of glutathione antioxidant system activity and diene conjugates content in rats liver and blood serum at the influence of melaksen and valdoxan under experimental hyperthyroidism (EG) has been revealed. It has been established that the activities of glutathione reductase (GR), glutathione peroxidase (GP) and glutathione transferase (GT), growing at pathological conditions, change to the side of control value at these substunces introduction. Reduced glutathione content (GSH) at melaxen and valdoxan action increased compared with values under the pathology, that, obviously, could be associated with a reduction of its spending on the detoxication of free radical oxidation (FRO) toxic products. Diene conjugates level in rats liver and blood serum, increasing at experimental hyperthyroidism conditions, under introduction of melatonin level correcting drugs, also approached to the control meaning. Results of the study indicate on positive effect of melaxen and valdoxan on free radical homeostasis, that appears to be accompanied by decrease of load on the glutathione antioxidant system in comparison with the pathology.

  14. The antitumour activity of alkylating agents is not correlated with the levels of glutathione, glutathione transferase and O6-alkylguanine-DNA-alkyltransferase of human tumour xenografts. EORTC SPG and PAMM Groups.

    PubMed

    D'Incalci, M; Bonfanti, M; Pifferi, A; Mascellani, E; Tagliabue, G; Berger, D; Fiebig, H H

    1998-10-01

    Twenty-three human xenografts, including five colon, five gastric, nine lung (three small cell lung cancer) and four breast carcinomas, were investigated for their sensitivity to nitrosoureas, dacarbazine (DTIC), cyclophosphamide (CTX) and cisplatin (DDP). In 12 cases, at least one of the drugs produced complete or partial remission, in 2, a minor regression was observed and in the other 9, treatment was ineffective. The level of sensitivity to each drug, using a score from 1 to 5, was correlated to three biochemical parameters reported to be involved in resistance to alkylating agents: glutathione (GSH), glutathione transferase (GST) and O6-alkylguanine-DNA-alkyltransferase (AGT). A wide variability was found in these parameters in the xenografts investigated. No correlation was found between any of the three parameters and sensitivity to the drugs used or between sensitivity to one drug and to any of the other drugs tested. These results illustrate the complexity of the question of resistance to alkylating agents and indicate that, at least in xenografts, the biochemical parameters examined are not predictive of response to alkylating agents.

  15. Chemical form of selenium affects its uptake, transport, and glutathione peroxidase activity in the human intestinal Caco-2 cell model.

    PubMed

    Zeng, Huawei; Jackson, Matthew I; Cheng, Wen-Hsing; Combs, Gerald F

    2011-11-01

    Determining the effect of selenium (Se) chemical form on uptake, transport, and glutathione peroxidase activity in human intestinal cells is critical to assess Se bioavailability at nutritional doses. In this study, we found that two sources of L-selenomethionine (SeMet) and Se-enriched yeast each increased intracellular Se content more effectively than selenite or methylselenocysteine (SeMSC) in the human intestinal Caco-2 cell model. Interestingly, SeMSC, SeMet, and digested Se-enriched yeast were transported at comparable efficacy from the apical to basolateral sides, each being about 3-fold that of selenite. In addition, these forms of Se, whether before or after traversing from apical side to basolateral side, did not change the potential to support glutathione peroxidase (GPx) activity. Although selenoprotein P has been postulated to be a key Se transport protein, its intracellular expression did not differ when selenite, SeMSC, SeMet, or digested Se-enriched yeast was added to serum-contained media. Taken together, our data show, for the first time, that the chemical form of Se at nutritional doses can affect the absorptive (apical to basolateral side) efficacy and retention of Se by intestinal cells; but that, these effects are not directly correlated to the potential to support GPx activity.

  16. 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.

  17. Co-administration of α-lipoic acid and glutathione is associated with no significant changes in serum bilirubin, alkaline phosphatase or γ-glutamyltranspeptidase levels during the treatment of neuroborreliosis with intravenous ceftriaxone.

    PubMed

    Puri, Basant K; Hakkarainen-Smith, Jaana S; Derham, Anne; Monro, Jean A

    2015-09-01

    While pharmacotherapy with intravenous ceftriaxone, a third-generation cephalosporin, is a potential treatment of Lyme neuroborreliosis, there is concern that it can cause the formation of biliary sludge, leading to hepatobiliary complications such as biliary colic, jaundice and cholelithiasis, which are reflected in changes in serum levels of bilirubin and markers of cholestatic liver injury (alkaline phosphatase and γ-glutamyltranspeptidase). It has been suggested that the naturally occurring substances α-lipoic acid and glutathione may be helpful in preventing hepatic disease. α-Lipoic acid exhibits antioxidant, anti-inflammatory and anti-apoptotic activities in the liver, while glutathione serves as a sulfhydryl buffer. The aim of this study was to determine whether co-administration of α-lipoic acid and glutathione is associated with significant changes in serum levels of bilirubin, alkaline phosphatase and γ-glutamyltranspeptidase during the treatment of Lyme neuroborreliosis with long-term intravenous ceftriaxone. Serum levels of bilirubin, alkaline phosphatase and γ-glutamyltranspeptidase were measured in 42 serologically positive Lyme neuroborreliosis patients before and after long-term treatment with intravenous ceftriaxone (2-4 g daily) with co-administration of oral/intravenous α-lipoic acid (600 mg daily) and glutathione (100 mg orally or 0.6-2.4 g intravenously daily). None of the patients developed biliary colic and there were no significant changes in serum bilirubin, alkaline phosphatase or γ-glutamyltranspeptidase levels over the course of the intravenous ceftriaxone treatment (mean length 75.0 days). Co-administration of α-lipoic acid and glutathione is associated with no significant changes in serum bilirubin, alkaline phosphatase or γ-glutamyltranspeptidase levels during the treatment of neuroborreliosis with intravenous ceftriaxone.

  18. Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers.

    PubMed

    Navarro, Gemma; Aguinaga, David; Moreno, Estefania; Hradsky, Johannes; Reddy, Pasham P; Cortés, Antoni; Mallol, Josefa; Casadó, Vicent; Mikhaylova, Marina; Kreutz, Michael R; Lluís, Carme; Canela, Enric I; McCormick, Peter J; Ferré, Sergi

    2014-11-20

    The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.

  19. A mesoporous nanocontainer gated by a stimuli-responsive peptide for selective triggering of intracellular drug release

    NASA Astrophysics Data System (ADS)

    Lee, Jeonghun; Oh, Eun-Taex; Yoon, Haerry; Kim, Hyunmi; Park, Heon Joo; Kim, Chulhee

    2016-04-01

    Mesoporous silica nanocontainers (MSNs) with biologically responsive gatekeepers have great potential for effective delivery of cargo molecules to the desired sites. For that purpose, peptides could be effective candidates as gatekeepers because of their bioresponsiveness and targeting capability. Taking advantage of the zinc finger domain peptide (CXXC), we designed a biocompatible all-peptide gatekeeper (WCGKC) with on-off gatekeeping capability through stimulus-responsive conformational conversion and the steric bulkiness of the tryptophan unit. The turn structure induced by an intramolecular disulfide bond of the peptide gatekeeper (WCGKC-SS) completely inhibited the release of the entrapped doxorubicin (DOX). However, upon reduction of the disulfide bond by glutathione (GSH), the peptide conformation was converted to a random structure, which opened the orifice of the mesopore leading to the release of DOX. The amine moiety of the lysine of the peptide gatekeeper was PEGylated to enhance dispersion stability and biocompatibility of the nanocontainer. Furthermore, the MSNs with the peptide gatekeeper (PEG-WCGKC-SS-Si) selectively released the entrapped DOX in A549 human lung cancer cells in a controlled manner triggered by intracellular GSH, but not in CCD normal lung cells containing a low intracellular GSH level. In A549 cells, DOX-loaded PEG-WCGKC-SS-Si exhibited about 10-times higher cytotoxicity induced by apoptosis than that in CCD cells.Mesoporous silica nanocontainers (MSNs) with biologically responsive gatekeepers have great potential for effective delivery of cargo molecules to the desired sites. For that purpose, peptides could be effective candidates as gatekeepers because of their bioresponsiveness and targeting capability. Taking advantage of the zinc finger domain peptide (CXXC), we designed a biocompatible all-peptide gatekeeper (WCGKC) with on-off gatekeeping capability through stimulus-responsive conformational conversion and the steric

  20. Glutathione metabolic status in the aged rabbit aorta.

    PubMed

    Lapenna, Domenico; Ciofani, Giuliano; Giamberardino, Maria Adele

    2017-05-01

    It is not known whether aging alters glutathione metabolic status of the mammalian arterial tissue favoring vascular oxidative stress and dysfunction. Thus we assessed total, reduced and oxidized glutathione (TG, GSH and GSSG, respectively), the glutathione redox ratio (GRR, namely [GSSG]/[GSH+2GSSG]×100), and the activities of the glutathione status-regulating enzymes glutathione reductase (GSSG-Red), γ-glutamylcysteine synthetase (γ-GCS) and γ-glutamyl transpeptidase (γ-GT) in the aortic tissue of 9 young adult control rabbits (YACR, about 4months old) and 9 aged rabbits (AR, about 4.5years old); aortic lipid and protein oxidation and H 2 O 2 were also determined as oxidative stress indicators. Vascular function was assessed on aortic ring preparations. TG and GSH concentrations, together with γ-GCS and γ-GT activities, were significantly lower, while GSSG content and the GRR higher, in the AR than in the YACR aortas; GSSG-Red activity did not differ significantly between the two groups. Heightened levels of lipid and protein oxidation and H 2 O 2 occurred in the AR aortas, indicating age-dependent vascular oxidative stress. Moreover, in the whole population of 18 rabbits, the aortic values of GSH and related enzyme activities were inversely and significantly correlated with those of lipid and protein oxidation and H 2 O 2 , highlighting the antioxidant role of GSH and related enzymes in the vascular tissue. Aortic endothelium-dependent vasodilation was lower in the AR than in the YACR. In conclusion, glutathione metabolic status is altered in the aged rabbit aorta reflecting depressed γ-GCS- and γ-GT-related GSH biosynthesis and GSSG burden eventually favoring vascular oxidative stress and dysfunction. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Understanding the degradation of ascorbic acid and glutathione in relation to the levels of oxidative stress biomarkers in broccoli (Brassica oleracea L. italica cv. Bellstar) during storage and mechanical processing.

    PubMed

    Raseetha, Siva; Leong, Sze Ying; Burritt, David John; Oey, Indrawati

    2013-06-01

    The purpose of this research was to understand the degradation of ascorbic acid and glutathione content in broccoli florets (Brassica oleracea L. italica cv. Bellstar) during prolonged storage and subsequent mechanical processing. The initial content of total ascorbic acid and glutathione in broccoli florets averaged at 5.18 ± 0.23 and 0.70 ± 0.03 μmol/g fresh weight, respectively. Results showed that the content of ascorbic acid and glutathione in broccoli degraded during storage at 23°C, for at least 4.5-fold after 6 days of storage. On each day of storage, broccoli florets were mechanically processed, but the content of total ascorbic acid and glutathione was not significantly affected. When the mechanically processed broccoli florets were further incubated for up to 6h, the amount of ascorbic acid was greatly reduced as compared to glutathione. To obtain an in-depth understanding on the degradation of ascorbic acid and glutathione, the activity of enzymes involved in plant antioxidative system via ascorbate-glutathione cycle, as a response towards oxidative stress that took place during storage was determined in this study. The content of total ascorbic acid and glutathione in broccoli florets before and after mechanical processing were found to decrease concurrently with the activity of ascorbic acid peroxidase and glutathione reductase over the experimental storage duration. Meanwhile, the effect of oxidative stress on the content of ascorbic acid and glutathione was apparent during the 6h of incubation after mechanical processing. This phenomenon was demonstrated by the level of oxidative stress biomarkers examined, in which the formation of lipid peroxides, protein carbonyls and DNA oxidised products was positively associated with the degradation of total ascorbic acid and glutathione. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Changes of reduced glutathion, glutathion reductase, and glutathione peroxidase after radiation in guinea pigs

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

    Erden, M.; Bor, N.M.

    1984-04-01

    In this series of experiments the protective action of reduced glutathion due to ionizing radiation has been studied. In the experimental group 18 guinea pigs were exposed to successive radiations of 150 rad 3 or 4 days apart. Total dose given amounted to 750 rad which is the LD50 for guinea pigs. Blood samples were taken 30 min after each exposure. The control series were sham radiated but otherwise treated identically. The cells of the removed blood samples were separated by centrifugation and were subjected to the reduced glutathion stability test. GSSGR, GPer, and LDH enzyme activities were also measuredmore » of which the latter served as a marked enzyme. It was found that LDH did not show any alteration after radiation. The reduced glutathion stability test showed a consistent but minor reduction (P greater than 0.05), in the experimental group. GSSGR enzyme activity on the other hand was reduced significantly (from 176.48 +/- 11.32 to 41.34 +/- 1.17 IU/ml of packed erythrocytes, P less than 0.001) in the same group. GPer activity showed a consistent but minor elevation during the early phase of the experimental group. It was later increased significantly beginning after 600 rad total radiation on the fourth session (P less than 0.050).« less

  3. Hybrid molecule from O2-(2,4-dinitrophenyl)diazeniumdiolate and oleanolic acid: a glutathione S-transferase π-activated nitric oxide prodrug with selective anti-human hepatocellular carcinoma activity and improved stability.

    PubMed

    Fu, Junjie; Liu, Ling; Huang, Zhangjian; Lai, Yisheng; Ji, Hui; Peng, Sixun; Tian, Jide; Zhang, Yihua

    2013-06-13

    A series of hybrids from O(2)-(2,4-dinitrophenyl)diazeniumdiolate and oleanolic acid (OA) were designed, synthesized, and biologically evaluated as novel nitric oxide (NO)-releasing prodrugs that could be activated by glutathione S-transferase π (GSTπ) overexpressed in a number of cancer cells. It was discovered that the most active compound, 21, released high levels of NO selectively in HCC cells but not in the normal cells and exhibited potent antiproliferative activity in vitro as well as remarkable tumor-retarding effects in vivo. Compared with the reported GSTπ-activated prodrugs JS-K and PABA/NO, 21 exhibited remarkably improved stability in the absence of GSTπ. Importantly, the decomposition of 21 occurred in the presence of GSTπ and was much more effective than in glutathione S-transferase α. Additionally, 21 induced apoptosis in HepG2 cells by arresting the cell cycle at the G2/M phase, activating both the mitochondrion-mediated pathway and the MAPK pathway and enhancing the intracellular production of ROS.

  4. Demonstration of nuclear compartmentalization of glutathione in hepatocytes.

    PubMed Central

    Bellomo, G; Vairetti, M; Stivala, L; Mirabelli, F; Richelmi, P; Orrenius, S

    1992-01-01

    The intracellular distribution of glutathione (GSH) in cultured hepatocytes has been investigated by using the compound monochlorobimane (BmCl), which interacts specifically with GSH to form a highly fluorescent adduct. Image analysis of BmCl-labeled hepatocytes predominantly localized the fluorescence in the nucleus; the nuclear/cytoplasmic concentration gradient was approximately three. This concentration gradient was collapsed by treatment of the cells with ATP-depleting agents. The uneven distribution of BmCl fluorescence was not attributable to (i) nonspecific interaction of BmCl with protein sulfhydryl groups, (ii) any selective nuclear localization of the GSH transferase(s) catalyzing formation of the GSH-BmCl conjugate, or (iii) any apparent alterations in cell morphology from culture conditions, suggesting that this distribution did, indeed, reflect a nuclear compartmentalization of GSH. That the nuclear pool of GSH was found more resistant to depletion by several agents than the cytoplasmic pool supports the assumption that GSH is essential in protecting DNA and other nuclear structures from chemical injury. Images PMID:1584774

  5. 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

  6. The effect of aluminium-stress and exogenous spermidine on chlorophyll degradation, glutathione reductase activity and the photosystem II D1 protein gene (psbA) transcript level in lichen Xanthoria parietina.

    PubMed

    Sen, Gulseren; Eryilmaz, Isil Ezgi; Ozakca, Dilek

    2014-02-01

    In this study, the effects of short-term aluminium toxicity and the application of spermidine on the lichen Xanthoria parietina were investigated at the physiological and transcriptional levels. Our results suggest that aluminium stress leads to physiological processes in a dose-dependent manner through differences in lipid peroxidation rate, chlorophyll content and glutathione reductase (EC 1.6.4.2) activity in aluminium and spermidine treated samples. The expression of the photosystem II D1 protein (psbA) gene was quantified using semi-quantitative RT-PCR. Increased glutathione reductase activity and psbA mRNA transcript levels were observed in the X. parietina thalli that were treated with spermidine before aluminium-stress. The results showed that the application of spermidine could mitigate aluminium-induced lipid peroxidation and chlorophyll degradation on lichen X. parietina thalli through an increase in psbA transcript levels and activity of glutathione reductase (GR) enzymes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Knockdown of Host Antioxidant Defense Genes Enhances the Effect of Glucantime on Intracellular Leishmania braziliensis in Human Macrophages.

    PubMed

    Téllez, Jair; Romero, Ibeth; Soares, Maurilio José; Steindel, Mario; Romanha, Alvaro José

    2017-07-01

    Leishmaniasis is a neglected tropical disease that affects millions of people worldwide and represents a major public health problem. Information on protein expression patterns and functional roles within the context of Leishmania -infected human monocyte-derived macrophages (MDMs) under drug treatment conditions is essential for understanding the role of these cells in leishmaniasis treatment. We analyzed functional changes in the expression of human MDM genes and proteins during in vitro infection by Leishmania braziliensis and treatment with Glucantime (Sb V ), using quantitative PCR (qPCR) arrays, Western blotting, confocal microscopy, and small interfering RNA (siRNA) human gene inhibition assays. Comparison of the results from gene transcription and protein expression analyses revealed that glutathione S -transferase π1 (GSTP1), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione synthetase (GSS), thioredoxin (TRX), and ATP-binding cassette, subfamily B, member 5 (ABCB5), were strongly upregulated at both the mRNA and protein levels in human MDMs that were infected and treated, compared to the control group. Subcellular localization studies showed a primarily phagolysosomal location for the ABCB5 transporter, indicating that this protein may be involved in the transport of Sb V By inducing a decrease in L. braziliensis intracellular survival in THP-1 macrophages, siRNA silencing of GSTP1 , GSS , and ABCB5 resulted in an increased leishmanicidal effect of Sb V exposure in vitro Our results suggest that human MDMs infected with L. braziliensis and treated with Sb V express increased levels of genes participating in antioxidant defense, whereas our functional analyses provide evidence for the involvement of human MDMs in drug detoxification. Therefore, we conclude that GSS, GSTP1, and ABCB5 proteins represent potential targets for enhancing the leishmanicidal activity of Glucantime. Copyright © 2017 American Society for

  8. Knockdown of Host Antioxidant Defense Genes Enhances the Effect of Glucantime on Intracellular Leishmania braziliensis in Human Macrophages

    PubMed Central

    Romero, Ibeth; Soares, Maurilio José; Romanha, Alvaro José

    2017-01-01

    ABSTRACT Leishmaniasis is a neglected tropical disease that affects millions of people worldwide and represents a major public health problem. Information on protein expression patterns and functional roles within the context of Leishmania-infected human monocyte-derived macrophages (MDMs) under drug treatment conditions is essential for understanding the role of these cells in leishmaniasis treatment. We analyzed functional changes in the expression of human MDM genes and proteins during in vitro infection by Leishmania braziliensis and treatment with Glucantime (SbV), using quantitative PCR (qPCR) arrays, Western blotting, confocal microscopy, and small interfering RNA (siRNA) human gene inhibition assays. Comparison of the results from gene transcription and protein expression analyses revealed that glutathione S-transferase π1 (GSTP1), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione synthetase (GSS), thioredoxin (TRX), and ATP-binding cassette, subfamily B, member 5 (ABCB5), were strongly upregulated at both the mRNA and protein levels in human MDMs that were infected and treated, compared to the control group. Subcellular localization studies showed a primarily phagolysosomal location for the ABCB5 transporter, indicating that this protein may be involved in the transport of SbV. By inducing a decrease in L. braziliensis intracellular survival in THP-1 macrophages, siRNA silencing of GSTP1, GSS, and ABCB5 resulted in an increased leishmanicidal effect of SbV exposure in vitro. Our results suggest that human MDMs infected with L. braziliensis and treated with SbV express increased levels of genes participating in antioxidant defense, whereas our functional analyses provide evidence for the involvement of human MDMs in drug detoxification. Therefore, we conclude that GSS, GSTP1, and ABCB5 proteins represent potential targets for enhancing the leishmanicidal activity of Glucantime. PMID:28461312

  9. Effect of glutathione on the cadmium chelation of EDTA in a patient with cadmium intoxication.

    PubMed

    Gil, Hyo-wook; Kang, Eun-jung; Lee, Kwon-hyun; Yang, Jong-oh; Lee, Eun-young; Hong, Sae-yong

    2011-01-01

    In order to evaluate the efficiency and renal protective effects of glutathione during Ca(++)-EDTA chelation therapy for chronic cadmium intoxication, we measured the renal excretion of cadmium, β(2)-microglobulin, proteinuria, and hematuria during intravenous administration of glutathione with Ca(++)-EDTA in a 54-year-old patient with chronic cadmium intoxication. We administered 500 mg of Ca(++)-EDTA and 50 mg/kg of glutathione alone or in 1 L of normal saline over the next 24 hours and repeated this over 12 consecutive days. During the first 3 days, the basal levels (only saline administration) were determined; during the second 3 days, Ca(++)-EDTA only was administered, for the third sequence of 3 days, Ca(++)-EDTA with glutathione was provided, and for the last 3 days, glutathione alone was given. One month later, the same protocol was repeated. There were six blood and urine samples to analyze in each group. The blood cadmium level was higher when the EDTA was infused together with glutathione (7.44 ± 0.73 µg/L, p < 0.01) compared to the basal level of 4.6 ± 0.44 µg/L. Also, the renal cadmium excretion was significantly higher in the EDTA with glutathione group than in the basal group (23.4 ± 15.81 µg/g creatinine vs 89.23 ± 58.52 µg/g creatinine, p < 0.01). There was no difference in the protein/creatinine and β(2)-microglobulin/creatinine ratio in the urine (p > 0.05) among the groups. Furthermore, microhematuria and proteinuria did not develop over the observation period of 6 months. These results suggest that glutathione administration with EDTA might be an effective treatment modality for patients with cadmium intoxication.

  10. [Establishment of oxygen and glucose deprive model of in vitro cultured hippocampal neuron and effect of ligustrazine on intracellular Ca+ level in model neurons].

    PubMed

    Wan, Hai-tong; Wang, Yu; Yang, Jie-hong

    2007-03-01

    To establish the oxygen and glucose deprive (OGD) model in cultured hippocampal neuron and study the effect of ligustrazine on intracellular Ca2+ level in the model neurons. The OGD model was established in cultured hippocampal neuron, and the intracellular Ca2+ level in it was detected by laser scanning confocal microscope (LSCM). The OGD model was successfully established in cultured hippocampal neurons; the intracellular Ca2+ level in the OGD model group was significantly higher than that in the blank control group (P < 0.05), and that in the nemodipine and high and medium dosage of ligustrazine treated groups was lower than that in the OGD model group (P < 0.05). Intracellular Ca2+ overload occurs in OGD model neuron, which could be antagonized by ligustrazine, indicating that ligustrazine has a protective effect on hippocampal neuron from hypoxic-ischemic injury.

  11. Arabidopsis GLUTATHIONE REDUCTASE1 plays a crucial role in leaf responses to intracellular hydrogen peroxide and in ensuring appropriate gene expression through both salicylic acid and jasmonic acid signaling pathways.

    PubMed

    Mhamdi, Amna; Hager, Jutta; Chaouch, Sejir; Queval, Guillaume; Han, Yi; Taconnat, Ludivine; Saindrenan, Patrick; Gouia, Houda; Issakidis-Bourguet, Emmanuelle; Renou, Jean-Pierre; Noctor, Graham

    2010-07-01

    Glutathione is a major cellular thiol that is maintained in the reduced state by glutathione reductase (GR), which is encoded by two genes in Arabidopsis (Arabidopsis thaliana; GR1 and GR2). This study addressed the role of GR1 in hydrogen peroxide (H(2)O(2)) responses through a combined genetic, transcriptomic, and redox profiling approach. To identify the potential role of changes in glutathione status in H(2)O(2) signaling, gr1 mutants, which show a constitutive increase in oxidized glutathione (GSSG), were compared with a catalase-deficient background (cat2), in which GSSG accumulation is conditionally driven by H(2)O(2). Parallel transcriptomics analysis of gr1 and cat2 identified overlapping gene expression profiles that in both lines were dependent on growth daylength. Overlapping genes included phytohormone-associated genes, in particular implicating glutathione oxidation state in the regulation of jasmonic acid signaling. Direct analysis of H(2)O(2)-glutathione interactions in cat2 gr1 double mutants established that GR1-dependent glutathione status is required for multiple responses to increased H(2)O(2) availability, including limitation of lesion formation, accumulation of salicylic acid, induction of pathogenesis-related genes, and signaling through jasmonic acid pathways. Modulation of these responses in cat2 gr1 was linked to dramatic GSSG accumulation and modified expression of specific glutaredoxins and glutathione S-transferases, but there is little or no evidence of generalized oxidative stress or changes in thioredoxin-associated gene expression. We conclude that GR1 plays a crucial role in daylength-dependent redox signaling and that this function cannot be replaced by the second Arabidopsis GR gene or by thiol systems such as the thioredoxin system.

  12. Neuroprotective effect of Aronia melanocarpa extract against glutamate-induced oxidative stress in HT22 cells.

    PubMed

    Lee, Hyeon Yong; Weon, Jin Bae; Ryu, Gahee; Yang, Woo Seung; Kim, Nam Young; Kim, Myong Ki; Ma, Choong Je

    2017-04-11

    Glutamate (an endogenous excitatory neurotransmitter) at high concentrations contributes to the development of neurodegenerative diseases. Aronia melanocarpa (A. melanocarpa) berries contain anthocyanins and have high antioxidant activities. In this study, we evaluated whether A. melanocarpa berries could protect neuronal cells against glutamate-induced oxidative stress. A. melanocarpa berries exerted a protective effect against cytotoxicity in HT22 mouse hippocampal cells by MTT assay. We evaluated oxidative stress parameters including ROS level, intracellular Ca 2+ level, glutathione level and antioxidant enzyme activity in HT22 cells to elucidate the mechanism of its neuroprotective effect. A. melanocarpa berries decreased glutamate-induced death of HT22 cells. In addition, A. melanocarpa berries reduced ROS and intracellular Ca 2+ levels. Glutathione level, antioxidant enzymes, glutathione reductase and glutathione peroxide activities and mitochondrial membrane potential were also increased in HT22 cells. These results suggested that A. melanocarpa berries protected HT22 cells by exerting an antioxidant effect.

  13. 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.

  14. Hemolytic anemia and metabolic acidosis: think about glutathione synthetase deficiency.

    PubMed

    Ben Ameur, Salma; Aloulou, Hajer; Nasrallah, Fehmi; Kamoun, Thouraya; Kaabachi, Naziha; Hachicha, Mongia

    2015-02-01

    Glutathione synthetase deficiency (GSSD) is a rare disorder of glutathione metabolism with varying clinical severity. Patients may present with hemolytic anemia alone or together with acidosis and central nervous system impairment. Diagnosis is made by clinical presentation and detection of elevated concentrations of 5-oxoproline in urine and low glutathione synthetase activity in erythrocytes or cultured skin fibroblasts. The prognosis seems to depend on early diagnosis and treatment. We report a 4 months old Tunisian male infant who presented with severe metabolic acidosis with high anion gap and hemolytic anemia. High level of 5-oxoproline was detected in her urine and diagnosis of GSSD was made. Treatment consists of the correction of acidosis, blood transfusion, and supplementation with antioxidants. He died of severe metabolic acidosis and sepsis at the age of 15 months.

  15. Cross-functioning between the extraneuronal monoamine transporter and multidrug resistance protein 1 in the uptake of adrenaline and export of 5-(glutathion-S-yl)adrenaline in rat cardiomyocytes.

    PubMed

    Costa, Vera Marisa; Ferreira, Lusa Maria; Branco, Paula Srio; Carvalho, Flix; Bastos, Maria Lourdes; Carvalho, Rui Albuquerque; Carvalho, Mrcia; Remio, Fernando

    2009-01-01

    Isolated heart cells are highly susceptible to the toxicity of catecholamine oxidation products, namely, to catecholamine-glutathione adducts. Although cellular uptake and/or efflux of these products may constitute a crucial step, the knowledge about the involvement of transporters is still very scarce. This work aimed to contribute to the characterization of membrane transport mechanisms, namely, extraneuronal monoamine transporter (EMT), the multidrug resistant protein 1 (MRP1), and P-glycoprotein (P-gp) in freshly isolated cardiomyocytes from adult rats. These transporters may be accountable for uptake and/or efflux of adrenaline and an adrenaline oxidation product, 5-(glutathion-S-yl)adrenaline, in cardiomyocyte suspensions. Our results showed that 5-(glutathion-S-yl)adrenaline efflux was mediated by MRP1. Additionally, we demonstrated that the adduct formation occurs within the cardiomyocytes, since EMT inhibition reduced the intracellular adduct levels. The classical uptake2 transport in rat myocardial cells was inhibited by the typical EMT inhibitor, corticosterone, and surprisingly was also inhibited by low concentrations of another drug, a well-known P-gp inhibitor, GF120918. The P-gp activity was absent in the cells since P-gp-mediated efflux of quinidine was not blocked by GF120918. In conclusion, this work showed that freshly isolated cardiomyocytes from adult rats constitute a good model for the study of catecholamines and catecholamines metabolites membrane transport. The cardiomyocytes maintain EMT and MRP1 fully active, and these transporters contribute to the formation and efflux of 5-(glutathion-S-yl)adrenaline. In the present experimental conditions, P-gp activity is absent in the isolated cardiomyocytes.

  16. 21 CFR 862.1365 - Glutathione test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ....1365 Glutathione test system. (a) Identification. A glutathione test system is a device intended to measure glutathione (the tripeptide of glycine, cysteine, and glutamic acid) in erythrocytes (red blood... I (general controls). The device is exempt from the premarket notification procedures in subpart E...

  17. 21 CFR 862.1365 - Glutathione test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ....1365 Glutathione test system. (a) Identification. A glutathione test system is a device intended to measure glutathione (the tripeptide of glycine, cysteine, and glutamic acid) in erythrocytes (red blood... I (general controls). The device is exempt from the premarket notification procedures in subpart E...

  18. 21 CFR 862.1365 - Glutathione test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ....1365 Glutathione test system. (a) Identification. A glutathione test system is a device intended to measure glutathione (the tripeptide of glycine, cysteine, and glutamic acid) in erythrocytes (red blood... I (general controls). The device is exempt from the premarket notification procedures in subpart E...

  19. Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

    PubMed

    Handlogten, Michael W; Lee-O'Brien, Allison; Roy, Gargi; Levitskaya, Sophia V; Venkat, Raghavan; Singh, Shailendra; Ahuja, Sanjeev

    2018-01-01

    A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally

  20. Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae

    PubMed Central

    Zechmann, Bernd; Liou, Liang-Chun; Koffler, Barbara E; Horvat, Lucija; Tomašić, Ana; Fulgosi, Hrvoje; Zhang, Zhaojie

    2011-01-01

    Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H2O2) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress. PMID:22093747

  1. Ethacrynic acid and a derivative enhance apoptosis in arsenic trioxide-treated myeloid leukemia and lymphoma cells: the role of glutathione S-transferase P1-1

    PubMed Central

    Wang, Rui; Liu, Changda; Xia, Lijuan; Zhao, Guisen; Gabrilove, Janice; Waxman, Samuel; Jing, Yongkui

    2012-01-01

    Purpose Arsenic trioxide (ATO) as a single agent is used for treatment of acute promyelocytic leukemia (APL) with minimal toxicity but therapeutic effect of ATO in other types of malignancies has not been achieved. We tested whether a combination with ethacrynic acid (EA), a glutathione S-transferase P1-1 (GSTP1-1) inhibitor and a reactive oxygen species (ROS) inducer will extend the therapeutic effect of ATO beyond APL. Experimental Design The combined apoptotic effects of ATO plus EA were tested in non-APL leukemia and lymphoma cell lines. The role of ROS, GSTP1-1, glutathione, and Mcl-1 in apoptosis was determined. The selective response to this combination of cells with and without GSTP1-1 expression was compared. Results ATO/EA combination synergistically induced apoptosis in myeloid leukemia and lymphoma cells. This treatment produced high ROS levels, activated c-jun-NH2-terminal kinase and reduced Mcl-1 protein. This led to the decrease of mitochondrial transmembrane potential, release of cytochrome c and, subsequently, to activation of caspase 3 and 9. Induction of apoptosis in leukemia and lymphoma cells expressing GSTP1-1 required that high EA concentrations be combined with ATO. Silencing of GSTP1 in leukemia cells sensitized them to ATO/EA-induced apoptosis. In a sub-group of B-cell lymphoma which do not express GSTP1-1, lower concentrations of EA and its more potent derivative, ethacrynic acid butyl-ester, decreased intracellular glutathione levels and synergistically induced apoptosis when combined with ATO. Conclusion B-cell lymphoma cells lacking GSTP1-1 are more sensitive than myeloid leukemia cells to ATO/EA-induced apoptosis. PMID:23082001

  2. Rice protein improves oxidative stress by regulating glutathione metabolism and attenuating oxidative damage to lipids and proteins in rats.

    PubMed

    Yang, Lin; Chen, Jia-Hou; Xu, Tong; Zhou, Ai-Shen; Yang, Hong-Kun

    2012-10-05

    To evaluate the effects of rice protein (RP) on glutathione metabolism and oxidative damage. Seven-week-old male Wistar rats were fed diets containing casein and RP without cholesterol for 3weeks. Plasma and liver lipid levels, hepatic accumulation of total glutathione (T-GSH), oxidized glutathione (GSSG), reduced glutathione (GSH), malondialdehyde (MDA) and protein carbonyl (PCO) were measured. In the liver, the total antioxidative capacity (T-AOC), mRNA levels of glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modulatory subunit (GCLM), and the activities of hepatic catalase (CAT), total superoxide dismutase (T-SOD), γ-glutamylcysteine synthetase (γ-GCS), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSHPx) were also measured. T-AOC, GCLC and GCLM mRNA levels, antioxidative enzyme activities (T-SOD and CAT) and glutathione metabolism related enzyme activities (γ-GCS, GST, GR and GSHPx) were effectively stimulated by RP feeding compared to casein, and RP significantly reduced the hepatic accumulation of MDA and PCO in rats. These results indicate that lipid-lowering activity was induced by RP feeding. The present study demonstrates that RP improves oxidative stress primarily through enzymatic and non-enzymatic antioxidative defense mechanisms, reflected by enhancing the antioxidative status and attenuating the oxidative damage to lipids and proteins. These results suggest that RP can prevent hyperlipidemia in part through modifying glutathione metabolism, and sulfur amino acids may be the main modulator of this antioxidative mechanism. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. 1-3-A Resolution Structure of Human Glutathione S-Transferase With S-Hexyl Glutathione Bound Reveals Possible Extended Ligandin Binding Site

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

    Trong, I.Le; Stenkamp, R.E.; Ibarra, C.

    2005-08-22

    Cytosolic glutathione S-transferases (GSTs) play a critical role in xenobiotic binding and metabolism, as well as in modulation of oxidative stress. Here, the high-resolution X-ray crystal structures of homodimeric human GSTA1-1 in the apo form and in complex with S-hexyl glutathione (two data sets) are reported at 1.8, 1.5, and 1.3A respectively. At this level of resolution, distinct conformations of the alkyl chain of S-hexyl glutathione are observed, reflecting the nonspecific nature of the hydrophobic substrate binding site (H-site). Also, an extensive network of ordered water, including 75 discrete solvent molecules, traverses the open subunit-subunit interface and connects the glutathionemore » binding sites in each subunit. In the highest-resolution structure, three glycerol moieties lie within this network and directly connect the amino termini of the glutathione molecules. A search for ligand binding sites with the docking program Molecular Operating Environment identified the ordered water network binding site, lined mainly with hydrophobic residues, suggesting an extended ligand binding surface for nonsubstrate ligands, the so-called ligandin site. Finally, detailed comparison of the structures reported here with previously published X-ray structures reveal a possible reaction coordinate for ligand-dependent conformational changes in the active site and the C-terminus.« less

  4. Determination of glutathione and glutathione disulfide in biological samples: an in-depth review.

    PubMed

    Monostori, Péter; Wittmann, Gyula; Karg, Eszter; Túri, Sándor

    2009-10-15

    Glutathione (GSH) is a thiol-containing tripeptide, which plays central roles in the defence against oxidative damage and in signaling pathways. Upon oxidation, GSH is transformed to glutathione disulfide (GSSG). The concentrations of GSH and GSSG and their molar ratio are indicators of cell functionality and oxidative stress. Assessment of redox homeostasis in various clinical states and medical applications for restoration of the glutathione status are of growing importance. This review is intended to provide a state-of-the-art overview of issues relating to sample pretreatment and choices for the separation and detection of GSH and GSSG. High-performance liquid chromatography, capillary electrophoresis and gas chromatography (as techniques with a separation step) with photometric, fluorimetric, electrochemical and mass spectrometric detection are discussed, stress being laid on novel approaches.

  5. Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1.

    PubMed

    Cooke, R J; Björnestedt, R; Douglas, K T; McKie, J H; King, M D; Coles, B; Ketterer, B; Mannervik, B

    1994-09-01

    The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher levels of radioisotope incorporation for the Mu than the Alpha families. Taking rat GST 3-3, 1.18 (+/- 0.05) mol of radiolabel from S-(2-nitro-4-azidophenyl)glutathione was incorporated per mol of dimeric enzyme, which could be blocked by the presence of the strong competitive inhibitor, S-tritylglutathione (Ki = 1.4 x 10(-7) M). Radiolabelling of the protein paralleled the loss of enzyme activity. Photoaffinity labelling by tritiated S-(2-nitro-4-azidophenyl)glutathione on a preparative scale (in the presence and absence of S-tritylglutathione) followed by tryptic digestion and purification of the labelled peptides indicated that GST 3-3 was specifically photolabelled; the labelled peptides were sequenced. Similarly, preparative photoaffinity labelling by S-(2-nitro-4-azidophenyl)glutathione of the rat liver 1-1 isoenzyme, the human GST A1-1 and the human-rat chimaeric GST, H1R1/1, was carried out with subsequent sequencing of radiolabelled h.p.l.c.-purified tryptic peptides. The results were interpreted by means of molecular-graphics analysis to locate photoaffinity-labelled peptides using the X-ray-crystallographic co-ordinates of rat GST 3-3 and human GST A1-1. The molecular-graphical analysis indicated that the labelled peptides are located within the immediate vicinity of the region occupied by S-substituted glutathione derivatives bound in the active-site cavity of the GSTs investigated.

  6. p-aminophenol nephrotoxicity: biosynthesis of toxic glutathione conjugates.

    PubMed

    Klos, C; Koob, M; Kramer, C; Dekant, W

    1992-07-01

    p-Aminophenol causes necrosis of the pars recta of the proximal tubules in rats, and its nephrotoxicity may be due to glutathione-dependent bioactivation reactions. We have investigated the hepatic metabolism of p-aminophenol in Wistar rats and the cytotoxicity of formed glutathione S-conjugates in rat renal epithelial cells. After ip application of p-aminophenol (100 mg/kg), the following metabolites were identified in rat bile: 4-amino-2-(glutathion-S-yl)phenol, 4-amino-3-(glutathion-S-yl)-phenol, 4-amino-2,5-bis(glutathion-S-yl)phenol, 4-amino-2,3,5(or 6)-tris(glutathion-S-yl)phenol, an aminophenol conjugate (likely a sulfate or glucuronide), acetaminophen glucuronide, and 3-(glutathion-S-yl)acetaminophen. 4-Amino-3-(glutathion-S-yl)phenol, 4-amino-2,5-bis(glutathion-S-yl)phenol, and 4-amino-2,3,5(or 6)-tris(glutathion-S-yl)phenol induced a dose- and time-dependent loss of cell viability in rat kidney cortical cells. Cell killing was significantly reduced by inhibition of gamma-glutamyl transpeptidase with Acivicin. p-Aminophenol was also toxic to renal epithelial cells. Coincubation of p-aminophenol with tetraethylammonium bromide, a competitive inhibitor of the organic cation transporter, and with SKF-525A, an inhibitor of cytochrome P450, protected cells from p-aminophenol-induced toxicity. p-Aminophenol would thus be accumulated in the kidney mainly by organic cation transport systems, which are concentrated in the S-1 segment of the proximal tubule. However, p-aminophenol toxicity in vivo is directed toward the S-2 and S-3 segments, which are rich in gamma-glutamyl transpeptidase. These results and the observation that biliary cannulation and glutathione depletion reduce p-aminophenol nephrotoxicity suggest that the biosynthesis of toxic glutathione conjugates is responsible for p-aminophenol nephrotoxicity in vivo. The aminophenol glutathione S-conjugates formed induce p-aminophenol nephrotoxicity by a pathway dependent on gamma-glutamyl transpeptidase.

  7. 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.

  8. 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.

  9. Transport via SLC5A8 (SMCT1) Is Obligatory for 2-Oxothiazolidine-4-Carboxylate to Enhance Glutathione Production in Retinal Pigment Epithelial Cells

    PubMed Central

    Babu, Ellappan; Ananth, Sudha; Veeranan-Karmegam, Rajalakshmi; Coothankandaswamy, Veena; Smith, Sylvia B.; Boettger, Thomas; Ganapathy, Vadivel

    2011-01-01

    Purpose. To evaluate the role of SLC5A8 in the transport of 2-oxothiazolidine-4-carboxylate (OTC) and to determine whether OTC augments glutathione production in RPE cells, thereby providing protection against oxidative stress. Methods. SLC5A8-mediated transport of OTC was monitored in Xenopus laevis oocytes by electrophysiological means. Saturation kinetics, Na+-activation kinetics, and inhibition by ibuprofen were analyzed by monitoring OTC-induced currents as a measure of transport activity. Oxidative stress was induced in ARPE-19 cells and primary RPE cells isolated from wild type and Slc5a8-/- mouse retinas using H2O2, and the effects of OTC on cell death and intracellular glutathione concentration were examined. Results. Heterologous expression of human SLC5A8 in X. laevis oocytes induced Na+-dependent inward currents in the presence of OTC under voltage-clamp conditions. The transport of OTC via SLC5A8 was saturable, with a Kt of 104 ± 3 μM. The Na+-activation kinetics was sigmoidal with a Hill coefficient of 1.9 ± 0.1, suggesting involvement of two Na+ in the activation process. Ibuprofen, a blocker of SLC5A8, inhibited SLC5A8-mediated OTC transport; the concentration necessary for half-maximal inhibition was 17 ± 1 μM. OTC increased glutathione levels and protected ARPE-19 and primary RPE cells isolated from wild type mouse retinas from H2O2-induced cell death. These effects were abolished in primary RPE isolated from Slc5a8-/- mouse retinas. Conclusions. OTC is a transportable substrate for SLC5A8. OTC augments glutathione production in RPE cells, thereby protecting them from oxidative damage. Transport via SLC5A8 is obligatory for this process. PMID:21508099

  10. Ascorbic acid glucoside reduces neurotoxicity and glutathione depletion in mouse brain induced by nitrotriazole radiosensitazer.

    PubMed

    Cherdyntseva, Nadezda V; Ivanova, Anna A; Ivanov, Vladimir V; Cherdyntsev, Evgeny; Nair, Cherupally Krishnan Krishnan; Kagiya, Tsutomu V

    2013-01-01

    To investigate the potential of the anti-oxidant ascorbic acid glucoside (AA-2G) to modulate neurotoxicity induced by high doses of nitrotriazole radiosensitizer. Male and female C56Bl/6xCBA hybrid mice aged 8-14 weeks (weight 18-24 g) were used. Nitrotriazole drug radiosensitizer sanazole at a high dose of 2, 1 g/kg was per os administered to induce neurotoxicity at mice. Ascorbic acid glucoside was given 30 min before the sanazole administration. Serum ascorbic acid, brain glutathione level, as well as behavioral performance using open field apparatus were measured. Administration of high (non-therapeutic) doses of the nitrotriazole drug sanazole results in neurotoxicity in mice as evidenced from behavioral performance, emotional activity and depletion of the cellular antioxidant, glutathione, in the brain. The serum levels of ascorbic acid was also found reduced in high dose sanazole treated animals. Per os administration of ascorbic acid glucoside significantly reduced the neurotoxicity. This effect was associated with the prevention of glutathione depletion in mouse brain and restoring the ascorbic acid level in serum. Administration of ascorbic acid glucoside, but not ascorbic acid, before sanazole administration protected from sanazole-induced neurotoxicity by preventing the decrease in the brain reduced glutathione level and providing high level of ascorbic acid in plasma.

  11. Glutathione regulation of redox-sensitive signals in tumor necrosis factor-{alpha}-induced vascular endothelial dysfunction

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

    Tsou, T.-C.; Yeh, S.C.; Tsai, F.-Y.

    2007-06-01

    We investigated the regulatory role of glutathione in tumor necrosis factor-alpha (TNF-{alpha})-induced vascular endothelial dysfunction as evaluated by using vascular endothelial adhesion molecule expression and monocyte-endothelial monolayer binding. Since TNF-{alpha} induces various biological effects on vascular cells, TNF-{alpha} dosage could be a determinant factor directing vascular cells into different biological fates. Based on the adhesion molecule expression patterns responding to different TNF-{alpha} concentrations, we adopted the lower TNF-{alpha} (0.2 ng/ml) to rule out the possible involvement of other TNF-{alpha}-induced biological effects. Inhibition of glutathione synthesis by L-buthionine-(S,R)-sulfoximine (BSO) resulted in down-regulations of the TNF-{alpha}-induced adhesion molecule expression and monocyte-endothelial monolayermore » binding. BSO attenuated the TNF-{alpha}-induced nuclear factor-kappaB (NF-{kappa}B) activation, however, with no detectable effect on AP-1 and its related mitogen-activated protein kinases (MAPKs). Deletion of an AP-1 binding site in intercellular adhesion molecule-1 (ICAM-1) promoter totally abolished its constitutive promoter activity and its responsiveness to TNF-{alpha}. Inhibition of ERK, JNK, or NF-{kappa}B attenuates TNF-{alpha}-induced ICAM-1 promoter activation and monocyte-endothelial monolayer binding. Our study indicates that TNF-{alpha} induces adhesion molecule expression and monocyte-endothelial monolayer binding mainly via activation of NF-{kappa}B in a glutathione-sensitive manner. We also demonstrated that intracellular glutathione does not modulate the activation of MAPKs and/or their downstream AP-1 induced by lower TNF-{alpha}. Although AP-1 activation by the lower TNF-{alpha} was not detected in our systems, we could not rule out the possible involvement of transiently activated MAPKs/AP-1 in the regulation of TNF-{alpha}-induced adhesion molecule expression.« less

  12. High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

    NASA Technical Reports Server (NTRS)

    Abukhalaf, Imad K.; Silvestrov, Natalia A.; Menter, Julian M.; von Deutsch, Daniel A.; Bayorh, Mohamed A.; Socci, Robin R.; Ganafa, Agaba A.

    2002-01-01

    A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

  13. Chrysin enhances doxorubicin-induced cytotoxicity in human lung epithelial cancer cell lines: The role of glutathione

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

    Brechbuhl, Heather M.; Kachadourian, Remy; Min, Elysia

    We hypothesized that flavonoid-induced glutathione (GSH) efflux through multi-drug resistance proteins (MRPs) and subsequent intracellular GSH depletion is a viable mechanism to sensitize cancer cells to chemotherapies. This concept was demonstrated using chrysin (5–25 μM) induced GSH efflux in human non-small cell lung cancer lines exposed to the chemotherapeutic agent, doxorubicin (DOX). Treatment with chrysin resulted in significant and sustained intracellular GSH depletion and the GSH enzyme network in the four cancer cell types was predictive of the severity of chrysin induced intracellular GSH depletion. Gene expression data indicated a positive correlation between basal MRP1, MRP3 and MRP5 expression andmore » total GSH efflux before and after chrysin exposure. Co-treating the cells for 72 h with chrysin (5–30 μM) and DOX (0.025–3.0 μM) significantly enhanced the sensitivity of the cells to DOX as compared to 72-hour DOX alone treatment in all four cell lines. The maximum decrease in the IC{sub 50} values of cells treated with DOX alone compared to co-treatment with chrysin and DOX was 43% in A549 cells, 47% in H157 and H1975 cells and 78% in H460 cells. Chrysin worked synergistically with DOX to induce cancer cell death. This approach could allow for use of lower concentrations and/or sensitize cancer cells to drugs that are typically resistant to therapy. -- Graphical abstract: Possible mechanisms by which chrysin enhances doxorubicin-induced toxicity in cancer cells. Highlights: ► Chyrsin sustains a significant depletion of GSH levels in lung cancer cells. ► Chyrsin synergistically potentiates doxorubicin-induced cancer cell cytotoxicity. ► Cancer cell sensitivity correlated with GSH and MRP gene network expression. ► This approach could allow for lower side effects and targeting resistant tumors.« less

  14. Glutathione in combination with trehalose has supplementary beneficial effects on cryopreserved red deer (cervus elaphus) sperm.

    PubMed

    Wang, Yan; Dong, Shude

    2017-01-01

    In this study, we evaluated the effects of glutathione in combination with trehalose addition to semen extenders on the quality parameters of frozen-thawed red deer (cervus elaphus) spermatozoa. The semen samples collected from six mature red deer once a week were diluted with Tris-egg yolk-based extenders. The diluted semen samples were supplemented with glutathione (8 mmol L -1 ) and or trehalose (5%, w/v), cryopreserved, thawed and then subjected to sperm quality parameter evaluation. Both glutathione and trehalose addition to the extender significantly improved progressive motility, acrosome integrity, membrane integrity, superoxide dismutase and glutathione peroxidase activity and decreased percentage abnormality and sperm malondialdehyde level compared with the control group (P<.05). Moreover, glutathione in combination with trehalose addition to semen extenders had higher efficiency compared with the glutathione or trehalose addition alone (P<.05). Therefore, glutathione in combination with trehalose could be a promising cryoprotectant for red deer sperm. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. The crucial protective role of glutathione against tienilic acid hepatotoxicity in rats

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

    Nishiya, Takayoshi; Mori, Kazuhiko; Hattori, Chiharu

    2008-10-15

    To investigate the hepatotoxic potential of tienilic acid in vivo, we administered a single oral dose of tienilic acid to Sprague-Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] andmore » phase II drug metabolism (glutathione S-transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3-6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.« less

  16. 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.

  17. 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

  18. Glutathione Preservation during Storage of Rat Lenses in Optisol-GS and Castor Oil

    PubMed Central

    Holm, Thomas; Brøgger-Jensen, Martin Rocho; Johnson, Leif; Kessel, Line

    2013-01-01

    Background Glutathione concentration in the lens decreases in aging and cataractous lenses, providing a marker for tissue condition. Experimental procedures requiring unfrozen lenses from donor banks rely on transportation in storage medium, affecting lens homeostasis and alterations in glutathione levels. The aim of the study was to examine the effects of Optisol-GS and castor oil on lens condition, determined from their ability to maintain glutathione concentrations. Methodology/Principal Findings Rat lenses were stored in the two types of storage media at varying time intervals up to 3 days. Glutathione concentration was afterwards determined in an enzymatic detection assay, specific for both reduced and oxidized forms. Lenses removed immediately after death exhibited a glutathione concentration of 4.70±0.29 mM. In vitro stored lenses in Optisol-GS lost glutathione quickly, ending with a concentration of 0.60±0.34 mM after 3 days while castor oil stored lenses exhibited a slower decline and ended at 3 times the concentration. A group of lenses were additionally stored under post mortem conditions within the host for 6 hours before its removal. Total glutathione after 6 hours was similar to that of lenses removed immediately after death, but with altered GSH and GSSG concentrations. Subsequent storage of these lenses in media showed changes similar to those in the first series of experiments, albeit to a lesser degree. Conclusions/Significance It was determined that storage in Optisol-GS resulted in a higher loss of glutathione than lenses stored in castor oil. Storage for more than 12 hours reduced glutathione to half its original concentration, and was considered unusable after 24 hours. PMID:24260265

  19. 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.

  20. Intracellular oxidant activity, antioxidant enzyme defense system, and cell senescence in fibroblasts with trisomy 21.

    PubMed

    Rodríguez-Sureda, Víctor; Vilches, Ángel; Sánchez, Olga; Audí, Laura; Domínguez, Carmen

    2015-01-01

    Down's syndrome (DS) is characterized by a complex phenotype associated with chronic oxidative stress and mitochondrial dysfunction. Overexpression of genes on chromosome-21 is thought to underlie the pathogenesis of the major phenotypic features of DS, such as premature aging. Using cultured fibroblasts with trisomy 21 (T21F), this study aimed to ascertain whether an imbalance exists in activities, mRNA, and protein expression of the antioxidant enzymes SOD1, SOD2, glutathione-peroxidase, and catalase during the cell replication process in vitro. T21F had high SOD1 expression and activity which led to an interenzymatic imbalance in the antioxidant defense system, accentuated with replicative senescence. Intracellular ROS production and oxidized protein levels were significantly higher in T21F compared with control cells; furthermore, a significant decline in intracellular ATP content was detected in T21F. Cell senescence was found to appear prematurely in DS cells as shown by SA-β-Gal assay and p21 assessment, though not apoptosis, as neither p53 nor the proapoptotic proteins cytochrome c and caspase 9 were altered in T21F. These novel findings would point to a deleterious role of oxidatively modified molecules in early cell senescence of T21F, thereby linking replicative and stress-induced senescence in cultured cells to premature aging in DS.

  1. Intracellular Oxidant Activity, Antioxidant Enzyme Defense System, and Cell Senescence in Fibroblasts with Trisomy 21

    PubMed Central

    Rodríguez-Sureda, Víctor; Vilches, Ángel; Sánchez, Olga; Audí, Laura; Domínguez, Carmen

    2015-01-01

    Down's syndrome (DS) is characterized by a complex phenotype associated with chronic oxidative stress and mitochondrial dysfunction. Overexpression of genes on chromosome-21 is thought to underlie the pathogenesis of the major phenotypic features of DS, such as premature aging. Using cultured fibroblasts with trisomy 21 (T21F), this study aimed to ascertain whether an imbalance exists in activities, mRNA, and protein expression of the antioxidant enzymes SOD1, SOD2, glutathione-peroxidase, and catalase during the cell replication process in vitro. T21F had high SOD1 expression and activity which led to an interenzymatic imbalance in the antioxidant defense system, accentuated with replicative senescence. Intracellular ROS production and oxidized protein levels were significantly higher in T21F compared with control cells; furthermore, a significant decline in intracellular ATP content was detected in T21F. Cell senescence was found to appear prematurely in DS cells as shown by SA-β-Gal assay and p21 assessment, though not apoptosis, as neither p53 nor the proapoptotic proteins cytochrome c and caspase 9 were altered in T21F. These novel findings would point to a deleterious role of oxidatively modified molecules in early cell senescence of T21F, thereby linking replicative and stress-induced senescence in cultured cells to premature aging in DS. PMID:25852816

  2. Variable Levels of Glutathione S-Transferases Are Responsible for the Differential Tolerance to Metolachlor between Maize (Zea mays) Shoots and Roots.

    PubMed

    Li, Dongzhi; Xu, Li; Pang, Sen; Liu, Zhiqian; Wang, Kai; Wang, Chengju

    2017-01-11

    Glutathione S-transferases (GSTs) play important roles in herbicide tolerance. However, studies on GST function in herbicide tolerance among plant tissues are still lacking. To explore the mechanism of metolachlor tolerance difference between maize shoots and roots, the effects of metolachlor on growth, GST activity, and the expression of the entire GST gene family were investigated. It was found that this differential tolerance to metolachlor was correlated with contrasting GST activity between the two tissues and can be eliminated by a GST inhibitor. An in vitro metolachlor-glutathione conjugation assay confirmed that the transformation of metolachlor is 2-fold faster in roots than in shoots. The expression analysis of the GST gene family revealed that most GST genes are expressed much higher in roots than shoots, both in control and in metolachlor-treated plants. Taken together, higher level expression of most GST genes, leading to higher GST activity and faster herbicide transformation, appears to be responsible for the higher tolerance to metolachlor of maize roots than shoots.

  3. Melatonin and nitric oxide modulate glutathione content and glutathione reductase activity in sunflower seedling cotyledons accompanying salt stress.

    PubMed

    Kaur, Harmeet; Bhatla, Satish C

    2016-09-30

    The present findings demonstrate significant modulation of total glutathione content, reduced glutathione (GSH) content, oxidized glutathione (GSSG) content, GSH/GSSG ratio and glutathione reductase (GR; EC 1.6.4.2) activity in dark-grown seedling cotyledons in response to salt-stress (120 mM NaCl) in sunflower (Helianthus annuus L.) seedlings. A differential spatial distribution of GR activity (monitored by confocal laser scanning microscopic (CLSM) imaging) is also evident. Melatonin and nitric oxide (NO) differentially ameliorate salt stress effect by modulating GR activity and GSH content in seedling cotyledons. Total glutathione content (GSH + GSSG) exhibit a seedling age-dependent increase in the cotyledons, more so in salt-stressed conditions and when subjected to melatonin treatment. Seedlings raised in presence of 15 μM of melatonin exhibit significant increase in GR activity in cotyledon homogenates (10,000 g supernatant) coinciding with significant increase in GSH content. GSSG content and GSH/GSSG ratio also increased due to melatonin treatment. A correlation is thus evident in NaCl-sensitized modulation of GSH content and GR activity by melatonin. GSH content is down regulated by NO provided as 250 μM of sodium nitroprusside (SNP) although total glutathione content remained in similar range. A reversal of response (enhanced total glutathione accumulation) by NO scavenger (cPTIO) highlights the critical role of NO in modulating glutathione homeostasis. SNP lowers the activity of hydroxyindole-O-methyltransferase (HIOMT) - a regulatory enzyme in melatonin biosynthesis in control seedlings whereas its activity is upregulated in salt-stressed seedling cotyledons. Melatonin content of seedling cotyledons is also modulated by NO. NO and melatonin thus seem to modulate GR activity and GSH content during seedling growth under salt stress. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. 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.

  5. Effects of Combined Low Glutathione with Mild Oxidative and Low Phosphorus Stress on the Metabolism of Arabidopsis thaliana

    PubMed Central

    Fukushima, Atsushi; Iwasa, Mami; Nakabayashi, Ryo; Kobayashi, Makoto; Nishizawa, Tomoko; Okazaki, Yozo; Saito, Kazuki; Kusano, Miyako

    2017-01-01

    Plants possess highly sensitive mechanisms that monitor environmental stress levels for a dose-dependent fine-tuning of their growth and development. Differences in plant responses to severe and mild abiotic stresses have been recognized. Although many studies have revealed that glutathione can contribute to plant tolerance to various environmental stresses, little is known about the relationship between glutathione and mild abiotic stress, especially the effect of stress-induced altered glutathione levels on the metabolism. Here, we applied a systems biology approach to identify key pathways involved in the gene-to-metabolite networks perturbed by low glutathione content under mild abiotic stress in Arabidopsis thaliana. We used glutathione synthesis mutants (cad2-1 and pad2-1) and plants overexpressing the gene encoding γ-glutamylcysteine synthetase, the first enzyme of the glutathione biosynthetic pathway. The plants were exposed to two mild stress conditions—oxidative stress elicited by methyl viologen and stress induced by the limited availability of phosphate. We observed that the mutants and transgenic plants showed similar shoot growth as that of the wild-type plants under mild abiotic stress. We then selected the synthesis mutants and performed multi-platform metabolomics and microarray experiments to evaluate the possible effects on the overall metabolome and the transcriptome. As a common oxidative stress response, several flavonoids that we assessed showed overaccumulation, whereas the mild phosphate stress resulted in increased levels of specific kaempferol- and quercetin-glycosides. Remarkably, in addition to a significant increased level of sugar, osmolytes, and lipids as mild oxidative stress-responsive metabolites, short-chain aliphatic glucosinolates over-accumulated in the mutants, whereas the level of long-chain aliphatic glucosinolates and specific lipids decreased. Coordinated gene expressions related to glucosinolate and flavonoid

  6. Self-Organization Processes at the Intracellular Level

    NASA Astrophysics Data System (ADS)

    Ponce Dawson, Silvina

    2003-03-01

    In spite of their relatively small sizes, cells are incredibly complex objects in which various sorts of self-organizing processes occur. Cell division is an example of a process that nearly all cells undergo in which a concerted sequence of events takes place. What are the signals that tell the cell to move along this sequence? Clearly, this is a self-organized process. Microtubules (long polymers that are part of the cytoskeleton) and calcium signals play a major role during cell division. In this course we will focus on some features of microtubule dynamics and calcium signals that are amenable to modeling. In both of these biological systems, behaviors at a single molecule level are key determinants of the self-organized dynamics that is observed at larger scales. Thus, the modeling of these systems presents interesting challenges which require novel strategies. Their study may not only provide answers for biologically motivated questions, but is also a natural setting in which the transition between particle-like and mean-field models can be explored. In this course we will first give a brief biological introduction on the structure of eukaryotic cells, microtubule dynamics and intracellular calcium signals. We will then describe some of the models that have been presented in the literature and discuss the spatio-temporal dynamics that they predict, comparing them with observed behaviors in vitro or in vivo. We will end with a discussion on the virtues and limitations of the various modeling strategies described.

  7. Glutathione Utilization by Candida albicans Requires a Functional Glutathione Degradation (DUG) Pathway and OPT7, an Unusual Member of the Oligopeptide Transporter Family

    PubMed Central

    Desai, Prashant Ramesh; Thakur, Anil; Ganguli, Dwaipayan; Paul, Sanjoy; Morschhäuser, Joachim; Bachhawat, Anand K.

    2011-01-01

    Candida albicans lacks the ability to survive within its mammalian host in the absence of endogenous glutathione biosynthesis. To examine the ability of this yeast to utilize exogenous glutathione, we exploited the organic sulfur auxotrophy of C. albicans met15Δ strains. We observed that glutathione is utilized efficiently by the alternative pathway of glutathione degradation (DUG pathway). The major oligopeptide transporters OPT1–OPT5 of C. albicans that were most similar to the known yeast glutathione transporters were not found to contribute to glutathione transport to any significant extent. A genomic library approach to identify the glutathione transporter of C. albicans yielded OPT7 as the primary glutathione transporter. Biochemical studies on OPT7 using radiolabeled GSH uptake revealed a Km of 205 μm, indicating that it was a high affinity glutathione transporter. OPT7 is unusual in several aspects. It is the most remote member to known yeast glutathione transporters, lacks the two highly conserved cysteines in the family that are known to be crucial in trafficking, and also has the ability to take up tripeptides. The transporter was regulated by sulfur sources in the medium. OPT7 orthologues were prevalent among many pathogenic yeasts and fungi and formed a distinct cluster quite remote from the Saccharomyces cerevisiae HGT1 glutathione transporter cluster. In vivo experiments using a systemic model of candidiasis failed to detect expression of OPT7 in vivo, and strains disrupted either in the degradation (dug3Δ) or transport (opt7Δ) of glutathione failed to show a defect in virulence. PMID:21994941

  8. 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.

  9. 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

  10. 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.

  11. Cadmium-Induced Hydrogen Sulfide Synthesis Is Involved in Cadmium Tolerance in Medicago sativa by Reestablishment of Reduced (Homo)glutathione and Reactive Oxygen Species Homeostases

    PubMed Central

    Cui, Weiti; Chen, Huiping; Zhu, Kaikai; Jin, Qijiang; Xie, Yanjie; Cui, Jin; Xia, Yan; Zhang, Jing; Shen, Wenbiao

    2014-01-01

    Until now, physiological mechanisms and downstream targets responsible for the cadmium (Cd) tolerance mediated by endogenous hydrogen sulfide (H2S) have been elusive. To address this gap, a combination of pharmacological, histochemical, biochemical and molecular approaches was applied. The perturbation of reduced (homo)glutathione homeostasis and increased H2S production as well as the activation of two H2S-synthetic enzymes activities, including L-cysteine desulfhydrase (LCD) and D-cysteine desulfhydrase (DCD), in alfalfa seedling roots were early responses to the exposure of Cd. The application of H2S donor sodium hydrosulfide (NaHS), not only mimicked intracellular H2S production triggered by Cd, but also alleviated Cd toxicity in a H2S-dependent fashion. By contrast, the inhibition of H2S production caused by the application of its synthetic inhibitor blocked NaHS-induced Cd tolerance, and destroyed reduced (homo)glutathione and reactive oxygen species (ROS) homeostases. Above mentioned inhibitory responses were further rescued by exogenously applied glutathione (GSH). Meanwhile, NaHS responses were sensitive to a (homo)glutathione synthetic inhibitor, but reversed by the cotreatment with GSH. The possible involvement of cyclic AMP (cAMP) signaling in NaHS responses was also suggested. In summary, LCD/DCD-mediated H2S might be an important signaling molecule in the enhancement of Cd toxicity in alfalfa seedlings mainly by governing reduced (homo)glutathione and ROS homeostases. PMID:25275379

  12. Quantitative real-time imaging of glutathione

    USDA-ARS?s Scientific Manuscript database

    Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe—designated as RealThiol (RT)—that can quantitatively monitor the real-time ...

  13. FMISO accumulation in tumor is dependent on glutathione conjugation capacity in addition to hypoxic state.

    PubMed

    Masaki, Yukiko; Shimizu, Yoichi; Yoshioka, Takeshi; Nishijima, Ken-Ichi; Zhao, Songji; Higashino, Kenichi; Numata, Yoshito; Tamaki, Nagara; Kuge, Yuji

    2017-10-01

    18 F-fluoromisonidazole (FMISO), a well-known PET imaging probe for diagnosis of hypoxia, is believed to accumulate in hypoxic cells via covalent binding with macromolecules after reduction of the nitro group. Previously, we showed the majority of 18 F-FMISO was incorporated into low-molecular-weight metabolites in hypoxic tumors, and the glutathione conjugate of reduced FMISO (amino-FMISO-GS) distributed in the tumor hypoxic regions as revealed by imaging mass spectrometry (IMS). The present study was conducted to clarify whether FMISO is metabolized to amino-FMISO-GS within tumor cells and how amino-FMISO-GS contributes to FMISO accumulation in hypoxic cells. We also evaluated the relationship between FMISO accumulation and the glutathione conjugation-related factors in the cells. Tumor cells (FaDu, LOVO, and T24) were treated with 18 F-FMISO and incubated under normoxic or hypoxic conditions for 4 h. The FMISO metabolites were analyzed with LC-ESI-MS. Several glutathione conjugation-related factors of tumor cells were evaluated in vitro. FaDu tumor-bearing mice were intravenously injected with 18 F-FMISO and the tumors were excised at 4 h post-injection. Autoradiography, IMS and histologic studies were performed. Amino-FMISO-GS was the main contributor to FMISO incorporated in hypoxic FaDu cells in vitro and in vivo. Total FMISO uptake levels and amino-FMISO-GS levels were highest in FaDu, followed by LOVO, and then T24 (total uptake: 0.851 ± 0.009 (FaDu), 0.617 ± 0.021 (LOVO) and 0.167 ± 0.006 (T24) % dose/mg protein; amino-FMISO-GS: 0.502 ± 0.035 (FaDu), 0.158 ± 0.013 (LOVO), and 0.007 ± 0.001 (T24) % dose/mg protein). The glutathione level of FaDu was significantly higher than those of LOVO and T24. The enzyme activity of glutathione-S-transferase catalyzing the glutathione conjugation reaction in FaDu was similar levels to that in LOVO, and was higher than that in T24. Quantitative RT-PCR analysis revealed that the expression levels of

  14. Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells.

    PubMed

    Wilmer, Martijn J G; de Graaf-Hess, Adriana; Blom, Henk J; Dijkman, Henry B P M; Monnens, Leo A; van den Heuvel, Lambertus P; Levtchenko, Elena N

    2005-11-18

    Cystinosis, the most frequent cause of inborn Fanconi syndrome, is characterized by the lysosomal cystine accumulation, caused by mutations in the CTNS gene. To elucidate the pathogenesis of cystinosis, we cultured proximal tubular cells from urine of cystinotic patients (n = 9) and healthy controls (n = 9), followed by immortalization with human papilloma virus (HPV E6/E7). Obtained cell lines displayed basolateral polarization, alkaline phosphatase activity, and presence of aminopeptidase N (CD-13) and megalin, confirming their proximal tubular origin. Cystinotic cell lines exhibited elevated cystine levels (0.86 +/- 0.95 nmol/mg versus 0.09 +/- 0.01 nmol/mg protein in controls, p = 0.03). Oxidized glutathione was elevated in cystinotic cells (1.16 +/- 0.83 nmol/mg versus 0.29 +/- 0.18 nmol/mg protein, p = 0.04), while total glutathione, free cysteine, and ATP contents were normal in these cells. In conclusion, elevated oxidized glutathione in cystinotic proximal tubular epithelial cell lines suggests increased oxidative stress, which may contribute to tubular dysfunction in cystinosis.

  15. Metabolic coupling of glutathione between mouse and quail cardiac myocytes and its protective role against oxidative stress.

    PubMed

    Nakamura, T Y; Yamamoto, I; Kanno, Y; Shiba, Y; Goshima, K

    1994-05-01

    Cultured quail myocytes were much more resistant to H2O2 toxicity than cultured mouse myocytes. The intracellular concentration of glutathione ([GSH]i) and the activity of gamma-glutamylcysteine synthetase (gamma-GCS) in quail heart cells were about five and three times higher, respectively, than in mouse heart cells, although catalase and glutathione peroxidase (GSHpx) activity was similar in both. Preloading of gamma-glutamylcysteine monoethyl ester (gamma-GCE), a membrane-permeating GSH precursor, increased the H2O2 resistance of cultured mouse myocytes. These observations suggest that the high [GSH]i and the high activity of gamma-GCS in quail myocytes are responsible for their high resistance to H2O2. Both H2O2 sensitivity and [GSH]i of mosaic sheets composed of equal amounts of mouse and quail myocytes approximated those of sheets composed entirely of quail myocytes. From these observations, it is hypothesized that GSH was transferred from quail myocytes to mouse myocytes, probably through gap junctions between them, and that quail myocytes resynthesized GSH by a feedback mechanism, thus maintaining their intracellular GSH levels. When the fluorescent dye lucifer yellow was injected into a beating quail myocyte in a mosaic sheet, it spread to neighboring mouse myocytes but not to neighboring L cells (a cell line derived from mouse connective tissue). These observations indicate that existence of gap junctions in the region of cell contact between mouse and quail myocytes but not between quail myocytes and L cells. When quail myocytes preloaded with [3H]gamma-GCE were cocultured with mouse myocytes and L cells, the radioactivity was transmitted to neighboring mouse myocytes but not L cells. These observations show that GSH and/or its precursors can be transmitted from quail myocytes to mouse myocytes through gap junctions and that this can protect mouse myocytes from H2O2 toxicity. Mouse myocyte sheets composed of 10(4) cells or more showed higher resistance

  16. A cadmium-sensitive, glutathione-deficient mutant of Arabidopsis thaliana.

    PubMed Central

    Howden, R; Andersen, C R; Goldsbrough, P B; Cobbett, C S

    1995-01-01

    The roots of the cadmium-sensitive mutant of Arabidopsis thaliana, cad1-1, become brown in the presence of cadmium. A new cadmium-sensitive mutant affected at a second locus, cad2, has been identified using this phenotype. Genetic analysis has grown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Assays of cadmium accumulation by intact plants indicated that the mutant is deficient in its ability to sequester cadmium. Undifferentiated callus tissue was also cadmium sensitive, suggesting that the mutant phenotype is expressed at the cellular level. The level of cadmium-binding complexes formed in vivo was decreased compared with the wild type and accumulation of phytochelatins was about 10% of that in the wild type. The level of glutathione, the substrate for phytochelatin biosynthesis, in tissues of the mutant was decreased to about 15 to 30% of that in the wild type. Thus, the deficiency in phytochelatin biosynthesis can be explained by a deficiency in glutathione. PMID:7770518

  17. Glutathione-responsive core cross-linked micelles for controlled cabazitaxel delivery

    NASA Astrophysics Data System (ADS)

    Han, Xiaoxiong; Gong, Feirong; Sun, Jing; Li, Yueqi; Liu, XiaoFei; Chen, Dan; Liu, Jianwen; Shen, Yaling

    2018-02-01

    Stimulus-responsive polymeric micelles (PMs) have recently received attention due to the controlled delivery of drug or gene for application in cancer diagnosis and treatment. In this work, novel glutathione-responsive PMs were prepared to encapsulate hydrophobic antineoplastic drug, cabazitaxel (CTX), to improve its solubility and toxicity. These CTX-loaded micelles core cross-linked by disulfide bonds (DCL-CTX micelles) were prepared by a novel copolymer, lipoic acid grafted mPEG-PLA. These micelles had regular spherical shape, homogeneous diameter of 18.97 ± 0.23 nm, and a narrow size distribution. The DCL-CTX micelles showed high encapsulation efficiency of 98.65 ± 1.77%, and the aqueous solubility of CTX was improved by a factor of 1:1200. In vitro release investigation showed that DCL-CTX micelles were stable in the medium without glutathione (GSH), whereas the micelles had burst CTX release in the medium with 10 mM GSH. Cell uptake results implied that DCL-CTX micelles were internalized into MCF-7 cells through clathrin-mediated endocytosis and released cargo more effectively than Jevtana (commercially available CTX) owing to GSH-stimulated degradation. In MTT assay against MCF-7 cells, these micelles inhibited tumor cell proliferation more effectively than Jevtana due to their GSH-responsive CTX release. All results revealed the potency of GSH-responsive DCL-CTX micelles for stable delivery in blood circulation and for intracellular GSH-trigged release of CTX. Therefore, DCL-CTX micelles show potential as safe and effective CTX delivery carriers and as a cancer chemotherapy formulation.

  18. Role of glutathione S-transferase Pi in cisplatin-induced nephrotoxicity.

    PubMed

    Townsend, Danyelle M; Tew, Kenneth D; He, Lin; King, Jarrod B; Hanigan, Marie H

    2009-02-01

    One of the dose-limiting toxicities of cisplatin is nephrotoxicity. Renal toxicity is localized to quiescent proximal tubule cells, where the formation of DNA-adducts cannot account for the dose-limiting toxicity. Our earlier results have shown that a glutathione conjugate of cisplatin is metabolized to a nephrotoxicant via gamma-glutamyl transpeptidase (GGT) and a cysteine S-conjugate beta-lyase. The present study was designed to evaluate the potential role of glutathione S-transferase Pi (GSTP) in the initial steps of the bioactivation of cisplatin. Wild-type mice and mice deficient in both murine GSTP genes (GstP1/P2) were treated with cisplatin. Toxicity in both male and female mice was evaluated 5 days after treatment and renal damage was most severe in wild-type male mice. Wild-type males have approximately 10-fold higher levels of GSTP expression in the liver than females, suggesting that hepatic GSTP in the wild-type males contributed to the formation of the nephrotoxic platinum-glutathione conjugate. In GstP1/P2 null mice the gender difference in toxicity was eliminated. Our data show that GSTP expression is a determinant in cisplatin-induced nephrotoxicity and its levels contribute to sex-dependent differences.

  19. Role of Glutathione S-Transferase Pi in Cisplatin Induced Nephrotoxicity

    PubMed Central

    Townsend, Danyelle M.; Tew, Kenneth D.; He, Lin; King, Jarrod B.; Hanigan, Marie H.

    2009-01-01

    SUMMARY One of the dose-limiting toxicities of cisplatin is nephrotoxicity. Renal toxicity is localized to quiescent proximal tubule cells, where the formation of DNA-adducts cannot account for the dose-limiting toxicity. Our earlier results have shown that a glutathione-conjugate of cisplatin is metabolized to a nephrotoxicant via gamma-glutamyltranspeptidase (GGT) and a cysteine S-conjugate beta-lyase. The present study was designed to evaluate the potential role of glutathione-S-transferase Pi (GSTP) in the initial steps of the bioactivation of cisplatin. Wild-type mice and mice deficient in both murine GSTP genes (GstP1/P2) were treated with cisplatin. Toxicity in both male and female mice was evaluated 5 days after treatment and renal damage was most severe in wild-type male mice. Wild-type males have ~10-fold higher levels of GSTP expression in the liver than females, suggesting that hepatic GSTP in the wild-type males contributed to the formation of the nephrotoxic platinum-glutathione conjugate. In GstP1/P2 null mice the gender difference in toxicity was eliminated. Our data show that GSTP expression is a determinant in cisplatin-induced nephrotoxicity and its levels contribute to sex-dependent differences. PMID:18819770

  20. The potential role of the antioxidant and detoxification properties of glutathione in autism spectrum disorders: a systematic review and meta-analysis

    PubMed Central

    2012-01-01

    Background Glutathione has a wide range of functions; it is an endogenous anti-oxidant and plays a key role in the maintenance of intracellular redox balance and detoxification of xenobiotics. Several studies have indicated that children with autism spectrum disorders may have altered glutathione metabolism which could play a key role in the condition. Methods A systematic literature review and meta-analysis was conducted of studies examining metabolites, interventions and/or genes of the glutathione metabolism pathways i.e. the γ-glutamyl cycle and trans-sulphuration pathway in autism spectrum disorders. Results Thirty nine studies were included in the review comprising an in vitro study, thirty two metabolite and/or co-factor studies, six intervention studies and six studies with genetic data as well as eight studies examining enzyme activity. Conclusions The review found evidence for the involvement of the γ-glutamyl cycle and trans-sulphuration pathway in autistic disorder is sufficiently consistent, particularly with respect to the glutathione redox ratio, to warrant further investigation to determine the significance in relation to clinical outcomes. Large, well designed intervention studies that link metabolites, cofactors and genes of the γ-glutamyl cycle and trans-sulphuration pathway with objective behavioural outcomes in children with autism spectrum disorders are required. Future risk factor analysis should include consideration of multiple nutritional status and metabolite biomarkers of pathways linked with the γ-glutamyl cycle and the interaction of genotype in relation to these factors. PMID:22524510

  1. Single-cell intracellular nano-pH probes†

    PubMed Central

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2016-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

  2. Single-cell intracellular nano-pH probes.

    PubMed

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

  3. Biatriosporin D displays anti-virulence activity through decreasing the intracellular cAMP levels

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

    Zhang, Ming; Chang, Wenqiang; Shi, Hongzhuo

    Candidiasis has long been a serious human health problem, and novel antifungal approaches are greatly needed. During both superficial and systemic infection, C. albicans relies on a battery of virulence factors, such as adherence, filamentation, and biofilm formation. In this study, we found that a small phenolic compound, Biatriosporin D (BD), isolated from an endolichenic fungus, Biatriospora sp., displayed anti-virulence activity by inhibiting adhesion, hyphal morphogenesis and biofilm formation of C. albicans. Of note is the high efficacy of BD in preventing filamentation with a much lower dose than its MIC value. Furthermore, BD prolonged the survival of worms infectedmore » by C. albicans in vivo. Quantitative real-time PCR analysis, exogenous cAMP rescue experiments and intracellular cAMP measurements revealed that BD regulates the Ras1-cAMP-Efg1 pathway by reducing cAMP levels to inhibit the hyphal formation. Further investigation showed that BD could upregulate Dpp3 to synthesize much more farnesol, which could inhibit the activity of Cdc35 and reduce the generation of cAMP. Taken together, these findings indicate that BD stimulates the expression of Dpp3 to synthesize more farnesol that directly inhibits the Cdc35 activity, reducing intracellular cAMP and thereby disrupting the morphologic transition and attenuating the virulence of C. albicans. Our study uncovers the underlying mechanism of BD as a prodrug in fighting against pathogenic C. albicans and provides a potential application of BD in fighting clinically relevant fungal infections by targeting fungal virulence. - Highlights: • BD inhibits the filamentation of C. albicans in multiple hypha-inducing conditions. • BD can prolong the survival of nematodes infected by C. albicans. • BD stimulates the expression of Dpp3 to synthesize more farnesol. • BD reduces intracellular cAMP and regulates Ras1-cAMP-PKA pathway.« less

  4. Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity.

    PubMed

    Okada, Kazushi; Fukui, Masayuki; Zhu, Bao-Ting

    2016-08-26

    Glutathione depletion is a distinct cause underlying many forms of pathogenesis associated with oxidative stress and cytotoxicity. Earlier studies showed that glutamate-induced glutathione depletion in immortalized murine HT22 hippocampal neuronal cells leads to accumulation of reactive oxygen species (ROS) and ultimately cell death, but the precise mechanism underlying these processes is not clear. Here we show that during the induction of glutathione depletion, nitric oxide (NO) accumulation precedes ROS accumulation. While neuronal NO synthase (nNOS) in untreated HT22 cells exists mostly as a monomer, glutathione depletion results in increased formation of the dimer nNOS, accompanied by increases in the catalytic activity. We identified that nNOS dimerization is catalyzed by protein disulfide isomerase (PDI). Inhibition of PDI's isomerase activity effectively abrogates glutathione depletion-induced conversion of monomer nNOS into dimer nNOS, accumulation of NO and ROS, and cytotoxicity. Furthermore, we found that PDI is present in untreated cells in an inactive S-nitrosylated form, which becomes activated following glutathione depletion via S-denitrosylation. These results reveal a novel role for PDI in mediating glutathione depletion-induced oxidative cytotoxicity, as well as its role as a valuable therapeutic target for protection against oxidative cytotoxicity. Copyright © 2016. Published by Elsevier Inc.

  5. 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.

  6. CalQuo: automated, simultaneous single-cell and population-level quantification of global intracellular Ca2+ responses.

    PubMed

    Fritzsche, Marco; Fernandes, Ricardo A; Colin-York, Huw; Santos, Ana M; Lee, Steven F; Lagerholm, B Christoffer; Davis, Simon J; Eggeling, Christian

    2015-11-13

    Detecting intracellular calcium signaling with fluorescent calcium indicator dyes is often coupled with microscopy techniques to follow the activation state of non-excitable cells, including lymphocytes. However, the analysis of global intracellular calcium responses both at the single-cell level and in large ensembles simultaneously has yet to be automated. Here, we present a new software package, CalQuo (Calcium Quantification), which allows the automated analysis and simultaneous monitoring of global fluorescent calcium reporter-based signaling responses in up to 1000 single cells per experiment, at temporal resolutions of sub-seconds to seconds. CalQuo quantifies the number and fraction of responding cells, the temporal dependence of calcium signaling and provides global and individual calcium-reporter fluorescence intensity profiles. We demonstrate the utility of the new method by comparing the calcium-based signaling responses of genetically manipulated human lymphocytic cell lines.

  7. GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerisation.

    PubMed

    Cai, Shijie; Alp, Nicholas J; McDonald, Denise; Smith, Ian; Kay, Jonathan; Canevari, Laura; Heales, Simon; Channon, Keith M

    2002-09-01

    Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation, by targeting the BH4 synthetic pathway as a novel strategy to modulate intracellular BH4 levels. We constructed a recombinant adenovirus, AdGCH, encoding human GTPCH. We infected human endothelial cells with AdGCH, investigated the changes in intracellular biopterin levels, and determined the effects on eNOS enzymatic activity, protein levels and dimerisation. GTPCH gene transfer in EAhy926 endothelial cells increased BH4 >10-fold compared with controls (cells alone or control adenovirus infection), and greatly enhanced NO production in a dose-dependent, eNOS-specific manner. We found that eNOS was principally monomeric in control cells, whereas GTPCH gene transfer resulted in a striking increase in eNOS homodimerisation. Furthermore, the total amounts of both native eNOS protein and a recombinant eNOS-GFP fusion protein were significantly increased following GTPCH gene transfer. These findings suggest that GTPCH gene transfer is a valid approach to increase BH4 levels in human endothelial cells, and provide new evidence for the relative importance of different mechanisms underlying BH4-mediated eNOS regulation in intact human endothelial cells. Additionally, these observations suggest that GTPCH may be a rational target to augment endothelial BH4 and normalise eNOS activity in endothelial dysfunction states.

  8. Beta-carotene reduces oxidative stress, improves glutathione metabolism and modifies antioxidant defense systems in lead-exposed workers

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

    Kasperczyk, Sławomir, E-mail: kaslav@mp.pl; Dobrakowski, Michał; Kasperczyk, Janusz

    2014-10-01

    The aim of this study was to determine whether beta-carotene administration reduces oxidative stress and influences antioxidant, mainly glutathione-related, defense systems in workers chronically exposed to lead. The population consisted of two randomly divided groups of healthy male volunteers exposed to lead. Workers in the first group (reference group) were not administered any antioxidants, while workers in the second group (CAR group) were treated orally with 10 mg of beta-carotene once a day for 12 weeks. Biochemical analysis included measuring markers of lead-exposure and oxidative stress in addition to the levels and activities of selected antioxidants. After treatment, levels ofmore » malondialdehyde, lipid hydroperoxides and lipofuscin significantly decreased compared with the reference group. However, the level of glutathione significantly increased compared with the baseline. Treatment with beta-carotene also resulted in significantly decreased glutathione peroxidase activity compared with the reference group, while the activities of other glutathione-related enzymes and of superoxide dismutase were not significantly changed. However, the activities of glucose-6-phosphate dehydrogenase and catalase, as well as the level of alpha-tocopherol, were significantly higher after treatment compared with the baseline. Despite controversy over the antioxidant properties of beta-carotene in vivo, our findings showed reduced oxidative stress after beta-carotene supplementation in chronic lead poisoning. - Highlights: • Beta-carotene reduces oxidative stress in lead-exposed workers. • Beta-carotene elevates glutathione level in lead-exposed workers. • Beta-carotene administration could be beneficial in lead poisoning.« less

  9. Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases.

    PubMed

    Castro, F A V; Herdeiro, R S; Panek, A D; Eleutherio, E C A; Pereira, M D

    2007-02-01

    Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione-GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.

  10. 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

  11. Monitoring Intracellular Oxygen Concentration: Implications for Hypoxia Studies and Real-Time Oxygen Monitoring.

    PubMed

    Potter, Michelle; Badder, Luned; Hoade, Yvette; Johnston, Iain G; Morten, Karl J

    2016-01-01

    The metabolic properties of cancer cells have been widely accepted as a hallmark of cancer for a number of years and have shown to be of critical importance in tumour development. It is generally accepted that tumour cells exhibit a more glycolytic phenotype than normal cells. In this study, we investigate the bioenergetic phenotype of two widely used cancer cell lines, RD and U87MG, by monitoring intracellular oxygen concentrations using phosphorescent Pt-porphyrin based intracellular probes. Our study demonstrates that cancer cell lines do not always exhibit an exclusively glycolytic phenotype. RD demonstrates a reliance on oxidative phosphorylation whilst U87MG display a more glycolytic phenotype. Using the intracellular oxygen sensing probe we generate an immediate readout of intracellular oxygen levels, with the glycolytic lines reflecting the oxygen concentration of the environment, and cells with an oxidative phenotype having significantly lower levels of intracellular oxygen. Inhibition of oxygen consumption in lines with high oxygen consumption increases intracellular oxygen levels towards environmental levels. We conclude that the use of intracellular oxygen probes provides a quantitative assessment of intracellular oxygen levels, allowing the manipulation of cellular bioenergetics to be studied in real time.

  12. Quantitation of protein S-glutathionylation by liquid chromatograph-tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

    USDA-ARS?s Scientific Manuscript database

    Protein S-glutathionylation is a posttranslational modification that links oxidative stimuli to reversible changes in cellular function. Protein-glutathione mixed disulfides (PSSG) are commonly quantified by the reduction of the disulfide and detection of the resultant glutathione species. This met...

  13. Nitric oxide alleviates aluminum-induced oxidative damage through regulating the ascorbate-glutathione cycle in roots of wheat.

    PubMed

    Sun, Chengliang; Liu, Lijuan; Yu, Yan; Liu, Wenjing; Lu, Lingli; Jin, Chongwei; Lin, Xianyong

    2015-06-01

    The possible association with nitric oxide (NO) and ascorbate-glutathione (AsA-GSH) cycle in regulating aluminum (Al) tolerance of wheat (Triticum aestivum L.) was investigated using two genotypes with different Al resistance. Exposure to Al inhibited root elongation, and triggered lipid peroxidation and oxidation of AsA to dehydroascorbate and GSH to glutathione disulfide in wheat roots. Exogenous NO significantly increased endogenous NO levels, and subsequently alleviated Al-induced inhibition of root elongation and oxidation of AsA and GSH to maintain the redox molecules in the reduced form in both wheat genotypes. Under Al stress, significantly increased activities and gene transcriptional levels of ascorbate peroxidase, glutathione reductase, and dehydroascorbate reductase, were observed in the root tips of the Al-tolerant genotype Jian-864. Nitric oxide application enhanced the activity and gene transcriptional level of these enzymes in both wheat genotypes. γ-Glutamylcysteine synthetase was not significantly affected by Al or NO, but NO treatments increased the activity of glutathione peroxidase and glutathione S-transferase to a greater extent than the Al-treated wheat seedlings. Proline was significantly decreased by Al, while it was not affected by NO. These results clearly suggest that NO protects wheat root against Al-induced oxidative stress, possibly through its regulation of the AsA-GSH cycle. © 2014 Institute of Botany, Chinese Academy of Sciences.

  14. Cortical glutathione levels in young people with bipolar disorder: a pilot study using magnetic resonance spectroscopy.

    PubMed

    Godlewska, Beata R; Yip, Sarah W; Near, Jamie; Goodwin, Guy M; Cowen, Philip J

    2014-01-01

    Glutathione (GSH) is a key scavenger for cellular free radicals, and patients with bipolar disorder may have lowered GSH levels in plasma and in post-mortem brain tissue. The objective of the current study was to use magnetic resonance spectroscopy (MRS) to measure cortical GSH levels in young people with bipolar disorder to determine if lowered GSH might be a useful biomarker of vulnerability to the illness. We studied 13 patients with DSM-IV bipolar disorder and 11 healthy age-matched controls using proton MRS in conjunction with the SPECIAL acquisition technique. Voxels were placed in prefrontal and occipital cortex. All patients were clinically euthymic at the time of study and unmedicated. GSH and other relevant neurometabolites were measured relative to creatinine. There was no difference in GSH levels between bipolar participants and controls in either prefrontal or occipital cortex. Similarly, participants showed no difference from controls in other measured cortical metabolites including γ-aminobutyric acid, glutamate and N-acetylaspartate. This pilot study suggests that levels of cortical GSH are unlikely to be a useful trait biomarker of bipolar disorder in young people with a history of relatively mild mood instability at an early stage of illness. Lowered GSH levels may be relevant to bipolar pathophysiology in more severely ill patients, particular those with significant current mood disturbance.

  15. The mitochondrial dicarboxylate and 2-oxoglutarate carriers do not transport glutathione

    PubMed Central

    Booty, Lee M.; King, Martin S.; Thangaratnarajah, Chancievan; Majd, Homa; James, Andrew M.; Kunji, Edmund R.S.; Murphy, Michael P.

    2015-01-01

    Glutathione carries out vital protective roles within mitochondria, but is synthesised in the cytosol. Previous studies have suggested that the mitochondrial dicarboxylate and 2-oxoglutarate carriers were responsible for glutathione uptake. We set out to characterise the putative glutathione transport by using fused membrane vesicles of Lactococcus lactis overexpressing the dicarboxylate and 2-oxoglutarate carriers. Although transport of the canonical substrates could be measured readily, an excess of glutathione did not compete for substrate uptake nor could transport of glutathione be measured directly. Thus these mitochondrial carriers do not transport glutathione and the identity of the mitochondrial glutathione transporter remains unknown. PMID:25637873

  16. 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

  17. Effects of glutamine supplementation on gut barrier, glutathione content and acute phase response in malnourished rats during inflammatory shock.

    PubMed

    Belmonte, Liliana; Coëffier, Moïse; Le Pessot, Florence; Miralles-Barrachina, Olga; Hiron, Martine; Leplingard, Antony; Lemeland, Jean-François; Hecketsweiler, Bernadette; Daveau, Maryvonne; Ducrotté, Philippe; Déchelotte, Pierre

    2007-05-28

    To evaluate the effect of glutamine on intestinal mucosa integrity, glutathione stores and acute phase response in protein-depleted rats during an inflammatory shock. Plasma acute phase proteins (APP), jejunal APP mRNA levels, liver and jejunal glutathione concentrations were measured before and one, three and seven days after turpentine injection in 4 groups of control, protein-restricted, protein-restricted rats supplemented with glutamine or protein powder. Bacterial translocation in mesenteric lymph nodes and intestinal morphology were also assessed. Protein deprivation and turpentine injection significantly reduced jejunal villus height, and crypt depths. Mucosal glutathione concentration significantly decreased in protein-restricted rats. Before turpentine oil, glutamine supplementation restored villus heights and glutathione concentration (3.24 +/- 1.05 vs 1.72 +/- 0.46 mumol/g tissue, P<0.05) in the jejunum, whereas in the liver glutathione remained low. Glutamine markedly increased jejunal alpha1-acid glycoprotein mRNA level after turpentine oil but did not affect its plasma concentration. Bacterial translocation in protein-restricted rats was not prevented by glutamine or protein powder supplementation. Glutamine restored gut glutathione stores and villus heights in malnourished rats but had no preventive effect on bacterial translocation in our model.

  18. [Glutathione participation in regulating the secretory function of the stomach].

    PubMed

    Shlygin, G K; Vasilevskaia, L S; Martinchik, A N; Ignatenko, L G

    1987-01-01

    In experiments on dogs with Pavlov's pouches it was shown that glutathione infusion into the blood produced a highly pronounced stimulating effect on the gastric secretion induced by pentagastrin. Endogenous glutathione produced similar effect. It was found that intake as a drink of mono-sodium glutamate led to a significant increase of glutathione concentration in the dogs' blood, that was, probably, the result of its intensified production in the intestinal wall and passing into the blood. The growth of glutathione concentration in the blood coincided with its stimulating effect on the gastric secretion. Glutathione administered separately into the blood, or intake of glutathione without pentagastrin did not produce stimulating effect on gastric secretion. The data presented have evidenced that glutathione, besides its known functions, plays a role of the factor engaged in the regulation of gastric secretion.

  19. Glutathione S-conjugates as prodrugs to target drug-resistant tumors

    PubMed Central

    Ramsay, Emma E.; Dilda, Pierre J.

    2014-01-01

    Living organisms are continuously exposed to xenobiotics. The major phase of enzymatic detoxification in many species is the conjugation of activated xenobiotics to reduced glutathione (GSH) catalyzed by the glutathione-S-transferase (GST). It has been reported that some compounds, once transformed into glutathione S-conjugates, enter the mercapturic acid pathway whose end products are highly reactive and toxic for the cell responsible for their production. The cytotoxicity of these GSH conjugates depends essentially on GST and gamma-glutamyl transferases (γGT), the enzymes which initiate the mercapturic acid synthesis pathway. Numerous studies support the view that the expression of GST and γGT in cancer cells represents an important factor in the appearance of a more aggressive and resistant phenotype. High levels of tumor GST and γGT expression were employed to selectively target tumor with GST- or γGT-activated drugs. This strategy, explored over the last two decades, has recently been successful using GST-activated nitrogen mustard (TLK286) and γGT-activated arsenic-based (GSAO and Darinaparsin) prodrugs confirming the potential of GSH-conjugates as anticancer drugs. PMID:25157234

  20. Antibacterial activity of exogenous glutathione and its synergism on antibiotics sensitize carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii.

    PubMed

    Alharbe, Roaa; Almansour, Ayidh; Kwon, Dong H

    2017-10-01

    A major clinical impact of A. baumannii is hospital-acquired infections including ventilator-associated pneumonia. The treatment of this pathogen is often difficult due to its innate and acquired resistance to almost all commercially available antibiotics. Infections with carbapenem-associated multidrug resistant A. baumannii is the most problematic. Glutathione is a tripeptide thiol-antioxidant and antibacterial activity of exogenous glutathione was reported in some bacteria. However, clinical relevance and molecular details of the antibacterial activity of glutathione are currently unclear. Seventy clinical isolates of A. baumannii including 63 carbapenem-associated multidrug resistant isolates and a type strain A. baumannii ATCC 19606 were used to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Fractional inhibitory concentration (FIC) and time-killing activity with meropenem and/or glutathione were also determined in the carbapenem-associated multidrug resistant isolates. In addition, the roles of exogenous glutathione in multidrug efflux pumps and β-lactamase production were examined. Levels of MIC and MBC were ranged from 10 to 15mM of exogenous glutathione. All tested carbapenem-associated multidrug resistant isolates were sensitized by all tested antibiotics in combination with subinhibitory concentrations of glutathione. FIC levels of glutathione with carbapenem (meropenem) were all<0.5 and the carbapenem-associated multidrug resistant isolates were killed by subinhibitory concentrations of both glutathione and meropenem at>2log10 within 12h, suggesting glutathione synergistically interacts with meropenem. The roles of multidrug efflux pumps and β-lactamase production were excluded for the glutathione-mediated antibiotic susceptibility. Overall results demonstrate that the antibacterial activity of glutathione is clinically relevant and its synergism on antibiotics sensitizes clinical isolates of A

  1. Arabidopsis GLUTATHIONE REDUCTASE1 Plays a Crucial Role in Leaf Responses to Intracellular Hydrogen Peroxide and in Ensuring Appropriate Gene Expression through Both Salicylic Acid and Jasmonic Acid Signaling Pathways1[C][W][OA

    PubMed Central

    Mhamdi, Amna; Hager, Jutta; Chaouch, Sejir; Queval, Guillaume; Han, Yi; Taconnat, Ludivine; Saindrenan, Patrick; Gouia, Houda; Issakidis-Bourguet, Emmanuelle; Renou, Jean-Pierre; Noctor, Graham

    2010-01-01

    Glutathione is a major cellular thiol that is maintained in the reduced state by glutathione reductase (GR), which is encoded by two genes in Arabidopsis (Arabidopsis thaliana; GR1 and GR2). This study addressed the role of GR1 in hydrogen peroxide (H2O2) responses through a combined genetic, transcriptomic, and redox profiling approach. To identify the potential role of changes in glutathione status in H2O2 signaling, gr1 mutants, which show a constitutive increase in oxidized glutathione (GSSG), were compared with a catalase-deficient background (cat2), in which GSSG accumulation is conditionally driven by H2O2. Parallel transcriptomics analysis of gr1 and cat2 identified overlapping gene expression profiles that in both lines were dependent on growth daylength. Overlapping genes included phytohormone-associated genes, in particular implicating glutathione oxidation state in the regulation of jasmonic acid signaling. Direct analysis of H2O2-glutathione interactions in cat2 gr1 double mutants established that GR1-dependent glutathione status is required for multiple responses to increased H2O2 availability, including limitation of lesion formation, accumulation of salicylic acid, induction of pathogenesis-related genes, and signaling through jasmonic acid pathways. Modulation of these responses in cat2 gr1 was linked to dramatic GSSG accumulation and modified expression of specific glutaredoxins and glutathione S-transferases, but there is little or no evidence of generalized oxidative stress or changes in thioredoxin-associated gene expression. We conclude that GR1 plays a crucial role in daylength-dependent redox signaling and that this function cannot be replaced by the second Arabidopsis GR gene or by thiol systems such as the thioredoxin system. PMID:20488891

  2. Glycation of human erythrocyte glutathione peroxidase: effect on the physical and kinetic properties.

    PubMed

    Suravajjala, Sreekanth; Cohenford, Menashi; Frost, Leslie R; Pampati, Praveen K; Dain, Joel A

    2013-06-05

    Glutathione peroxidase (GPx) is a significant antioxidant enzyme that plays a key role in protecting the body from reactive oxygen species (ROS) and their toxicity. As a biocatalyst, the enzyme has been shown to reduce hydrogen peroxide to water and lipid hydroperoxides to their respective alcohols. The increased levels of ROS in patients with diabetes have been speculated to arise, in part, from alterations in the activity of glutathione antioxidant enzymes, perhaps, by mechanisms such as the glycation of the protein, in vivo. Under physiological conditions of temperature and pH, we investigated the susceptibility of human glutathione peroxidase to glycation, determined the effects of glycation on the physical and kinetic properties of the enzyme, and identified the protein's vulnerable amino acid sites of glycation. Circular dichroism, UV and mass spectrometry studies revealed that methylglyoxal and DL-glyceraldehyde are potent glycators of glutathione peroxidase; destabilizing its structure, altering its pH activity and stability profiles and increasing its Km value. In comparison to DL-glyceraldehyde, methylglyxol was a more potent glycator of the enzyme and was found to nonenzymatically condense with Arg-177, located near the glutathione binding site of GPx. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. 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.

  4. The intracellular carboxyl tail of the PAR-2 receptor controls intracellular signaling and cell death.

    PubMed

    Zhu, Zhihui; Stricker, Rolf; Li, Rong yu; Zündorf, Gregor; Reiser, Georg

    2015-03-01

    The protease-activated receptors are a group of unique G protein-coupled receptors, including PAR-1, PAR-2, PAR-3 and PAR-4. PAR-2 is activated by multiple trypsin-like serine proteases, including trypsin, tryptase and coagulation proteases. The clusters of phosphorylation sites in the PAR-2 carboxyl tail are suggested to be important for the binding of adaptor proteins to initiate intracellular signaling to Ca(2+) and mitogen-activated protein kinases. To explore the functional role of PAR-2 carboxyl tail in controlling intracellular Ca(2+), ERK and AKT signaling, a series of truncated mutants containing different clusters of serines/threonines were generated and expressed in HEK293 cells. Firstly, we observed that lack of the complete C-terminus of PAR-2 in a mutated receptor gave a relatively low level of localization on the cell plasma membrane. Secondly, the shortened carboxyl tail containing 13 amino acids was sufficient for receptor internalization. Thirdly, the cells expressing truncation mutants showed deficits in their capacity to couple to intracellular Ca(2+) and ERK and AKT signaling upon trypsin challenge. In addition, HEK293 cells carrying different PAR-2 truncation mutants displayed decreased levels of cell survival after long-lasting trypsin stimulation. In summary, the PAR-2 carboxyl tail was found to control the receptor localization, internalization, intracellular Ca(2+) responses and signaling to ERK and AKT. The latter can be considered to be important for cell death control.

  5. The mitochondrial dicarboxylate and 2-oxoglutarate carriers do not transport glutathione.

    PubMed

    Booty, Lee M; King, Martin S; Thangaratnarajah, Chancievan; Majd, Homa; James, Andrew M; Kunji, Edmund R S; Murphy, Michael P

    2015-02-27

    Glutathione carries out vital protective roles within mitochondria, but is synthesised in the cytosol. Previous studies have suggested that the mitochondrial dicarboxylate and 2-oxoglutarate carriers were responsible for glutathione uptake. We set out to characterise the putative glutathione transport by using fused membrane vesicles of Lactococcus lactis overexpressing the dicarboxylate and 2-oxoglutarate carriers. Although transport of the canonical substrates could be measured readily, an excess of glutathione did not compete for substrate uptake nor could transport of glutathione be measured directly. Thus these mitochondrial carriers do not transport glutathione and the identity of the mitochondrial glutathione transporter remains unknown. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  6. 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.

  7. Seasonal- and temperature-dependent variation in CNS ascorbate and glutathione levels in anoxia-tolerant turtles.

    PubMed

    Pérez-Pinzón, M A; Rice, M E

    1995-12-24

    We determined the ascorbic acid (ascorbate) and glutathione (GSH) contents of eight regions of the CNS from anoxia-tolerant turtles collected in summer and in winter. Ascorbate was of special interest because it is found in exceptionally high levels in the turtle CNS. The temperature-dependence of CNS ascorbate content was established by comparing levels in animals collected from two geographic zones with different average winter temperatures and in animals re-acclimated to different temperatures in the laboratory. The analytical method was liquid chromatography with electrochemical detection. Turtle ascorbate levels were 30-40% lower in animals acclimatized to winter (2 degrees C) than to summer (23 degrees C) in all regions of the CNS. Similarly, GSH levels were 20-30% lower in winter than in summer. Winter ascorbate levels were higher in turtles from Louisiana (19 degrees C) than in turtles acclimatized to winter in Wisconsin (2 degrees C). Summer and winter levels of ascorbate could be reversed by re-acclimating animals to cold (1 degree C) or warm (23 degrees C) temperatures for at least one week. CNS water content did not differ between cold- and warm-acclimated turtles. Taken together, the data indicated that ascorbate and GSH undergo significant seasonal variation and that the catalyst for change is environmental temperature. Steady-state ascorbate content showed a linear dependence on temperature, with a slope of 1.5% per degree C that was independent of CNS region. Lower levels of cerebral antioxidants in turtles exposed to colder temperatures were consistent with the decreased rate of cerebral metabolism that accompanies winter hibernation. Cerebral ascorbate and GSH levels in the turtle remained similar to or higher than those in mammals, even during winter, however. These findings support the notion that unique mechanisms of antioxidant regulation in the turtle contribute to their tolerance of the hypoxia-reoxygenation that characterizes diving

  8. The antioxidant master glutathione and periodontal health

    PubMed Central

    Bains, Vivek Kumar; Bains, Rhythm

    2015-01-01

    Glutathione, considered to be the master antioxidant (AO), is the most-important redox regulator that controls inflammatory processes, and thus damage to the periodontium. Periodontitis patients have reduced total AO capacity in whole saliva, and lower concentrations of reduced glutathione (GSH) in serum and gingival crevicular fluid, and periodontal therapy restores the redox balance. Therapeutic considerations for the adjunctive use of glutathione in management of periodontitis, in limiting the tissue damage associated with oxidative stress, and enhancing wound healing cannot be underestimated, but need to be evaluated further through multi-centered randomized controlled trials. PMID:26604952

  9. Safety of long-term feeding of dl-alpha-lipoic acid and its effect on reduced glutathione:oxidized glutathione ratios in beagles.

    PubMed

    Zicker, Steven C; Hagen, Tory M; Joisher, Neha; Golder, Christina; Joshi, Dinesh K; Miller, E Phillip

    2002-01-01

    Alpha-lipoic acid is touted as a powerful antioxidant and possibly a conditionally essential nutrient in older mammals. The safety and efficacy of dl-alpha-lipoic acid was evaluated in 30 adult beagles that were evenly randomized into five groups, each of which was fed one of five different foods with varying inclusion rates of dl-alpha-lipoic acid (0, 150, 1500, 3000, and 4500 ppm). All dogs were fed their respective portion of food daily as their sole source of nutrition for 6 months. Evaluations included general health, body weight, food intake, hematologic and serum biochemical parameters, and glutathione:oxidized glutathione (GSH:GSSG) ratios in lymphocytes. No signs of toxicity were observed at any except the highest level of dl-alpha-lipoic acid inclusion, and no consistent abnormalities were noted in hematologic or biochemical measures at any level. There was a significant overall effect (P< .05) of food on the difference of GSH:GSSG ratio between Day 84 and Day 0. All inclusions of dl-alpha-lipoic acid increased the ratio of GSH:GSSG with the largest numeric improvement occurring at the lowest inclusion rate (150 ppm).

  10. Impaired synthesis and antioxidant defense of glutathione in the cerebellum of autistic subjects: alterations in the activities and protein expression of glutathione-related enzymes.

    PubMed

    Gu, Feng; Chauhan, Ved; Chauhan, Abha

    2013-12-01

    Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence in autism suggests a deficit in glutathione (GSH), a major endogenous antioxidant. It is not known whether the synthesis, consumption, and/or regeneration of GSH is affected in autism. In the cerebellum tissues from autism (n=10) and age-matched control subjects (n=10), the activities of GSH-related enzymes glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glutamate cysteine ligase (GCL) involved in antioxidant defense, detoxification, GSH regeneration, and synthesis, respectively, were analyzed. GCL is a rate-limiting enzyme for GSH synthesis, and the relationship between its activity and the protein expression of its catalytic subunit GCLC and its modulatory subunit GCLM was also compared between the autistic and the control groups. Results showed that the activities of GPx and GST were significantly decreased in autism compared to that of the control group (P<0.05). Although there was no significant difference in GR activity between autism and control groups, 40% of autistic subjects showed lower GR activity than 95% confidence interval (CI) of the control group. GCL activity was also significantly reduced by 38.7% in the autistic group compared to the control group (P=0.023), and 8 of 10 autistic subjects had values below 95% CI of the control group. The ratio of protein levels of GCLC to GCLM in the autism group was significantly higher than that of the control group (P=0.022), and GCLM protein levels were reduced by 37.3% in the autistic group compared to the control group. A positive strong correlation was observed between GCL activity and protein levels of GCLM (r=0.887) and GCLC (r=0.799) subunits in control subjects but not in autistic subjects, suggesting that regulation of GCL activity is affected in autism. These results suggest that enzymes involved in GSH homeostasis have impaired

  11. Characterization of intracellular and extracellular saxitoxin levels in both field and cultured Alexandrium spp. samples from Sequim Bay, Washington.

    PubMed

    Lefebvre, Kathi A; Bill, Brian D; Erickson, Aleta; Baugh, Keri A; O'Rourke, Lohna; Costa, Pedro R; Nance, Shelly; Trainer, Vera L

    2008-05-14

    Traditionally, harmful algal bloom studies have primarily focused on quantifying toxin levels contained within the phytoplankton cells of interest. In the case of paralytic shellfish poisoning toxins (PSTs), intracellular toxin levels and the effects of dietary consumption of toxic cells by planktivores have been well documented. However, little information is available regarding the levels of extracellular PSTs that may leak or be released into seawater from toxic cells during blooms. In order to fully evaluate the risks of harmful algal bloom toxins in the marine food web, it is necessary to understand all potential routes of exposure. In the present study, extracellular and intracellular PST levels were measured in field seawater samples (collected weekly from June to October 2004-2007) and in Alexandrium spp. culture samples isolated from Sequim Bay, Washington. Measurable levels of intra- and extra-cellular toxins were detected in both field and culture samples via receptor binding assay (RBA) and an enzyme-linked immunosorbent assay (ELISA). Characterization of the PST toxin profile in the Sequim Bay isolates by pre-column oxidation and HPLC-fluorescence detection revealed that gonyautoxin 1 and 4 made up 65 +/- 9.7% of the total PSTs present. Collectively, these data confirm that extracellular PSTs are present during blooms of Alexandrium spp. in the Sequim Bay region.

  12. Conjugations with glutathione. The enzymic conjugation of some chlorocyclohexenes

    PubMed Central

    Sims, P.; Grover, P. L.

    1965-01-01

    1. α-3,4,5,6-Tetrachlorocyclohex-1-ene and γ-2,3,4,5,6-pentachlorocyclohex-1-ene are conjugated with glutathione in vitro by a rat-liver enzyme that is probably glutathione S-aryltransferase. 2. Chlorocyclohexane and the α-, β-, γ- and δ-isomers of hexachlorocyclohexane were not substrates for rat-liver glutathione S-aryltransferase. 3. Glutathione-S-aryltransferase activity was present in tissue preparations of houseflies of insecticide-resistant and -susceptible strains. More activity was found in a dieldrin-resistant strain of houseflies fed on dieldrin than in either a dieldrin-resistant strain not fed on dieldrin or a control strain of dieldrin-susceptible houseflies. 4. Housefly soluble supernatant preparations converted S-(2-chloro-4-nitrophenyl)glutathione into the corresponding cysteine and mercapturic acid derivatives. PMID:14333551

  13. Effects of glutamine supplementation on gut barrier, glutathione content and acute phase response in malnourished rats during inflammatory shock

    PubMed Central

    Belmonte, Liliana; Coëffier, Moïse; Pessot, Florence Le; Miralles-Barrachina, Olga; Hiron, Martine; Leplingard, Antony; Lemeland, Jean-François; Hecketsweiler, Bernadette; Daveau, Maryvonne; Ducrotté, Philippe; Déchelotte, Pierre

    2007-01-01

    AIM: To evaluate the effect of glutamine on intestinal mucosa integrity, glutathione stores and acute phase response in protein-depleted rats during an inflammatory shock. METHODS: Plasma acute phase proteins (APP), jejunal APP mRNA levels, liver and jejunal glutathione concentrations were measured before and one, three and seven days after turpentine injection in 4 groups of control, protein-restricted, protein-restricted rats supplemented with glutamine or protein powder. Bacterial translocation in mesenteric lymph nodes and intestinal morphology were also assessed. RESULTS: Protein deprivation and turpentine injection significantly reduced jejunal villus height, and crypt depths. Mucosal glutathione concentration significantly decreased in protein-restricted rats. Before turpentine oil, glutamine supplementation restored villus heights and glutathione concentration (3.24 ± 1.05 vs 1.72 ± 0.46 μmol/g tissue, P < 0.05) in the jejunum, whereas in the liver glutathione remained low. Glutamine markedly increased jejunal α1-acid glycoprotein mRNA level after turpentine oil but did not affect its plasma concentration. Bacterial translocation in protein-restricted rats was not prevented by glutamine or protein powder supplementation. CONCLUSION: Glutamine restored gut glutathione stores and villus heights in malnourished rats but had no preventive effect on bacterial translocation in our model. PMID:17569119

  14. Intracellular redox state determines whether nitric oxide is toxic or protective to rat oligodendrocytes in culture.

    PubMed

    Rosenberg, P A; Li, Y; Ali, S; Altiok, N; Back, S A; Volpe, J J

    1999-08-01

    We found that several nitric oxide donors had similar potency in killing mature and immature forms of oligodendrocytes (OLs). Because of the possibility of interaction of nitric oxide with intracellular thiols, we tested the effect of the nitrosonium ion donor S-nitrosylglutathione (SNOG) in OL cultures in the setting of cystine deprivation, which has been shown to cause intracellular glutathione depletion. Surprisingly, the presence of 200 microM SNOG completely protected OLs against the toxicity of cystine depletion. This protection appeared to be due to nitric oxide, because it could be blocked by hemoglobin and potentiated by inclusion of superoxide dismutase. We tested the effect of three additional NO* donors and found that protection was not seen with diethylamine NONOate, a donor with a half-life measured in minutes, but was seen with dipropylenetriamine NONOate and diethylaminetriamine NONOate, donors with half-lives measured in hours. This need for donors with longer half-lives for the protective effect suggested that NO* was required when intracellular thiol concentrations were falling, a process evolving over hours in medium depleted of cystine. These studies suggest a novel protective role for nitric oxide in oxidative stress injury and raise the possibility that intracerebral nitric oxide production might be a mechanism of defense against oxidative stress injury in OLs.

  15. Novosphingobium aromaticivorans uses a Nu-class glutathione S-transferase as a glutathione lyase in breaking the β-aryl ether bond of lignin

    PubMed Central

    Kontur, Wayne S.; Bingman, Craig A.; Olmsted, Charles N.; Wassarman, Douglas R.; Ulbrich, Arne; Gall, Daniel L.; Smith, Robert W.; Yusko, Larissa M.; Fox, Brian G.; Noguera, Daniel R.; Coon, Joshua J.; Donohue, Timothy J.

    2018-01-01

    As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the β-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the β-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-β-guaiacyl ether (GGE) and found that Novosphingobium aromaticivorans metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate β-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione S-transferase NaGSTNu is the only enzyme needed to remove glutathione from both (R)- and (S)-GS-HPV in N. aromaticivorans. We solved the crystal structure of NaGSTNu and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism (Thr51, Tyr166, and Tyr224) were found to be critical for the lyase reaction. We also found that Nu-class GSTs from Sphingobium sp. SYK-6 (which can also break the β-aryl ether bond) and Escherichia coli (which cannot break the β-aryl ether bond) can also cleave (R)- and (S)-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione S-transferases. PMID:29449375

  16. CMOS nanoelectrode array for all-electrical intracellular electrophysiological imaging

    NASA Astrophysics Data System (ADS)

    Abbott, Jeffrey; Ye, Tianyang; Qin, Ling; Jorgolli, Marsela; Gertner, Rona S.; Ham, Donhee; Park, Hongkun

    2017-05-01

    Developing a new tool capable of high-precision electrophysiological recording of a large network of electrogenic cells has long been an outstanding challenge in neurobiology and cardiology. Here, we combine nanoscale intracellular electrodes with complementary metal-oxide-semiconductor (CMOS) integrated circuits to realize a high-fidelity all-electrical electrophysiological imager for parallel intracellular recording at the network level. Our CMOS nanoelectrode array has 1,024 recording/stimulation 'pixels' equipped with vertical nanoelectrodes, and can simultaneously record intracellular membrane potentials from hundreds of connected in vitro neonatal rat ventricular cardiomyocytes. We demonstrate that this network-level intracellular recording capability can be used to examine the effect of pharmaceuticals on the delicate dynamics of a cardiomyocyte network, thus opening up new opportunities in tissue-based pharmacological screening for cardiac and neuronal diseases as well as fundamental studies of electrogenic cells and their networks.

  17. 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.

  18. 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

  19. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

  20. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

  1. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

  2. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

  3. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

  4. Mechanism-based biomarker gene sets for glutathione depletion-related hepatotoxicity in rats

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

    Gao Weihua; Mizukawa, Yumiko; Nakatsu, Noriyuki

    Chemical-induced glutathione depletion is thought to be caused by two types of toxicological mechanisms: PHO-type glutathione depletion [glutathione conjugated with chemicals such as phorone (PHO) or diethyl maleate (DEM)], and BSO-type glutathione depletion [i.e., glutathione synthesis inhibited by chemicals such as L-buthionine-sulfoximine (BSO)]. In order to identify mechanism-based biomarker gene sets for glutathione depletion in rat liver, male SD rats were treated with various chemicals including PHO (40, 120 and 400 mg/kg), DEM (80, 240 and 800 mg/kg), BSO (150, 450 and 1500 mg/kg), and bromobenzene (BBZ, 10, 100 and 300 mg/kg). Liver samples were taken 3, 6, 9 andmore » 24 h after administration and examined for hepatic glutathione content, physiological and pathological changes, and gene expression changes using Affymetrix GeneChip Arrays. To identify differentially expressed probe sets in response to glutathione depletion, we focused on the following two courses of events for the two types of mechanisms of glutathione depletion: a) gene expression changes occurring simultaneously in response to glutathione depletion, and b) gene expression changes after glutathione was depleted. The gene expression profiles of the identified probe sets for the two types of glutathione depletion differed markedly at times during and after glutathione depletion, whereas Srxn1 was markedly increased for both types as glutathione was depleted, suggesting that Srxn1 is a key molecule in oxidative stress related to glutathione. The extracted probe sets were refined and verified using various compounds including 13 additional positive or negative compounds, and they established two useful marker sets. One contained three probe sets (Akr7a3, Trib3 and Gstp1) that could detect conjugation-type glutathione depletors any time within 24 h after dosing, and the other contained 14 probe sets that could detect glutathione depletors by any mechanism. These two sets, with appropriate

  5. 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

  6. The anthocyanin cyanidin-3-O-β-glucoside, a flavonoid, increases hepatic glutathione synthesis and protects hepatocytes against reactive oxygen species during hyperglycemia: Involvement of a cAMP-PKA-dependent signaling pathway.

    PubMed

    Zhu, Wei; Jia, Qianju; Wang, Yun; Zhang, Yuhua; Xia, Min

    2012-01-15

    Enhanced oxidative stress due to high glucose contributes to pathological changes in diabetes-related liver complications. Reducing oxidative stress may alleviate these pathogenic processes. Anthocyanin, a natural antioxidant, has been reported to reduce intracellular reactive oxygen species (ROS) levels but the mechanism of this reduction is not fully understood. The glutathione (GSH) antioxidant system is critical for counteracting oxidative stress-induced intracellular injury. In this study, we evaluated the mechanism of the anthocyanin-mediated regulation of GSH synthesis and reduction in intracellular ROS levels. We observed that treatment of human HepG2 cells with the anthocyanin C3G significantly reduced ROS levels induced by high glucose. C3G incubation increased glutamate-cysteine ligase expression, which in turn mediated the reduction in ROS levels. However, the upregulation of glutamate-cysteine ligase catalytic subunit (Gclc) expression by C3G occurred independent of the Nrf1/2 transcription factors. Notably, the cAMP-response element binding protein (CREB) was identified as the target transcription factor involved in the C3G-mediated upregulation of Gclc expression. C3G increased phosphorylation of CREB through protein kinase A (PKA) activation, which induced a CREB-mediated upregulation of Gclc transcription. In vivo, treatment with C3G increased the GSH synthesis in the liver of diabetic db/db mice through PKA-CREB-dependent induction of Gclc expression. Finally, oxidative stress determined by lipid peroxidation, neutrophil infiltration, and hepatic steatosis was attenuated in C3G-treated db/db mice. Our results demonstrate that the anthocyanin C3G has an effect of activating GSH synthesis through a novel antioxidant defense mechanism against excessive ROS production, contributing to the prevention of hyperglycemia-induced hepatic oxidative damage. Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.

  7. Properties of glutathione release observed during reduction of organic hydroperoxide, demethylation of aminopyrine and oxidation of some substances in perfused rat liver, and their implications for the physiological function of catalase.

    PubMed Central

    Oshino, N; Chance, B

    1977-01-01

    The enhanced reduction of t-butyl hydroperoxide by glutathione peroxidase is accompanied by a decrease in the cellular concentration of both glutathione and NADPH in isolated liver cells, resulting in the release of GSSG (oxidized glutathione) from the perfused rat liver. This phenomenon, first reported by H. Sies, C. Gerstenecker, H. Menzel & L. Flohé (1972) (FEBS Lett. 27, 171-175), can be observed under a variety of conditions, not only with the acceleration of the glutathione peroxidase reaction by organic peroxides, but also during the oxidation of glycollate and benzylamine, during demethylation of aminopyrine in the liver of the phenobarbital-pretreated rat and during oxidation of uric acid in the liver of the starved rat pretreated with 3-amino-1,2,4-triazole. The rate of release of GSSG is altered markedly by changes in the metabolic conditions which affect the rate of hepatic NADPH generation. Thus, regardless of whether achieved by enhanced oxidation of glutathione by glutathione peroxidase or by oxidation of NADPH through other metabolic pathways, an increase in the cellular concentration of GSSG appears to facilitate its release. It has been found that, in addition to the hexose monophosphate shunt, the mitochondrial NADH-NADP+ transhydrogenase reaction plays an important role in supplying reducing equivalents to the glutathione peroxidase reaction and in maintaining the cellular oxidation-reduction state of the nicotinamide nucleotides. Spectrophotometric analysis of the steady-state concentration of the catalase-H2O2 intermediate with simultaneous measurement of the rate of release of GSSG leads to the conclusion that intracellular compartmentation of catalase in the peroxisomes and glutathione peroxidase in the cytosol and mitochondria distinguishes the reactivities of these enzymes one from the other, and facilitates their effective cooperation in hydroperoxide metabolism in the liver. PMID:17386

  8. Lymphocyte DNA damage and plasma antioxidant status in Korean subclinical hypertensive patients by glutathione S-transferase polymorphism

    PubMed Central

    Han, Jeong-Hwa; Lee, Hye-Jin; Choi, Hee Jeong; Yun, Kyung Eun

    2017-01-01

    BACKGROUND/OBJECTIVES Glutathione S-transferase (GST) forms a multigene family of phase II detoxification enzymes which are involved in the detoxification of xenobiotics by conjugating substances with glutathione. The aim of this study is to assess the antioxidative status and the degree of DNA damage in the subclinical hypertensive patients in Korea using glutathione S-transferase polymorphisms. SUBJECTS/METHODS We examined whether DNA damage and antioxidative status show a difference between GSTM1 or GSTT1 genotype in 227 newly diagnosed, untreated (systolic blood pressure (BP) ≥ 130 mmHg or diastolic BP ≥ 85 mmHg) subclinical hypertensive patients and 130 normotensive subjects (systolic BP < 120 mmHg and diastolic BP < 80 mmHg). From the blood of the subjects, the degree of the DNA damage in lymphocyte, the activities of erythrocyte superoxide dismutase, the catalase, and the glutathione peroxidase, the level of glutathione, plasma total radical-trapping antioxidant potential (TRAP), anti-oxidative vitamins, as well as plasma lipid profiles and conjugated diene (CD) were analyzed. RESULTS Of the 227 subjects studied, 68.3% were GSTM1 null genotype and 66.5% were GSTT1 null genotype. GSTM1 null genotype had an increased risk of hypertension (OR: 2.104, CI: 1.38-3.35), but no significant association in GSTT1 null genotype (OR 0.982, CI: 0.62-1.55). No difference in erythrocyte activities of superoxide dismutase, catalase, or glutathione peroxidase, and plasma TRAP, CD, lipid profiles, and GSH levels were observed between GSTM1 or GSTT1 genotype. Plasma levels of α-tocopherol increased significantly in GSTT1 wild genotype (P < 0.05); however, plasma level of β-carotene increased significantly in GSTT1 null genotype (P < 0.01). DNA damage assessed by the Comet assay was significantly higher in GSTM1 null genotype than wild genotype (P < 0.05). CONCLUSIONS These results confirm the association between GSTM1 null genotype and risk of hypertension as they suggest

  9. Induction of glutathione synthesis and glutathione reductase activity by abiotic stresses in maize and wheat.

    PubMed

    Kocsy, Gábor; Szalai, Gabriella; Galiba, Gábor

    2002-06-21

    The effect of different abiotic stresses (extreme temperatures and osmotic stress) on the synthesis of glutathione and hydroxymethylglutathione, on the ratio of the reduced to oxidised forms of these thiols (GSH/GSSG, hmGSH/hmGSSG), and on the glutathione reductase (GR) activity was studied in maize and wheat genotypes having different sensitivity to low temperature stress. Cold treatment induced a greater increase in total glutathione (TG) content and in GR activity in tolerant genotypes of both species than in sensitive ones. The GSH/GSSG and hmGSH/hmGSSG ratios were increased by this treatment only in the frost-tolerant wheat variety. High-temperature stress increased the TG content and the GSH/GSSG ratio only in the chilling-sensitive maize genotype, but GR activity was greater after this treatment in both maize genotypes. Osmotic stress resulted in a great increase in the TG content in wheat and the GR activity in maize. The amount of total hydroxymethylglutathione increased following all stress treatments. These results indicate the involvement of these antioxidants in the stress responses of wheat and maize.

  10. Interaction of glutathione with bovine serum albumin: Spectroscopy and molecular docking.

    PubMed

    Jahanban-Esfahlan, Ali; Panahi-Azar, Vahid

    2016-07-01

    This study aims to investigate the interaction between glutathione and bovine serum albumin (BSA) using ultraviolet-visible (UV-vis) absorption, fluorescence spectroscopies under simulated physiological conditions (pH 7.4) and molecular docking methods. The results of fluorescence spectroscopy indicated that the fluorescence intensity of BSA was decreased considerably upon the addition of glutathione through a static quenching mechanism. The fluorescence quenching obtained was related to the formation of BSA-glutathione complex. The values of KSV, Ka and Kb for the glutathione and BSA interaction were in the order of 10(5). The thermodynamic parameters including enthalpy change (ΔH), entropy change (ΔS) and also Gibb's free energy (ΔG) were determined using Van't Hoff equation. These values showed that hydrogen bonding and van der Waals forces were the main interactions in the binding of glutathione to BSA and the stabilization of the complex. Also, the interaction of glutathione and BSA was spontaneous. The effects of glutathione on the BSA conformation were determined using UV-vis spectroscopy. Moreover, glutathione was docked in BSA using ArgusLab as a molecular docking program. It was recognized that glutathione binds within the sub-domain IIA pocket in domain II of BSA. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. [Association between intracellular zinc levels and nutritional status in HIV-infected and uninfected children exposed to the virus].

    PubMed

    Gómez G, Erika María; Maldonado C, María Elena; Rojas L, Mauricio; Posada J, Gladys

    2015-01-01

    Malnutrition, growth retardation and opportunistic infections outlast the metabolic, immune and gastrointestinal disorders produced by HIV. Zinc deficiency has been associated with deteriorating nutritional status, growth failure, and risk of infection. The aim of this study is to determine the association between zinc levels in peripheral blood mononuclear cells (PBMC) and the nutritional status of HIV-infected and uninfected children exposed to the virus. An analytical, observational, cross-sectional study was conducted on 17 infected and 17 exposed children, aged 2-10 years. Anthropometric measurements, clinical and nutritional history, 24h recall, measurement of physical activity, and zinc in PBMC by flow cytometry analysis were recorded. Height according to age, energy consumption and adequacy of energy, protein and dietary zinc were significantly higher in children exposed to the virus compared to those infected with HIV (P <.05). No significant differences were found in BMI, levels of zinc in monocytes, CD4 + and CD4- lymphocytes between the two study groups (P >.05). However, the median levels of zinc in monocytes of infected patients was higher (218.6) compared to the control group (217.0). No association was found between zinc intake and levels of intracellular zinc. The deterioration of nutritional status and growth retardation in children were associated with HIV, but not with the levels of intracellular zinc. The dietary intake of this nutrient was not associated with levels of zinc in monocytes or CD4 + and CD4- lymphocytes. Copyright © 2015. Publicado por Elsevier España, S.L.U.

  12. Characterization of Intracellular and Extracellular Saxitoxin Levels in Both Field and Cultured Alexandrium spp. Samples from Sequim Bay, Washington

    PubMed Central

    Lefebvre, Kathi A.; Bill, Brian D.; Erickson, Aleta; Baugh, Keri A.; O’Rourke, Lohna; Costa, Pedro R.; Nance, Shelly; Trainer, Vera L.

    2008-01-01

    Traditionally, harmful algal bloom studies have primarily focused on quantifying toxin levels contained within the phytoplankton cells of interest. In the case of paralytic shellfish poisoning toxins (PSTs), intracellular toxin levels and the effects of dietary consumption of toxic cells by planktivores have been well documented. However, little information is available regarding the levels of extracellular PSTs that may leak or be released into seawater from toxic cells during blooms. In order to fully evaluate the risks of harmful algal bloom toxins in the marine food web, it is necessary to understand all potential routes of exposure. In the present study, extracellular and intracellular PST levels were measured in field seawater samples (collected weekly from June to October 2004–2007) and in Alexandrium spp. culture samples isolated from Sequim Bay, Washington. Measurable levels of intra- and extra-cellular toxins were detected in both field and culture samples via receptor binding assay (RBA) and an enzyme-linked immunosorbent assay (ELISA). Characterization of the PST toxin profile in the Sequim Bay isolates by pre-column oxidation and HPLC-fluorescence detection revealed that gonyautoxin 1 and 4 made up 65 ± 9.7 % of the total PSTs present. Collectively, these data confirm that extracellular PSTs are present during blooms of Alexandrium spp. in the Sequim Bay region. PMID:18728762

  13. Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476.

    PubMed

    Srivastava, Preeti; Deb, J K

    2002-07-02

    A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coli was observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.

  14. Chronic aspartame intake causes changes in the trans-sulphuration pathway, glutathione depletion and liver damage in mice.

    PubMed

    Finamor, Isabela; Pérez, Salvador; Bressan, Caroline A; Brenner, Carlos E; Rius-Pérez, Sergio; Brittes, Patricia C; Cheiran, Gabriele; Rocha, Maria I; da Veiga, Marcelo; Sastre, Juan; Pavanato, Maria A

    2017-04-01

    No-caloric sweeteners, such as aspartame, are widely used in various food and beverages to prevent the increasing rates of obesity and diabetes mellitus, acting as tools in helping control caloric intake. Aspartame is metabolized to phenylalanine, aspartic acid, and methanol. Our aim was to study the effect of chronic administration of aspartame on glutathione redox status and on the trans-sulphuration pathway in mouse liver. Mice were divided into three groups: control; treated daily with aspartame for 90 days; and treated with aspartame plus N-acetylcysteine (NAC). Chronic administration of aspartame increased plasma alanine aminotransferase (ALT) and aspartate aminotransferase activities and caused liver injury as well as marked decreased hepatic levels of reduced glutathione (GSH), oxidized glutathione (GSSG), γ-glutamylcysteine ​​(γ-GC), and most metabolites of the trans-sulphuration pathway, such as cysteine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine ​​(SAH). Aspartame also triggered a decrease in mRNA and protein levels of the catalytic subunit of glutamate cysteine ligase (GCLc) and cystathionine γ-lyase, and in protein levels of methionine adenosyltransferase 1A and 2A. N-acetylcysteine prevented the aspartame-induced liver injury and the increase in plasma ALT activity as well as the decrease in GSH, γ-GC, cysteine, SAM and SAH levels and GCLc protein levels. In conclusion, chronic administration of aspartame caused marked hepatic GSH depletion, which should be ascribed to GCLc down-regulation and decreased cysteine levels. Aspartame triggered blockade of the trans-sulphuration pathway at two steps, cystathionine γ-lyase and methionine adenosyltransferases. NAC restored glutathione levels as well as the impairment of the trans-sulphuration pathway. Copyright © 2017. Published by Elsevier B.V.

  15. Glutathione Decrement Drives Thermogenic Program In Adipose Cells.

    PubMed

    Lettieri Barbato, Daniele; Tatulli, Giuseppe; Maria Cannata, Stefano; Bernardini, Sergio; Aquilano, Katia; Ciriolo, Maria R

    2015-08-11

    Adipose tissue metabolically adapts to external stimuli. We demonstrate that the induction of the thermogenic program in white adipocytes, through cold exposure in mice or in vitro adrenergic stimulation, is accompanied by a decrease in the intracellular content of glutathione (GSH). Moreover, the treatment with a GSH depleting agent, buthionine sulfoximine (BSO), recapitulates the effect of cold exposure resulting in the induction of thermogenic program. In particular, BSO treatment leads to enhanced uncoupling respiration as demonstrated by increased expression of thermogenic genes (e.g. Ucp1, Ppargc1a), augmented oxygen consumption and decreased mitochondrial transmembrane potential. Buffering GSH decrement by pre-treatment with GSH ester prevents the up-regulation of typical markers of uncoupling respiration. We demonstrate that FoxO1 activation is responsible for the conversion of white adipocytes into a brown phenotype as the "browning" effects of BSO are completely abrogated in cells down-regulating FoxO1. In mice, the BSO-mediated up-regulation of uncoupling genes results in weight loss that is at least in part ascribed to adipose tissue mass reduction. The induction of thermogenic program has been largely proposed to counteract obesity-related diseases. Based on these findings, we propose GSH as a novel therapeutic target to increase energy expenditure in adipocytes.

  16. Glutathione Decrement Drives Thermogenic Program In Adipose Cells

    PubMed Central

    Lettieri Barbato, Daniele; Tatulli, Giuseppe; Maria Cannata, Stefano; Bernardini, Sergio; Aquilano, Katia; Ciriolo, Maria R.

    2015-01-01

    Adipose tissue metabolically adapts to external stimuli. We demonstrate that the induction of the thermogenic program in white adipocytes, through cold exposure in mice or in vitro adrenergic stimulation, is accompanied by a decrease in the intracellular content of glutathione (GSH). Moreover, the treatment with a GSH depleting agent, buthionine sulfoximine (BSO), recapitulates the effect of cold exposure resulting in the induction of thermogenic program. In particular, BSO treatment leads to enhanced uncoupling respiration as demonstrated by increased expression of thermogenic genes (e.g. Ucp1, Ppargc1a), augmented oxygen consumption and decreased mitochondrial transmembrane potential. Buffering GSH decrement by pre-treatment with GSH ester prevents the up-regulation of typical markers of uncoupling respiration. We demonstrate that FoxO1 activation is responsible for the conversion of white adipocytes into a brown phenotype as the “browning” effects of BSO are completely abrogated in cells down-regulating FoxO1. In mice, the BSO-mediated up-regulation of uncoupling genes results in weight loss that is at least in part ascribed to adipose tissue mass reduction. The induction of thermogenic program has been largely proposed to counteract obesity-related diseases. Based on these findings, we propose GSH as a novel therapeutic target to increase energy expenditure in adipocytes. PMID:26260892

  17. The Triple Roles of Glutathione for a DNA-Cleaving DNAzyme and Development of a Fluorescent Glutathione/Cu2+-Dependent DNAzyme Sensor for Detection of Cu2+ in Drinking Water.

    PubMed

    Wang, Shijin; Liu, Chengcheng; Li, Guiying; Sheng, Yongjie; Sun, Yanhong; Rui, Hongyue; Zhang, Jin; Xu, Jiacui; Jiang, Dazhi

    2017-03-24

    Pistol-like DNAzyme (PLDz) is an oxidative DNA-cleaving catalytic DNA with ascorbic acid as cofactor. Herein, glutathione was induced into the reaction system to maintain reduced ascorbic acid levels for higher efficient cleavage. However, data indicated that glutathione played triple roles in PLDz-catalyzed reactions. Glutathione alone had no effect on PLDz, and showed inhibitory effect on ascorbic acid-induced PLDz catalysis, but exhibited stimulating effect on Cu 2+ -promoted self-cleavage of PLDz. Further analysis of the effect of glutathione/Cu 2+ on PLDz indicated that H 2 O 2 played a key role in PLDz catalysis. Finally, we developed a fluorescent Cu 2+ sensor (PL-Cu 1.0) based on the relationship between glutathione/Cu 2+ and catalytic activity of PLDz. The fluorescent intensity showed a linear response toward the logarithm concentration of Cu 2+ over the range from 80 nM to 30 μM, with a detection limit of 21.1 nM. PL-Cu 1.0 provided only detection of Cu 2+ over other divalent metal ions. Ca 2+ and Mg 2+ could not interfere with Cu 2+ detection even at a 1000-fold concentration. We further applied PL-Cu 1.0 for Cu 2+ detection in tap and bottled water. Water stored in copper taps overnight had relatively high Cu 2+ concentrations, with a maximum 22.3 μM. Trace Cu 2+ (52.2 nM) in deep spring was detected among the tested bottled water. Therefore, PL-Cu 1.0 is feasible to detect Cu 2+ in drinking water, with a practical application.

  18. Unearthing the secrets of mitochondrial ROS and glutathione in bioenergetics.

    PubMed

    Mailloux, Ryan J; McBride, Skye L; Harper, Mary-Ellen

    2013-12-01

    During the cellular oxidation of fuels, electrons are used to power the proton pumps of the mitochondrial electron transport chain (ETC) and ultimately drive ATP synthesis and the reduction of molecular oxygen to water. During these oxidative processes, some electrons can 'spin off' during fuel oxidation and electron transport to univalently reduce O2, forming reactive oxygen species (ROS). In excess, ROS can be detrimental; however, at low concentrations oxyradicals are essential signaling molecules. Mitochondria thus use a battery of systems to finely control types and levels of ROS, including antioxidants. Several antioxidant systems depend on glutathione. Here, we review mitochondrial ROS homeostatic systems, including emerging knowledge about roles of glutathione in redox balance and the control of protein function by post-translational modification. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. 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.

  20. Functional Analysis of Arabidopsis Mutants Points to Novel Roles for Glutathione in Coupling H2O2 to Activation of Salicylic Acid Accumulation and Signaling

    PubMed Central

    Han, Yi; Chaouch, Sejir; Mhamdi, Amna; Queval, Guillaume; Zechmann, Bernd

    2013-01-01

    Abstract Aims: Through its interaction with H2O2, glutathione is a candidate for transmission of signals in plant responses to pathogens, but identification of signaling roles is complicated by its antioxidant function. Using a genetic approach based on a conditional catalase-deficient Arabidopsis mutant, cat2, this study aimed at establishing whether GSH plays an important functional role in the transmission of signals downstream of H2O2. Results: Introducing the cad2 or allelic mutations in the glutathione synthesis pathway into cat2 blocked H2O2-triggered GSH oxidation and accumulation. While no effects on NADP(H) or ascorbate were observed, and H2O2-induced decreases in growth were maintained, blocking GSH modulation antagonized salicylic acid (SA) accumulation and SA-dependent responses. Other novel double and triple mutants were produced and compared with cat2 cad2 at the levels of phenotype, expression of marker genes, nontargeted metabolite profiling, accumulation of SA, and bacterial resistance. Most of the effects of the cad2 mutation on H2O2-triggered responses were distinct from those produced by mutations for GLUTATHIONE REDUCTASE1 (GR1) or NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), and were linked to compromised induction of ISOCHORISMATE SYNTHASE1 (ICS1) and ICS1-dependent SA accumulation. Innovation: A novel genetic approach was used in which GSH content or antioxidative capacity was independently modified in an H2O2 signaling background. Analysis of new double and triple mutants allowed us to infer previously undescribed regulatory roles for GSH. Conclusion: In parallel to its antioxidant role, GSH acts independently of NPR1 to allow increased intracellular H2O2 to activate SA signaling, a key defense response in plants. Antioxid. Redox Signal. 18, 2106–2121. PMID:23148658

  1. Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft.

    PubMed

    Yamamoto, Yutaro; Tomiyama, Arata; Sasaki, Nobuyoshi; Yamaguchi, Hideki; Shirakihara, Takuya; Nakashima, Katsuhiko; Kumagai, Kosuke; Takeuchi, Satoru; Toyooka, Terushige; Otani, Naoki; Wada, Kojiro; Narita, Yoshitaka; Ichimura, Koichi; Sakai, Ryuichi; Namba, Hiroki; Mori, Kentaro

    2018-01-01

    Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MβCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MβCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MβCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Dimethylfumarate induces cell cycle arrest and apoptosis via regulating intracellular redox systems in HeLa cells.

    PubMed

    Han, Guocan; Zhou, Qiang

    2016-12-01

    Dimethylfumarate (DMF) is cytotoxic to several kinds of cells and serves as an anti-tumor drug. This study was designed to investigate the effects and underlying mechanism of DMF on cervical cancer cells. HeLa cells were cultured and treated with 0, 50, 100, 150, and 200 μM DMF, respectively. After 24 h, cell growth was evaluated using Cell Counting Kit-8 (CCK-8) assay and the cell cycle was examined using flow cytometry. In addition, cell apoptosis was detected by Annexin V/propidium iodide (PI) staining and the expressions of caspase-3 and poly-ADP-ribose polymerase (PARP) were detected using western blotting. The redox-related factors were then assessed. Furthermore, all of the indicators were detected in HeLa cells after combined treatment of DMF and N-acetyl-L-cysteine (NAC, an oxygen-free radical scavenger). The cell number and cell growth of HeLa were obviously inhibited by DMF in a dose-dependent manner, as the cell cycle was arrested at G0/G1 phase (P < 0.05). The apoptotic HeLa cells were markedly increased, and the expression levels of caspase-3 and PARP were significantly increased in a DMF concentration-dependent way (P < 0.05). Meanwhile, loss of △Ψm, increase in reactive oxygen species and O 2 ·- , and the decrease in catalase activity and glutathione (GSH) level were found after DMF treatment (P < 0.05). All these changes were significantly attenuated and even completely disappeared by adding NAC (P < 0.05). In conclusion, the cytotoxicity of DMF on cell proliferation and apoptosis of HeLa cells was mainly related to the intracellular redox systems by depletion of intracellular GSH.

  3. Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability.

    PubMed

    Axarli, Irine; Muleta, Abdi W; Chronopoulou, Evangelia G; Papageorgiou, Anastassios C; Labrou, Nikolaos E

    2017-01-01

    Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher k cat /K m and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Šand 2.3Šresolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. DNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. Such an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Structure-activity relationships for the impact of selected isothiazol-3-one biocides on glutathione metabolism and glutathione reductase of the human liver cell line Hep G2.

    PubMed

    Arning, Jürgen; Dringen, Ralf; Schmidt, Maike; Thiessen, Anette; Stolte, Stefan; Matzke, Marianne; Bottin-Weber, Ulrike; Caesar-Geertz, Birgit; Jastorff, Bernd; Ranke, Johannes

    2008-04-18

    To investigate the toxic mode of action of isothiazol-3-one biocides the four compounds N-methylisothiazol-3-one (MIT), 5-chloro-N-methylisothiazol-3-one (CIT), N-octylisothiazol-3-one (OIT) and 4,5-dichloro-N-octylisothiazol-3-one (DCOIT) were purified and tested as single chemical entities for their effects on the human hepatoblastoma cell line Hep G2 and on isolated and cellular glutathione reductase GR). The two chlorinated substances CIT and DCOIT significantly decreased the amount of total cellular glutathione (GSx) in a dose and time dependent manner. Concomitantly, an increase in the level of oxidised glutathione (GSSG) was observed. The resulting shift in the GSH/GSSG ratio entailing the breakdown of the cellular thiol reduction potential was accompanied by necrotic morphological changes like swelling of the plasma membrane and subsequent lysis of the cells. Additionally, CIT and DCOIT were found to inhibit cellular GR in the cells in a concentration dependent manner. The T-SAR-based (thinking in terms of structure-activity relationships) comparison of the chlorine-substituted structures CIT and DCOIT with their non-chlorinated and less active analogues MIT and OIT identified the chlorine substituents and the resulting reaction mechanisms to be the key structural mediators of the observed toxic effects. Furthermore, differences in the activity of both chlorinated substances could be explained using the T-SAR approach to link the lipophilicity and the intrinsic glutathione-reactivity of the compounds to the expected target site concentrations inside the cells.

  5. Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level.

    PubMed

    Banerjee, S; Ecavade, A; Rao, A R

    1993-02-01

    The effect of the oil from the wood of Santalum album on glutathione S-transferase (GST) activity and acid soluble sulphydryl (SH) levels in the liver of adult male Swiss albino mice was investigated. Oral feeding by gavage to mice each day with 5 and 15 microliters sandalwood oil for 10 and 20 days exhibited an increase in GST activity in time- and dose-responsive manners. Feeding a dose of 5 microliters sandalwood oil for 10 and 20 days caused, respectively, a 1.80-fold (P < 0.001) and 1.93-fold (P < 0.001) increase in GST enzyme activity, while feeding a dose of 15 microliters of the oil per day for 10 and 20 days induced, respectively, 4.73-fold (P < 0.001) and 6.10-fold (P < 0.001) increases in the enzyme's activity. In addition, there were 1.59-fold (P < 0.001) and 1.57 (P < 0.001) increases in acid-soluble SH levels in the hepatic tissue of the mice following feeding of the oil at the dose levels of 5 and 15 microliters for 10 days. Furthermore, mice fed on a diet containing 1% 2(3)-butyl-4-hydroxyanisole (positive control) also showed an increase in hepatic GST activity and SH levels. Enhancement of GST activity and acid-soluble SH levels are suggestive of a possible chemopreventive action of sandalwood oil on carcinogenesis through a blocking mechanism.

  6. Effect of fish oil on glutathione redox system in multiple sclerosis

    PubMed Central

    Sorto-Gomez, Tania E; Ortiz, Genaro G; Pacheco-Moises, Fermín P; Torres-Sanchez, Erandis D; Ramirez-Ramirez, Viridiana; Macias-Islas, Miguel A; de la Rosa, Alfredo Celis; Velázquez-Brizuela, Irma E

    2016-01-01

    Multiple sclerosis (MS) is a chronic, inflammatory and autoimmune disease of the central nervous system. Dysregulation of glutathione homeostasis and alterations in glutathione-dependent enzyme activities are implicated in the induction and progression of MS. Evidence suggests that Omega-3 polyunsaturated fatty acids (PUFAs) have anti-inflammatory, antioxidant and neuroprotective effects. The aim of the present work was to evaluate the effect of fish oil on the activity of glutathione reductase (GR), content of reduced and oxidized glutathione, and GSH/GSSG ratio in MS. 50 patients with relapsing-remitting MS were enrolled. The experimental group received orally 4 g/day of fish oil for 12 months. Fish oil supplementation resulted in a significant increase in n-3 fatty acids and a decrease n-6 fatty acids. No differences in glutathione reductase activity, content of reduced and oxidized glutathione, and GSH/GSSG ratio were found. Conclusion: Glutathione reductase activity was not significantly different between the groups; however, fish oil supplementation resulted in smaller increase in GR compared with control group, suggesting a possible effect on antioxidant defence mechanisms. PMID:27335704

  7. Butyrate influences intracellular levels of adenine and adenine derivatives in the fungus Penicillium restrictum.

    PubMed

    Zutz, Christoph; Chiang, Yi Ming; Faehnrich, Bettina; Bacher, Markus; Hellinger, Roland; Kluger, Bernhard; Wagner, Martin; Strauss, Joseph; Rychli, Kathrin

    2017-04-01

    Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians including the inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence that butyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation. Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linked to its toxicity at a high concentration. In fungi there are indications that butyrate induces the production of secondary metabolites potentially via inhibition of histone deacetylases. However, information about the influence of butyrate on growth, primary metabolite production and metabolism, besides lipid catabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as a susceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activity was detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrate ranging from low (0.001mM) to high (30mM), potentially toxic, concentrations on biomass and antimicrobial activity. Butyrate at high concentrations (3 and 30mM) significantly reduced the fungal biomass. In contrast P. restrictum treated with 0.03mM of butyrate showed the highest antimicrobial activity. We isolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictum cellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivate adenosine. Production of all three compounds was increased at low butyrate concentrations. Furthermore we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP, an important signalling molecule in fungi and various organisms. In conclusion butyrate treatment increases the intracellular levels of adenine and its respective derivatives. Copyright © 2017 Elsevier GmbH. All rights reserved.

  8. Glutathione and glutamate in schizophrenia: a 7T MRS study.

    PubMed

    Kumar, Jyothika; Liddle, Elizabeth B; Fernandes, Carolina C; Palaniyappan, Lena; Hall, Emma L; Robson, Siân E; Simmonite, Molly; Fiesal, Jan; Katshu, Mohammad Z; Qureshi, Ayaz; Skelton, Michael; Christodoulou, Nikolaos G; Brookes, Matthew J; Morris, Peter G; Liddle, Peter F

    2018-06-22

    In schizophrenia, abnormal neural metabolite concentrations may arise from cortical damage following neuroinflammatory processes implicated in acute episodes. Inflammation is associated with increased glutamate, whereas the antioxidant glutathione may protect against inflammation-induced oxidative stress. We hypothesized that patients with stable schizophrenia would exhibit a reduction in glutathione, glutamate, and/or glutamine in the cerebral cortex, consistent with a post-inflammatory response, and that this reduction would be most marked in patients with "residual schizophrenia", in whom an early stage with positive psychotic symptoms has progressed to a late stage characterized by long-term negative symptoms and impairments. We recruited 28 patients with stable schizophrenia and 45 healthy participants matched for age, gender, and parental socio-economic status. We measured glutathione, glutamate and glutamine concentrations in the anterior cingulate cortex (ACC), left insula, and visual cortex using 7T proton magnetic resonance spectroscopy (MRS). Glutathione and glutamate were significantly correlated in all three voxels. Glutamine concentrations across the three voxels were significantly correlated with each other. Principal components analysis (PCA) produced three clear components: an ACC glutathione-glutamate component; an insula-visual glutathione-glutamate component; and a glutamine component. Patients with stable schizophrenia had significantly lower scores on the ACC glutathione-glutamate component, an effect almost entirely leveraged by the sub-group of patients with residual schizophrenia. All three metabolite concentration values in the ACC were significantly reduced in this group. These findings are consistent with the hypothesis that excitotoxicity during the acute phase of illness leads to reduced glutathione and glutamate in the residual phase of the illness.

  9. Glutathione protects Candida albicans against horseradish volatile oil.

    PubMed

    Bertóti, Regina; Vasas, Gábor; Gonda, Sándor; Nguyen, Nhat Minh; Szőke, Éva; Jakab, Ágnes; Pócsi, István; Emri, Tamás

    2016-10-01

    Horseradish essential oil (HREO; a natural mixture of different isothiocyanates) had strong fungicide effect against Candida albicans both in volatile and liquid phase. In liquid phase this antifungal effect was more significant than those of its main components allyl, and 2-phenylethyl isothiocyanate. HREO, at sublethal concentration, induced oxidative stress which was characterized with elevated superoxide content and up-regulated specific glutathione reductase, glutathione peroxidase, catalase and superoxide dismutase activities. Induction of specific glutathione S-transferase activities as marker of glutathione (GSH) dependent detoxification was also observed. At higher concentration, HREO depleted the GSH pool, increased heavily the superoxide production and killed the cells rapidly. HREO and the GSH pool depleting agent, 1-chlore-2,4-dinitrobenzene showed strong synergism when they were applied together to kill C. albicans cells. Based on all these, we assume that GSH metabolism protects fungi against isothiocyanates. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Rutin improves spatial memory in Alzheimer's disease transgenic mice by reducing Aβ oligomer level and attenuating oxidative stress and neuroinflammation.

    PubMed

    Xu, Peng-Xin; Wang, Shao-Wei; Yu, Xiao-Lin; Su, Ya-Jing; Wang, Teng; Zhou, Wei-Wei; Zhang, He; Wang, Yu-Jiong; Liu, Rui-Tian

    2014-05-01

    Alzheimer's disease (AD) is a progressive, neurodegenerative disease characterized by extracellular β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles in the brain. Aβ aggregation is closely associated with neurotoxicity, oxidative stress, and neuronal inflammation. The soluble Aβ oligomers are believed to be the most neurotoxic form among all forms of Aβ aggregates. We have previously reported a polyphenol compound rutin that could inhibit Aβ aggregation and cytotoxicity, attenuate oxidative stress, and decrease the production of nitric oxide and proinflammatory cytokines in vitro. In the current study, we investigated the effect of rutin on APPswe/PS1dE9 transgenic mice. Results demonstrated that orally administered rutin significantly attenuated memory deficits in AD transgenic mice, decreased oligomeric Aβ level, increased super oxide dismutase (SOD) activity and glutathione (GSH)/glutathione disulfide (GSSG) ratio, reduced GSSG and malondialdehyde (MDA) levels, downregulated microgliosis and astrocytosis, and decreased interleukin (IL)-1β and IL-6 levels in the brain. These results indicated that rutin is a promising agent for AD treatment because of its antioxidant, anti-inflammatory, and reducing Aβ oligomer activities. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Effect of glutathione aerosol on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis.

    PubMed

    Borok, Z; Buhl, R; Grimes, G J; Bokser, A D; Hubbard, R C; Holroyd, K J; Roum, J H; Czerski, D B; Cantin, A M; Crystal, R G

    1991-07-27

    Idiopathic pulmonary fibrosis (IPF) is characterised by alveolar inflammation, exaggerated release of oxidants, and subnormal concentrations of the antioxidant glutathione in respiratory epithelial lining fluid (ELF). Glutathione (600 mg twice daily for 3 days) was given by aerosol to 10 patients with IPF. Total ELF glutathione rose transiently, ELF oxidised glutathione concentrations increased, and there was a decrease in spontaneous superoxide anion release by alveolar macrophages. Thus, glutathione by aerosol could be a means of reversing the oxidant-antioxidant imbalance in IPF.

  12. 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

  13. Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids.

    PubMed

    Treyer, Andrea; Mateus, André; Wiśniewski, Jacek R; Boriss, Hinnerk; Matsson, Pär; Artursson, Per

    2018-06-04

    Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability ( F ic ) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F ic . The induction of NL did not further increase drug binding but led to altered F ic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.

  14. ChaC2, an Enzyme for Slow Turnover of Cytosolic Glutathione*

    PubMed Central

    Kaur, Amandeep; Gautam, Ruchi; Srivastava, Ritika; Chandel, Avinash; Kumar, Akhilesh; Karthikeyan, Subramanian; Bachhawat, Anand Kumar

    2017-01-01

    Glutathione degradation plays an important role in glutathione and redox homeostasis, and thus it is imperative to understand the enzymes and the mechanisms involved in glutathione degradation in detail. We describe here ChaC2, a member of the ChaC family of γ-glutamylcyclotransferases, as an enzyme that degrades glutathione in the cytosol of mammalian cells. ChaC2 is distinct from the previously described ChaC1, to which ChaC2 shows ∼50% sequence identity. Human and mouse ChaC2 proteins purified in vitro show 10–20-fold lower catalytic efficiency than ChaC1, although they showed comparable Km values (Km of 3.7 ± 0.4 mm and kcat of 15.9 ± 1.0 min−1 toward glutathione for human ChaC2; Km of 2.2 ± 0.4 mm and kcat of 225.2 ± 15 min−1 toward glutathione for human ChaC1). The ChaC1 and ChaC2 proteins also shared the same specificity for reduced glutathione, with no activity against either γ-glutamyl amino acids or oxidized glutathione. The ChaC2 proteins were found to be expressed constitutively in cells, unlike the tightly regulated ChaC1. Moreover, lower eukaryotes have a single member of the ChaC family that appears to be orthologous to ChaC2. In addition, we determined the crystal structure of yeast ChaC2 homologue, GCG1, at 1.34 Å resolution, which represents the first structure of the ChaC family of proteins. The catalytic site is defined by a fortuitous benzoic acid molecule bound to the crystal structure. The mechanism for binding and catalytic activity of this new enzyme of glutathione degradation, which is involved in continuous but basal turnover of cytosolic glutathione, is proposed. PMID:27913623

  15. 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.

  16. Glutathione Metabolism and Parkinson’s Disease

    PubMed Central

    Smeyne, Michelle

    2013-01-01

    It has been established that oxidative stress, defined as the condition when the sum of free radicals in a cell exceeds the antioxidant capacity of the cell, contributes to the pathogenesis of Parkinson’s disease. Glutathione is a ubiquitous thiol tripeptide that acts alone, or in concert with enzymes within cells to reduce superoxide radicals, hydroxyl radicals and peroxynitrites. In this review, we examine the synthesis, metabolism and functional interactions of glutathione, and discuss how this relates to protection of dopaminergic neurons from oxidative damage and its therapeutic potential in Parkinson’s disease. PMID:23665395

  17. Methods for the determination of intracellular levels of ribose phosphates.

    PubMed

    Camici, Marcella; Tozzi, Maria Grazia; Ipata, Piero Luigi

    2006-10-31

    Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway or stem from the phosphorolytic cleavage of the N-glycosidic bond of ribonucleosides. The two major pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, can be readily interconverted by phosphopentomutase. Ribose-5-phosphate is also the direct precursor of 5-phosphoribosyl-1-pyrophosphate, which is used for both de novo and salvage synthesis of nucleotides. On the other hand, the phosphorolysis of deoxyribonucleosides is the major source of deoxyribose phosphates. While the destiny of the nucleobase stemming from nucleoside phosphorolysis has been extensively investigated, the fate of the sugar moiety has been somehow neglected. However, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. Nevertheless, many aspects of pentose phosphate metabolism, and the possible involvement of these compounds in a number of cellular processes still remain obscure. The comprehension of the role played by pentose phosphates may be greatly facilitated by the knowledge of their steady-state intracellular levels and of their changes in response to variations of intra- and extracellular signals.

  18. Avocado oil induces long-term alleviation of oxidative damage in kidney mitochondria from type 2 diabetic rats by improving glutathione status.

    PubMed

    Ortiz-Avila, Omar; Figueroa-García, María Del Consuelo; García-Berumen, Claudia Isabel; Calderón-Cortés, Elizabeth; Mejía-Barajas, Jorge A; Rodriguez-Orozco, Alain R; Mejía-Zepeda, Ricardo; Saavedra-Molina, Alfredo; Cortés-Rojo, Christian

    2017-04-01

    Hyperglycemia and mitochondrial ROS overproduction have been identified as key factors involved in the development of diabetic nephropathy. This has encouraged the search for strategies decreasing glucose levels and long-term improvement of redox status of glutathione, the main antioxidant counteracting mitochondrial damage. Previously, we have shown that avocado oil improves redox status of glutathione in liver and brain mitochondria from streptozotocin-induced diabetic rats; however, the long-term effects of avocado oil and its hypoglycemic effect cannot be evaluated because this model displays low survival and insulin depletion. Therefore, we tested during 1 year the effects of avocado oil on glycemia, ROS levels, lipid peroxidation and glutathione status in kidney mitochondria from type 2 diabetic Goto-Kakizaki rats. Diabetic rats exhibited glycemia of 120-186 mg/dL the first 9 months with a further increase to 250-300 mg/dL. Avocado oil decreased hyperglycemia at intermediate levels between diabetic and control rats. Diabetic rats displayed augmented lipid peroxidation and depletion of reduced glutathione throughout the study, while increased ROS generation was observed at the 3rd and 12th months along with diminished content of total glutathione at the 6th and 12th months. Avocado oil ameliorated all these defects and augmented the mitochondrial content of oleic acid. The beneficial effects of avocado oil are discussed in terms of the hypoglycemic effect of oleic acid and the probable dependence of glutathione transport on lipid peroxidation and thiol oxidation of mitochondrial carriers.

  19. Protoapigenone, a natural derivative of apigenin, induces mitogen-activated protein kinase-dependent apoptosis in human breast cancer cells associated with induction of oxidative stress and inhibition of glutathione S-transferase π.

    PubMed

    Chen, Wen-Ying; Hsieh, Yu-An; Tsai, Ching-I; Kang, Ya-Fei; Chang, Fang-Rong; Wu, Yang-Chang; Wu, Chin-Chung

    2011-12-01

    Protoapigenone, a natural derivative of the flavonoid apigenin, has been shown to exhibit potent antitumor activity in vitro and in vivo; the precise mechanism of action, however, is not fully elucidated. In this study, we investigated and compared the mechanisms by which protoapigenone and apigenin caused cell death in the human breast cancer MDA-MB-231 cells. Flow cytometry analysis revealed that protoapigenone induced apoptosis with 10-fold greater potency than apigenin. Cancer cells treated with protoapigenone resulted in persistent activation of mitogen-activated protein kinase (MAPK) ERK, JNK, and p38, hyperphosphorylation of Bcl-2 and Bcl-xL, and loss of mitochondrial membrane potential (MMP). The MAPK inhibitors effectively prevented the loss of MMP and apoptosis induced by protoapigenone. Treatment of cells with protoapigenone led to increased levels of reactive oxygen species (ROS) and decreased levels of intracellular glutathione. The thiol-antioxidant N-acetylcysteine abolished protoapigenone-induced MAPK activation, mitochondrial dysfunction, and apoptosis. These results suggest that the induction of oxidative stress preceding the activation of MAPK is required to initiate the mitochondria-mediated apoptosis induced by protoapigenone. Additionally, protoapigenone-induced JNK activation was linked to thiol modification of glutathione S-transferase π (GSTpi), which impeded GSTpi inhibition of JNK. In contrast to protoapigenone, apigenin-induced apoptosis was neither dependent on ROS nor on MAPK. Structure-activity relationship studies suggested that the thiol reacting effect of protoapigenone might be associated with an α, β-unsaturated ketone moiety in the structure of ring B.

  20. Bergamot essential oil differentially modulates intracellular Ca2+ levels in vascular endothelial and smooth muscle cells: a new finding seen with fura-2.

    PubMed

    You, Ji H; Kang, Purum; Min, Sun Seek; Seol, Geun Hee

    2013-04-01

    In this study, we compared the effect of the essential oil of Citrus bergamia Risso [bergamot, bergamot essential oil (BEO)] on the intracellular Ca levels in vascular endothelial (EA) and mouse vascular smooth muscle (MOVAS) cells, using the fura-2 fluorescence technique. BEO caused an initial transient increase in intracellular Ca concentration ([Ca]i) in EA cells, followed by a decrease, whereas it induced a sustained increase in [Ca]i in MOVAS cells. Linalyl acetate (LA) as a major component of BEO-induced [Ca]i mobilization was similar to BEO in EA cells. The increase of [Ca]i by LA was higher in EA cells than in MOVAS cells. [Ca]i rise induced by extracellular Ca application was significantly blocked by BEO or LA in EA cells but not in MOVAS cells, suggesting that BEO and LA block Ca influx in EA cells. The present results suggest that BEO and LA differentially modulate intracellular Ca levels in vascular endothelial and smooth muscle cells. In addition, blockade of Ca influx by BEO and LA in EA cells may explain the protective effects of BEO on endothelial dysfunction associated with cardiovascular disease.

  1. Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions.

    PubMed

    Zhao, Chunhua; Lin, Zhao; Dong, Hongjun; Zhang, Yanping; Li, Yin

    2017-06-01

    Pyruvate kinase is one of the three rate-limiting glycolytic enzymes that catalyze the last step of glycolysis, conversion of phosphoenolpyruvate (PEP) into pyruvate, which is associated with ATP generation. Two isozymes of pyruvate kinase, PykF and PykA, are identified in Escherichia coli PykF is considered important, whereas PykA has a less-defined role. Prior studies inactivated the pykA gene to increase the level of its substrate, PEP, and thereby increased the yield of end products derived from PEP. We were surprised when we found a pykA ::Tn 5 mutant in a screen for increased yield of an end product derived from pyruvate ( n -butanol), suggesting that the role of PykA needs to be reexamined. We show that the pykA mutant exhibited elevated intracellular ATP levels, biomass concentrations, glucose consumption, and n -butanol production. We also discovered that the pykA mutant expresses higher levels of a presumed pyruvate transporter, YhjX, permitting the mutant to recapture and metabolize excreted pyruvate. Furthermore, we demonstrated that the nucleotide diphosphate kinase activity of PykA leads to negative regulation of the intracellular ATP levels. Taking the data together, we propose that inactivation of pykA can be considered a general strategy to enhance the production of pyruvate-derived metabolites under anaerobic conditions. IMPORTANCE This study showed that knocking out pykA significantly increased the intracellular ATP level and thus significantly increased the levels of glucose consumption, biomass formation, and pyruvate-derived product formation under anaerobic conditions. pykA was considered to be encoding a dispensable pyruvate kinase; here we show that pykA negatively regulates the anaerobic glycolysis rate through regulating the energy distribution. Thus, knocking out pykA can be used as a general strategy to increase the level of pyruvate-derived fermentative products. Copyright © 2017 American Society for Microbiology.

  2. Amodiaquine failure associated with erythrocytic glutathione in Plasmodium falciparum malaria

    PubMed Central

    Zuluaga, Lina; Pabón, Adriana; López, Carlos; Ochoa, Aleida; Blair, Silvia

    2007-01-01

    Objective To establish the relationship between production of glutathione and the therapeutic response to amodiaquine (AQ) monotherapy in Plasmodium falciparum non-complicated malaria patients. Methodology Therapeutic response to AQ was evaluated in 32 patients with falciparum malaria in two townships of Antioquia, Colombia, and followed-up for 28 days. For every patient, total glutathione and enzymatic activity (glutathione reductase, GR, and γ-glutamylcysteine synthetase, γ-GCS) were determined in parasitized erythrocytes, non-infected erythrocytes and free parasites, on the starting day (day zero, before ingestion of AQ) and on the day of failure (in case of occurrence). Results There was found an AQ failure of 31.25%. Independent of the therapeutic response, on the starting day and on the day of failure, lower total glutathione concentration and higher GR activities in parasitized erythrocytes were found, compared with non-infected erythrocytes (p < 0.003). In addition, only on the day of failure, γ-GCS activity of parasitized erythrocytes was higher, compared with that of healthy erythrocytes (p = 0.01). Parasitized and non-parasitized erythrocytes in therapeutic failure patients (TF) had higher total glutathione on the starting day compared with those of adequate clinical response (ACR) (p < 0.02). Parasitized erythrocytes of TF patients showed lower total glutathione on the failure day, compared with starting day (p = 0.017). No differences was seen in the GR and γ-GCS activities by compartment, neither between the two therapeutic response groups nor between the two treatment days. Conclusion This study is a first approach to explaining P. falciparum therapeutic failure in humans through differences in glutathione metabolism in TF and ACR patients. These results suggest a role for glutathione in the therapeutic failure to antimalarials. PMID:17451604

  3. Effect of catalase inactivation on levels of inorganic peroxides, superoxide dismutase, glutathione, oxygen consumption and life span in adult houseflies (Musca domestica).

    PubMed Central

    Allen, R G; Farmer, K J; Sohal, R S

    1983-01-01

    The effects of total inhibition of catalase, induced by 3-amino-1,2,4-triazole, on the adult housefly (Musca domestica) were examined. The lack of catalase activity had no effect on the longevity of the houseflies. Inorganic-peroxide concentration was elevated at younger ages, but declined in older flies. The rate of oxygen consumption by the flies was greatly decreased and the levels of oxidized as well as reduced glutathione were augmented. Superoxide dismutase activity showed a slight increase. This study suggests that loss of catalase activity does not affect survival of houseflies due to adaptive responses. PMID:6661212

  4. Drought stress in maize causes differential acclimation responses of glutathione and sulfur metabolism in leaves and roots.

    PubMed

    Ahmad, Nisar; Malagoli, Mario; Wirtz, Markus; Hell, Ruediger

    2016-11-09

    Drought is the most important environmental stress that limits crop yield in a global warming world. Despite the compelling evidence of an important role of oxidized and reduced sulfur-containing compounds during the response of plants to drought stress (e.g. sulfate for stomata closure or glutathione for scavenging of reactive oxygen species), the assimilatory sulfate reduction pathway is almost not investigated at the molecular or at the whole plant level during drought. In the present study, we elucidated the role of assimilatory sulfate reduction in roots and leaves of the staple crop maize after application of drought stress. The time-resolved dynamics of the adaption processes to the stress was analyzed in a physiological relevant situation -when prolonged drought caused significant oxidation stress but root growth should be maintained. The allocation of sulfate was significantly shifted to the roots upon drought and allowed for significant increase of thiols derived from sulfate assimilation in roots. This enabled roots to produce biomass, while leaf growth was stopped. Accumulation of harmful reactive oxygen species caused oxidation of the glutathione pool and decreased glutathione levels in leaves. Surprisingly, flux analysis using [ 35 S]-sulfate demonstrated a significant down-regulation of sulfate assimilation and cysteine synthesis in leaves due to the substantial decrease of serine acetyltransferase activity. The insufficient cysteine supply caused depletion of glutathione pool in spite of significant transcriptional induction of glutathione synthesis limiting GSH1. Furthermore, drought impinges on transcription of membrane-localized sulfate transport systems in leaves and roots, which provides a potential molecular mechanism for the reallocation of sulfur upon prolonged water withdrawal. The study demonstrated a significant and organ-specific impact of drought upon sulfate assimilation. The sulfur metabolism related alterations at the transcriptional

  5. 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.

  6. 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

  7. ChaC2, an Enzyme for Slow Turnover of Cytosolic Glutathione.

    PubMed

    Kaur, Amandeep; Gautam, Ruchi; Srivastava, Ritika; Chandel, Avinash; Kumar, Akhilesh; Karthikeyan, Subramanian; Bachhawat, Anand Kumar

    2017-01-13

    Glutathione degradation plays an important role in glutathione and redox homeostasis, and thus it is imperative to understand the enzymes and the mechanisms involved in glutathione degradation in detail. We describe here ChaC2, a member of the ChaC family of γ-glutamylcyclotransferases, as an enzyme that degrades glutathione in the cytosol of mammalian cells. ChaC2 is distinct from the previously described ChaC1, to which ChaC2 shows ∼50% sequence identity. Human and mouse ChaC2 proteins purified in vitro show 10-20-fold lower catalytic efficiency than ChaC1, although they showed comparable K m values (K m of 3.7 ± 0.4 mm and k cat of 15.9 ± 1.0 min -1 toward glutathione for human ChaC2; K m of 2.2 ± 0.4 mm and k cat of 225.2 ± 15 min -1 toward glutathione for human ChaC1). The ChaC1 and ChaC2 proteins also shared the same specificity for reduced glutathione, with no activity against either γ-glutamyl amino acids or oxidized glutathione. The ChaC2 proteins were found to be expressed constitutively in cells, unlike the tightly regulated ChaC1. Moreover, lower eukaryotes have a single member of the ChaC family that appears to be orthologous to ChaC2. In addition, we determined the crystal structure of yeast ChaC2 homologue, GCG1, at 1.34 Å resolution, which represents the first structure of the ChaC family of proteins. The catalytic site is defined by a fortuitous benzoic acid molecule bound to the crystal structure. The mechanism for binding and catalytic activity of this new enzyme of glutathione degradation, which is involved in continuous but basal turnover of cytosolic glutathione, is proposed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Glutathione suppresses the enzymatic and non-enzymatic browning in grape juice.

    PubMed

    Wu, Shengjun

    2014-10-01

    Browning tends to occur in grape juice during processing and storage and decreases the commercial value of it. Thus, browning inhibition is an important objective for manufacturers. This study aims to investigate the efficacy of glutathione as a browning inhibitor for use on grape juice. Grape juice browning treated with glutathione was monitored during processing and accelerated browning. 0.04% of glutathione inhibited 99.4% of the polyphenoloxidase activity in the grape juice. Consequently, during processing at room temperature and accelerated browning at 80 °C, the browning in the grape juice treated with glutathione was significantly lower than that in the control (p<0.05). The results indicate that glutathione is a promising browning inhibitor used in grape juice. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Extraction of glutathione from EFB fermentation waste using methanol with sonication process

    NASA Astrophysics Data System (ADS)

    Muryanto, Muryanto; Alvin, Nurdin, Muhammad; Hanifah, Ummu; Sudiyani, Yanni

    2017-11-01

    Glutathione is important compound on the human body. Glutathione have a widely use at pharmacy and cosmetics as detoxification, skin whitening agent, antioxidant and many other. This study aims to obtain glutathione from Saccharomyces cerevisiae in fermentation waste of second generation bioethanol. The remaining yeast in the empty fruit bunch (EFB) fermentation was separated from the fermentation solution use centrifugation process and then extracted using a methanol-water solution. The extraction process was done by maceration which was assisted by sonication process. Solvent concentration and time of sonication were varied to see its effect on glutathione concentration. The concentration of glutathione from the extraction process was analyzed using alloxan method with UV-Vis spectrophotometer. The results show that the highest glutathione concentration was approximately 1.32 g/L obtained with methanol solvent at 90 minutes of maceration following with 15 minutes sonication.

  10. [Induction of glutathione and activation of immune functions by low-dose, whole-body irradiation with gamma-rays].

    PubMed

    Kojima, Shuji

    2006-10-01

    We first examined the relation between the induction of glutathione and immune functions in mice after low-dose gamma-ray irradiation. Thereafter, inhibition of tumor growth by radiation was confirmed in Ehrlich solid tumor (EST)-bearing mice. The total glutathione level of the splenocytes transiently increased soon after irradiation and reached a maximum at around 4 h postirradiation. Thereafter, the level reverted to the 0 h value by 24 h postirradiation. A significantly high splenocyte proliferative response was also recognized 4 h postirradiation. Natural killer (NK) activity was also increased significantly in a similar manner. The time at which the response reached the maximum coincided well with that of maximum total glutathione levels of the splenocytes in the gamma-ray-irradiated mice. Reduced glutathione exogenously added to splenocytes obtained from normal mice enhanced the proliferative response and NK activity in a dose-dependent manner. The inhibitory effects of radiation on tumor growth was then examined in EST-bearing mice. Repeated low-dose irradiation (0.5 Gy, four times, before and within an early time after inoculation) significantly delayed the tumor growth. Finally, the effect of single low-dose (0.5 Gy), whole-body gamma-ray irradiation on immune balance was examined to elucidate the mechanism underlying the antitumor immunity. The percentage of B cells in blood lymphocytes was selectively decreased after radiation, concomitant with an increase in that of the helper T cell population. The IFN-gamma level in splenocyte culture prepared from EST-bearing mice was significantly increased 48 h after radiation, although the level of IL-4 was unchanged. IL-12 secretion from macrophages was also enhanced by radiation. These results suggest that low-dose gamma-rays induce Th1 polarization and enhance the activities of tumoricidal effector cells, leading to an inhibition of tumor growth.

  11. Effect of antioxidant-enriched diets on glutathione redox status in tissue homogenates and mitochondria of the senescence-accelerated mouse

    PubMed Central

    Rebrin, Igor; Zicker, Steven; Wedekind, Karen J.; Paetau-Robinson, Inke; Packer, Lester; Sohal, Rajindar S.

    2010-01-01

    The main purpose of this study was to investigate whether consumption of diets enriched in antioxidants attenuates the level of oxidative stress in the senescence-accelerated mouse (SAM). In separate and independent studies, two different dietary mixtures, one enriched with vitamin E, vitamin C, L-carnitine, and lipoic acid (Diet I) and another diet including vitamins E and C and 13 additional ingredients containing micronutrients with bioflavonoids, polyphenols, and carotenoids (Diet II), were fed for 8 and 10 months, respectively. The amounts of glutathione (GSH) and glutathione disulfides (GSSG) and GSH:GSSG ratios were determined in plasma, tissue homogenates, and mitochondria isolated from five different tissues of SAM (P8) mice. Both diets had a reductive effect in plasma; however Diet I had relatively little effect on the glutathione redox status in tissue homogenates or mitochondria. Remarkably, Diet II caused a large increase in the amount of glutathione and a marked reductive shift in glutathione redox state in mitochondria. Overall, the effects of Diet II were tissue and gender specific. Results indicated that the glutathione redox state in mitochondria and tissues can be altered by supplemental intake of a relatively complex mixture of dietary antioxidants that contains substances known to induce phase 2 enzymes, glutathione, and antioxidant defenses. Whether corresponding attenuations occur in age-associated deleterious changes in physiological functions or life span remains unknown. PMID:16043026

  12. Effect of antioxidant-enriched diets on glutathione redox status in tissue homogenates and mitochondria of the senescence-accelerated mouse.

    PubMed

    Rebrin, Igor; Zicker, Steven; Wedekind, Karen J; Paetau-Robinson, Inke; Packer, Lester; Sohal, Rajindar S

    2005-08-15

    The main purpose of this study was to investigate whether consumption of diets enriched in antioxidants attenuates the level of oxidative stress in the senescence-accelerated mouse (SAM). In separate and independent studies, two different dietary mixtures, one enriched with vitamin E, vitamin C, L-carnitine, and lipoic acid (Diet I) and another diet including vitamins E and C and 13 additional ingredients containing micronutrients with bioflavonoids, polyphenols, and carotenoids (Diet II), were fed for 8 and 10 months, respectively. The amounts of glutathione (GSH) and glutathione disulfides (GSSG) and GSH:GSSG ratios were determined in plasma, tissue homogenates, and mitochondria isolated from five different tissues of SAM (P8) mice. Both diets had a reductive effect in plasma; however Diet I had relatively little effect on the glutathione redox status in tissue homogenates or mitochondria. Remarkably, Diet II caused a large increase in the amount of glutathione and a marked reductive shift in glutathione redox state in mitochondria. Overall, the effects of Diet II were tissue and gender specific. Results indicated that the glutathione redox state in mitochondria and tissues can be altered by supplemental intake of a relatively complex mixture of dietary antioxidants that contains substances known to induce phase 2 enzymes, glutathione, and antioxidant defenses. Whether corresponding attenuations occur in age-associated deleterious changes in physiological functions or life span remains unknown.

  13. 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

  14. HIV-1 activation of innate immunity depends strongly on the intracellular level of TREX1 and sensing of incomplete reverse transcription products.

    PubMed

    Kumar, Swati; Morrison, James H; Dingli, David; Poeschla, Eric

    2018-05-16

    TREX1 has been reported to degrade cytosolic immune-stimulatory DNA, including viral DNA generated during HIV-1 infection, but the dynamic range of its capacity to suppress innate immune stimulation is unknown and its full role in the viral life cycle remains unclear. A main purpose of our study was to determine how the intracellular level of TREX1 affects HIV-1 activation and avoidance of innate immunity. Using stable over-expression and CRISPR-mediated gene disruption, we engineered a range of TREX1 levels in human THP-1 monocytes. Increasing the level of TREX1 dramatically suppressed HIV-1 induction of interferon-stimulated genes (ISGs). Productive infection and integrated proviruses were equal to increased. Knocking out TREX1 impaired viral infectivity, increased early viral cDNA and caused ten-fold or greater increases in HIV-1 ISG induction. Knockout of cyclic GMP-AMP synthase (cGAS) abrogated all ISG induction. Moreover, cGAS knockout produced no increase in single cycle infection, establishing that HIV-1 DNA-triggered signaling is not rapid enough to impair the initial ISG-triggering infection cycle. Disruption of the HIV-1 capsid by PF74 also induced ISGs and this was TREX1 level-dependent, required reverse transcriptase catalysis, and was eliminated by cGAS gene knockout. Thus, the intracellular level of TREX1 pivotally modulates innate immune induction by HIV-1. Partial HIV-1 genomes are the TREX1 target and are sensed by cGAS. The nearly complete lack of innate immune induction despite equal to increased viral integration observed when the TREX1 protein level is experimentally elevated indicates that integration-competent genomes are shielded from cytosolic sensor-effectors during uncoating and transit to the nucleus. IMPORTANCE Much remains unknown about how TREX1 influences HIV-1 replication, whether it targets full-length viral DNA versus partial intermediates, how intracellular TREX1 protein levels correlate with ISG induction, and whether TREX1

  15. Expression of glutathione peroxidase I gene in selenium-deficient rats.

    PubMed Central

    Reddy, A P; Hsu, B L; Reddy, P S; Li, N Q; Thyagaraju, K; Reddy, C C; Tam, M F; Tu, C P

    1988-01-01

    We have characterized a cDNA pGPX1211 encoding rat glutathione peroxidase I. The selenocysteine in the protein corresponded to a TGA codon in the coding region of the cDNA, similar to earlier findings in mouse and human genes, and a gene encoding the formate dehydrogenase from E. coli, another selenoenzyme. The rat GSH peroxidase I has a calculated subunit molecular weight of 22,155 daltons and shares 95% and 86% sequence homology with the mouse and human subunits, respectively. The 3'-noncoding sequence (greater than 930 bp) in pGPX1211 is much longer than that of the human sequences. We found that glutathione peroxidase I mRNA, but not the polypeptide, was expressed under nutritional stress of selenium deficiency where no glutathione peroxidase I activity can be detected. The failure of detecting any apoprotein for the glutathione peroxidase I under selenium deficiency and results published from other laboratories supports the proposal that selenium may be incorporated into the glutathione peroxidase I co-translationally. Images PMID:2838821

  16. Glutathione S-transferase pi isoform (GSTP1) expression in murine retina increases with developmental maturity.

    PubMed

    Lee, Wen-Hsiang; Joshi, Pratibha; Wen, Rong

    2014-01-01

    Glutathione S-transferase pi isoform (GSTP1) is an intracellular detoxification enzyme that catalyzes reduction of chemically reactive electrophiles and is a zeaxanthin-binding protein in the human macula. We have previously demonstrated that GSTP1 levels are decreased in human age-related macular degeneration (AMD) retina compared to normal controls (Joshi et al., Invest Ophthalmol Vis Sci, e-abstract, 2009). We also showed that GSTP1 levels parallel survival of human retinal pigment epithelial (RPE) cells exposed to ultraviolet (UV) light, and GSTP1 over-expression protects them against UV light damage (Joshi et al., Invest Ophthalmol Vis Sci, e-abstract, 2010). In the present work, we determined the developmental time course of GSTP1 expression in murine retina and in response to light challenge. Eyes from BALB/c mice at postnatal day 20, 1 month, and 2 months of age were prepared for retinal protein extraction and cryo sectioning, and GSTP1 levels in the retina were analyzed by Western blot and immunohistochemistry (IHC). Another group of BALB/c mice with the same age ranges was exposed to 1000 lx of white fluorescent light for 24 h, and their retinas were analyzed for GSTP1 expression by Western blot and IHC in a similar manner. GSTP1 levels in the murine retina increased in ascending order from postnatal day 20, 1 month, and 2 months of age. Moreover, GSTP1 expression in murine retina at postnatal day 20, 1 month, and 2 months of age increased in response to brief light exposure compared to age-matched controls under normal condition. GSTP1 expression in retina increases with developmental age in mice and accompanies murine retinal maturation. Brief exposure to light induces GSTP1 expression in the murine retina across various developmental ages. GSTP1 induction may be a protective response to light-induced oxidative damage in the murine retina.

  17. Glutathione S-Transferase Pi Isoform (GSTP1) Expression in Murine Retina Increases with Developmental Maturity

    PubMed Central

    Lee, Wen-Hsiang; Joshi, Pratibha; Wen, Rong

    2014-01-01

    Background and Aims Glutathione S-transferase pi isoform (GSTP1) is an intracellular detoxification enzyme that catalyzes reduction of chemically reactive electrophiles and is a zeaxanthin-binding protein in the human macula. We have previously demonstrated that GSTP1 levels are decreased in human age-related macular degeneration (AMD) retina compared to normal controls [1]. We also showed that GSTP1 levels parallel survival of human retinal pigment epithelial (RPE) cells exposed to UV light, and GSTP1 over-expression protects them against UV light damage [2]. In the present work, we determined the developmental time course of GSTP1 expression in murine retina and in response to light challenge. Methods Eyes from BALB/c mice at post-natal day 20, 1 month, and 2 months of age were prepared for retinal protein extraction and cryo sectioning, and GSTP1 levels in the retina were analyzed by Western blot and immunohistochemistry (IHC). Another group of BALB/c mice with the same age ranges was exposed to 1000 lux of white fluorescent light for 24 hours, and their retinas were analyzed for GSTP1 expression by Western blot and IHC in a similar manner. Results GSTP1 levels in the murine retina increased in ascending order from post-natal day 20, 1 month, and 2 months of age. Moreover, GSTP1 expression in murine retina at post-natal day 20, 1 month, and 2 months of age increased in response to brief light exposure compared to age-matched controls under normal condition. Conclusions GSTP1 expression in retina increases with developmental age in mice and accompanies murine retinal maturation. Brief exposure to light induces GSTP1 expression in the murine retina across various developmental ages. GSTP1 induction may be a protective response to light-induced oxidative damage in the murine retina. PMID:24664677

  18. Two Naturally Occurring Terpenes, Dehydrocostuslactone and Costunolide, Decrease Intracellular GSH Content and Inhibit STAT3 Activation

    PubMed Central

    Butturini, Elena; Cavalieri, Elisabetta; Carcereri de Prati, Alessandra; Darra, Elena; Rigo, Antonella; Shoji, Kazuo; Murayama, Norie; Yamazaki, Hiroshi; Watanabe, Yasuo; Suzuki, Hisanori; Mariotto, Sofia

    2011-01-01

    The main purpose of the present study is to envisage the molecular mechanism of inhibitory action ofdehydrocostuslactone (DCE) andcostunolide (CS), two naturally occurring sesquiterpene lactones, towards the activation of signal transducer and activator of transcription 3 (STAT3). We report that, in human THP-1 cell line, they inhibit IL-6-elicited tyrosine phosphorylation of STAT3 and its DNA binding activity with EC50 of 10 µM with concomitantdown-regulation ofthe phosphorylation of the tyrosine Janus kinases JAK1, JAK2 and Tyk2. Furthermore, these compounds that contain an α-β-unsatured carbonyl moiety and function as potent Michael reaction acceptor, induce a rapid drop in intracellular glutathione (GSH) concentration by direct interaction with it, thereby triggering S-glutathionylation of STAT3. Dehydrocostunolide (HCS), the reduced form of CS lacking only the α-β-unsaturated carbonyl group, fails to exert any inhibitory action. Finally, the glutathione ethylene ester (GEE), the cell permeable GSH form, reverts the inhibitory action of DCE and CS on STAT3 tyrosine phosphorylation. We conclude that these two sesquiterpene lactones are able to induce redox-dependent post-translational modification of cysteine residues of STAT3 protein in order to regulate its function. PMID:21625597

  19. Polymorphisms of glutathione S-transferase Mu 1, glutathione S-transferase theta 1 and glutathione S-transferase Pi 1 genes in Hodgkin's lymphoma susceptibility and progression.

    PubMed

    Lourenço, Gustavo J; Néri, Iramaia A; Sforni, Vitor C S; Kameo, Rodolfo; Lorand-Metze, Irene; Lima, Carmen S P

    2009-06-01

    We tested in this study whether the polymorphisms of the glutathione S-transferase Mu1 (GSTM1), glutathione S-transferase Theta 1 (GSTT1) and glutathione S-transferase Pi 1 (GSTP1), involved in metabolism of chemical agents, cell proliferation and cell survival, alter the risk for Hodgkin lymphoma (HL). Genomic DNA from 110 consecutive patients with HL and 226 controls was analysed by polymerase chain reaction and restriction digestion for the polymorphism analyses. Similar frequencies of the GSTM1 and GSTT1 genotypes were seen in patients and controls. In contrast, the frequency of the GSTP1 wild genotype (59.1%versus 36.3%, P = 0.004) was higher in patients than in controls. Individuals with the wild genotype had a 2.68 (95%CI: 1.38-5.21)-fold increased risk for the disease than others. An excess of the GSTP1 wild genotype was also observed in patients with tumors of stages III + IV when compared with those with tumors of stages I + II (39.1%versus 20.0%, P = 0.03). These results suggest that the wild allele of the GSTP1 gene is linked to an increased risk and high aggressiveness of the HL in our cases but they should be confirmed by further studies with larger cohorts of patients and controls.

  20. Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

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

    Arai, Roberto J.; Ogata, Fernando T.; Batista, Wagner L.

    2008-12-01

    Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects ofmore » GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.« less

  1. 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.

  2. Effects of Ozone Oxidative Preconditioning on TNF-α Release and Antioxidant-Prooxidant Intracellular Balance in Mice During Endotoxic Shock

    PubMed Central

    Zamora, Zullyt B.; Borrego, Aluet; López, Orlay Y.; Delgado, René; González, Ricardo; Menéndez, Silvia; Hernández, Frank; Schulz, Siegfried

    2005-01-01

    Ozone oxidative preconditioning is a prophylactic approach, which favors the antioxidant-prooxidant balance for preservation of cell redox state by the increase of antioxidant endogenous systems in both in vivo and in vitro experimental models. Our aim is to analyze the effect of ozone oxidative preconditioning on serum TNF-α levels and as a modulator of oxidative stress on hepatic tissue in endotoxic shock model (mice treated with lipopolysaccharide (LPS)). Ozone/oxygen gaseous mixture which was administered intraperitoneally (0.2, 0.4, and 1.2 mg/kg) once daily for five days before LPS (0.1 mg/kg, intraperitoneal). TNF-α was measured by cytotoxicity on L-929 cells. Biochemical parameters such as thiobarbituric acid reactive substances (TBARS), enzymatic activity of catalase, glutathione peroxidase, and glutathione-S transferase were measured in hepatic tissue. One hour after LPS injection there was a significant increase in TNF-α levels in mouse serum. Ozone/oxygen gaseous mixture reduced serum TNF-α levels in a dose-dependent manner. Statistically significant decreases in TNF-α levels after LPS injection were observed in mice pretreated with ozone intraperitoneal applications at 0.2 (78%), 0.4 (98%), and 1.2 (99%). Also a significant increase in TBARS content was observed in the hepatic tissue of LPS-treated mice, whereas enzymatic activity of glutathion-S transferase and glutathione peroxidase was decreased. However in ozone-treated animals a significant decrease in TBARS content was appreciated as well as an increase in the activity of antioxidant enzymes. These results indicate that ozone oxidative preconditioning exerts inhibitory effects on TNF-α production and on the other hand it exerts influence on the antioxidant-prooxidant balance for preservation of cell redox state by the increase of endogenous antioxidant systems. PMID:15770062

  3. Investigation into the Effects of Boron on Liver Tissue Protein Carbonyl, MDA, and Glutathione Levels in Endotoxemia.

    PubMed

    Balabanlı, Barbaros; Balaban, Tuba

    2015-10-01

    Endotoxin has been known to cause the formation and damage of free radical. The importance of boron for human life is increasing each passing day, and its consuming fields are continuing to expand due to the advances in science and technology. Therefore, in our study, we intended to investigate into the effects of boron on liver tissue oxidative events. Eighteen male Wistar albino rats were randomly separated into three equal groups in the experiments; control group, boron + endotoxin group, and endotoxin group. Dissolved in distilled water, boric acid (100 mg/kg) was administered to boron + endotoxin group via gavage procedure for 28 days. Only distilled water was administered to control and endotoxin groups via gavage procedure for 28 days. Then 4 mg/kg endotoxin (LPS; Escherichia coli 0111:B4) was intraperitoneally (ip) administered to boron + endotoxin and endotoxin groups on the 28th day. Sterile saline was injected into control group on the 28th day (ip). Malondialdehyde (MDA), which is the end product of lipid peroxidation in liver tissues, protein carbonyl compounds (PC), which are protein oxidization markers, and glutathione (GSH) levels were measured spectrophotometrically. The results were compared with Mann-Whitney U test. When boron + endotoxin group is compared with endotoxin group, PC levels of endotoxin group showed a significant increase. When GSH levels are compared, GSH level in boron + endotoxin group decreased according to endotoxin group. Variations among all groups in MDA levels were found to be statistically insignificant. We are of the opinion that endotoxin affects the proteins by forming free radicals, and boron may also cause the structural and/or functional changes in proteins in order to protect proteins from oxidization.

  4. Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin.

    PubMed

    Wu, Sau-Ching; Wong, Sui-Lam

    2002-03-01

    Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.

  5. 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.

  6. The Roles of Glutathione Peroxidases during Embryo Development

    PubMed Central

    Ufer, Christoph; Wang, Chi Chiu

    2011-01-01

    Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency – in contrast to all other GPx family members – leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis

  7. The Roles of Glutathione Peroxidases during Embryo Development.

    PubMed

    Ufer, Christoph; Wang, Chi Chiu

    2011-01-01

    Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency - in contrast to all other GPx family members - leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis on

  8. 3-Sulfanyl-4-methylpentan-1-ol in Dry-Hopped Beers: First Evidence of Glutathione S-Conjugates in Hop (Humulus lupulus L.).

    PubMed

    Kankolongo Cibaka, Marie-Lucie; Decourrière, Laura; Lorenzo-Alonso, Celso-José; Bodart, Etienne; Robiette, Raphaël; Collin, Sonia

    2016-11-16

    Monovarietal dry-hopped beers were produced with the dual-purpose hop cultivars Amarillo, Hallertau Blanc, and Mosaic. The grapefruit-like 3-sulfanyl-4-methylpentan-1-ol was found in all three beers at concentrations much higher than expected on the basis of the free thiol content in hop. Even cysteinylated precursors proved unable to explain our results. As observed in wine, the occurrence of S-glutathione precursors was therefore suspected in hop. The analytical standards of S-3-(4-methyl-1-hydroxypentyl)glutathione, never described before, and of S-3-(1-hydroxyhexyl)glutathione, previously evidenced in grapes, were chemically synthesized. An optimized extraction of glutathionylated precursors was then applied to Amarillo, Hallertau Blanc, and Mosaic hop samples. HPLC-ESI(+)MS/MS revealed, for the first time, the occurrence of S-3-(1-hydroxyhexyl)glutathione and S-3-(4-methyl-1-hydroxypentyl)glutathione in hop, at levels well above those reported for their cysteinylated counterparts. S-3-(1-Hydroxyhexyl)glutathione emerged in all cases as the major adduct in hop. Yet, although 3-sulfanylhexan-1-ol seems relatively ubiquitous in free, cysteinylated, and glutathionylated forms, the glutathione adduct of 3-sulfanyl-4-methylpentan-1-ol, never evidenced in other plants up to now, was found only in the Hallertau Blanc variety.

  9. Glutathione depletion triggers actin cytoskeleton changes via actin-binding proteins.

    PubMed

    Zepeta-Flores, Nahum; Valverde, Mahara; Lopez-Saavedra, Alejandro; Rojas, Emilio

    2018-06-04

    The importance of glutathione (GSH) in alternative cellular roles to the canonically proposed, were analyzed in a model unable to synthesize GSH. Gene expression analysis shows that the regulation of the actin cytoskeleton pathway is strongly impacted by the absence of GSH. To test this hypothesis, we evaluate the effect of GSH depletion via buthionine sulfoximine (5 and 12.5 mM) in human neuroblastoma MSN cells. In the present study, 70% of GSH reduction did not induce reactive oxygen species, lipoperoxidation, or cytotoxicity, which enabled us to evaluate the effect of glutathione in the absence of oxidative stress. The cells with decreasing GSH levels acquired morphology changes that depended on the actin cytoskeleton and not on tubulin. We evaluated the expression of three actin-binding proteins: thymosin β4, profilin and gelsolin, showing a reduced expression, both at gene and protein levels at 24 hours of treatment; however, this suppression disappears after 48 hours of treatment. These changes were sufficient to trigger the co-localization of the three proteins towards cytoplasmic projections. Our data confirm that a decrease in GSH in the absence of oxidative stress can transiently inhibit the actin binding proteins and that this stimulus is sufficient to induce changes in cellular morphology via the actin cytoskeleton.

  10. Enalapril and captopril enhance glutathione-dependent antioxidant defenses in mouse tissues.

    PubMed

    de Cavanagh, E M; Inserra, F; Ferder, L; Fraga, C G

    2000-03-01

    The effect of enalapril and captopril on total glutathione content (GSSG + GSH) and selenium-dependent glutathione peroxidase (Se-GPx) and glutathione reductase (GSSG-Rd) activities was investigated in mouse tissues. CF-1 mice (4-mo-old females) received water containing enalapril (20 mg/l) or captopril (50 mg/l) for 11 wk. Enalapril increased GSSG + GSH content (P < 0.05) in erythrocytes (147%), brain (112%), and lung (67%), and captopril increased GSSG + GSH content in erythrocytes (190%) and brain (132%). Enalapril enhanced Se-GPx activity in kidney cortex (42%) and kidney medulla (23%) and captopril in kidney cortex (30%). GSSG-Rd activity was enhanced by enalapril in erythrocytes (21%), brain (21%), liver (18%), and kidney cortex (53%) and by captopril in erythrocytes (25%), brain (19%), and liver (34%). In vitro erythrocyte oxidant stress was evaluated by thiobarbituric acid-reactive substances (TBARS) production (control 365 +/- 11, enalapril 221 +/- 26, captopril 206 +/- 17 nmol TBARS x g Hb(-1) x h(-1); both P < 0.05 vs. control) and phenylhydrazine-induced methemoglobin (MetHb) formation (control 66.5 +/- 3.5, enalapril 52.9 +/- 0.4, captopril: 56.4 +/- 2.9 micromol MetHb/g Hb; both P < 0.05 vs. control). Both angiotensin-converting enzyme inhibitor treatments were associated with increased nitric oxide production, as assessed by plasma NO-(3) + NO-(2) level determination (control 9.22 +/- 0.64, enalapril 13.7 +/- 1.9, captopril 17.3 +/- 3.0 micromol NO-(3) + NO-(2)/l plasma; both P < 0.05 vs. control). These findings support our previous reports on the enalapril- and captopril-induced enhancement of endogenous antioxidant defenses and include new data on glutathione-dependent defenses, thus furthering current knowledge on the association of ACE inhibition and antioxidants.

  11. 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

  12. A Western diet induced NAFLD in LDLR(-/)(-) mice is associated with reduced hepatic glutathione synthesis.

    PubMed

    Li, Ling; Zhang, Guo-Fang; Lee, Kwangwon; Lopez, Rocio; Previs, Stephen F; Willard, Belinda; McCullough, Arthur; Kasumov, Takhar

    2016-07-01

    Oxidative stress plays a key role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Glutathione is the major anti-oxidant involved in cellular oxidative defense, however there are currently no simple non-invasive methods for assessing hepatic glutathione metabolism in patients with NAFLD. As a primary source of plasma glutathione, liver plays an important role in interorgan glutathione homeostasis. In this study, we have tested the hypothesis that measurements of plasma glutathione turnover could be used to assess the hepatic glutathione metabolism in LDLR(-/)(-) mice, a mouse model of diet-induced NAFLD. Mice were fed a standard low fat diet (LFD) or a high fat diet containing cholesterol (a Western type diet (WD)). The kinetics of hepatic and plasma glutathione were quantified using the (2)H2O metabolic labeling approach. Our results show that a WD leads to reduced fractional synthesis rates (FSR) of hepatic (25%/h in LFD vs. 18%/h in WD, P<0.05) and plasma glutathione (43%/h in LFD vs. 21%/h in WD, P<0.05), without any significant effect on their absolute production rates (PRs). WD-induced concordant changes in both hepatic and plasma glutathione turnover suggest that the plasma glutathione turnover measurements could be used to assess hepatic glutathione metabolism. The safety, simplicity, and low cost of the (2)H2O-based glutathione turnover approach suggest that this method has the potential for non-invasive probing of hepatic glutathione metabolism in patients with NAFLD and other diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Protection against oxidative DNA damage and stress in human prostate by glutathione S-transferase P1

    PubMed Central

    Kanwal, Rajnee; Pandey, Mitali; Bhaskaran, Natarajan; MacLennan, Gregory T; Fu, Pingfu; Ponsky, Lee E; Gupta, Sanjay

    2014-01-01

    The pi-class glutathione S-transferase (GSTP1) actively protect cells from carcinogens and electrophilic compounds. Loss of GSTP1 expression via promoter hypermethylation is the most common epigenetic alteration observed in human prostate cancer. Silencing of GSTP1 can increase generation of reactive oxygen species (ROS) and DNA damage in cells. In this study we investigated whether loss of GSTP1 contributes to increased DNA damage that may predispose men to a higher risk of prostate cancer. We found significantly elevated (103%; P<0.0001) levels of 8-oxo-2′-deoxogunosine (8-OHdG), an oxidative DNA damage marker, in adenocarcinomas, compared to benign counterparts, which positively correlated (r=0.2) with loss of GSTP1 activity (34%; P<0.0001). Silencing of GSTP1 using siRNA approach in normal human prostate epithelial RWPE1 cells caused increased intracellular production of ROS and higher susceptibility of cells to H2O2-mediated oxidative stress. Additionally, human prostate carcinoma LNCaP cells, which contain a silenced GSTP1 gene, were genetically modified to constitutively express high levels of GSTP1. Induction of GSTP1 activity lowered endogenous ROS levels in LNCaP-pLPCX-GSTP1 cells, and when exposed to H2O2, these cells exhibited significantly reduced production of ROS and 8-OHdG levels, compared to vector control LNCaP-pLPCX cells. Furthermore, exposure of LNCaP cells to green tea polyphenols caused re-expression of GSTP1, which protected the cells from H2O2-mediated DNA damage through decreased ROS production compared to non-exposed cells. These results suggest that loss of GSTP1 expression in human prostate cells, a process that increases their susceptibility to oxidative stress-induced DNA damage, may be an important target for primary prevention of prostate cancer. PMID:22833520

  14. Glutathione Transferase from Trichoderma virens Enhances Cadmium Tolerance without Enhancing Its Accumulation in Transgenic Nicotiana tabacum

    PubMed Central

    Dixit, Prachy; Mukherjee, Prasun K.; Ramachandran, V.; Eapen, Susan

    2011-01-01

    Background Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST) are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. Results Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST) showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. Conclusion The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for developing Cd tolerance and

  15. Atomic force microscopy for cellular level manipulation: imaging intracellular structures and DNA delivery through a membrane hole.

    PubMed

    Afrin, Rehana; Zohora, Umme Salma; Uehara, Hironori; Watanabe-Nakayama, Takahiro; Ikai, Atsushi

    2009-01-01

    The atomic force microscope (AFM) is a versatile tool for imaging, force measurement and manipulation of proteins, DNA, and living cells basically at the single molecular level. In the cellular level manipulation, extraction, and identification of mRNA's from defined loci of a cell, insertion of plasmid DNA and pulling of membrane proteins, for example, have been reported. In this study, AFM was used to create holes at defined loci on the cell membrane for the investigation of viability of the cells after hole creation, visualization of intracellular structure through the hole and for targeted gene delivery into living cells. To create large holes with an approximate diameter of 5-10 microm, a phospholipase A(2) coated bead was added to the AFM cantilever and the bead was allowed to touch the cell surface for approximately 5-10 min. The evidence of hole creation was obtained mainly from fluorescent image of Vybrant DiO labeled cell before and after the contact with the bead and the AFM imaging of the contact area. In parallel, cells with a hole were imaged by AFM to reveal intracellular structures such as filamentous structures presumably actin fibers and mitochondria which were identified with fluorescent labeling with rhodamine 123. Targeted gene delivery was also attempted by inserting an AFM probe that was coated with the Monster Green Fluorescent Protein phMGFP Vector for transfection of the cell. Following targeted transfection, the gene expression of green fluorescent protein (GFP) was observed and confirmed by the fluorescence microscope. Copyright (c) 2009 John Wiley & Sons, Ltd.

  16. Intracellular diffusion of oxygen and hypoxic sensing: role of mitochondrial respiration.

    PubMed

    Takahashi, Eiji; Sato, Michihiko

    2010-01-01

    In vivo, diffusional O(2) gradients from the capillary blood to the intracellular space determine O(2) availability at the O(2) sensing molecules in the cell. With a novel technique for imaging intracellular O(2) levels using green fluorescent protein (GFP), we examined the possibility that diffusional O(2) concentration gradients might be involved in the cellular hypoxic sensing in cultured Hep3B cells. In the present study, we failed to demonstrate significant gradients of intracellular O(2) when mitochondrial respiration was maximally elevated by an uncoupler of oxidative phosphorylation. Thus, we conclude that intracellular O(2) gradients may be negligible at normal mitochondrial O(2) demand in these cells.

  17. Manganese (Mn) Oxidation Increases Intracellular Mn in Pseudomonas putida GB-1

    PubMed Central

    Banh, Andy; Chavez, Valarie; Doi, Julia; Nguyen, Allison; Hernandez, Sophia; Ha, Vu; Jimenez, Peter; Espinoza, Fernanda; Johnson, Hope A.

    2013-01-01

    Bacterial manganese (Mn) oxidation plays an important role in the global biogeochemical cycling of Mn and other compounds, and the diversity and prevalence of Mn oxidizers have been well established. Despite many hypotheses of why these bacteria may oxidize Mn, the physiological reasons remain elusive. Intracellular Mn levels were determined for Pseudomonas putida GB-1 grown in the presence or absence of Mn by inductively coupled plasma mass spectrometry (ICP-MS). Mn oxidizing wild type P. putida GB-1 had higher intracellular Mn than non Mn oxidizing mutants grown under the same conditions. P. putida GB-1 had a 5 fold increase in intracellular Mn compared to the non Mn oxidizing mutant P. putida GB-1-007 and a 59 fold increase in intracellular Mn compared to P. putida GB-1 ∆2665 ∆2447. The intracellular Mn is primarily associated with the less than 3 kDa fraction, suggesting it is not bound to protein. Protein oxidation levels in Mn oxidizing and non oxidizing cultures were relatively similar, yet Mn oxidation did increase survival of P. putida GB-1 when oxidatively stressed. This study is the first to link Mn oxidation to Mn homeostasis and oxidative stress protection. PMID:24147089

  18. High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila

    PubMed Central

    Chiaraviglio, Lucius

    2015-01-01

    Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche. PMID:26392509

  19. FRET sensor-based quantification of intracellular trehalose in mammalian cells.

    PubMed

    Kikuta, Shingo; Hou, Bi-Huei; Sato, Ryoichi; Frommer, Wolf B; Kikawada, Takahiro

    2016-01-01

    Trehalose acts as a stress protectant and an autophagy inducer in mammalian cells. The molecular mechanisms of action remain obscure because intracellular trehalose at micromolar level is difficult to quantitate. Here, we show a novel trehalose monitoring technology based on FRET. FLIP-suc90μ∆1Venus sensor expressed in mammalian cells enables to quickly and non-destructively detect an infinitesimal amount of intracellular trehalose.

  20. Glutathione in the human brain: Review of its roles and measurement by magnetic resonance spectroscopy.

    PubMed

    Rae, Caroline D; Williams, Stephen R

    2017-07-15

    We review the transport, synthesis and catabolism of glutathione in the brain as well as its compartmentation and biochemistry in different brain cells. The major reactions involving glutathione are reviewed and the factors limiting its availability in brain cells are discussed. We also describe and critique current methods for measuring glutathione in the human brain using magnetic resonance spectroscopy, and review the literature on glutathione measurements in healthy brains and in neurological, psychiatric, neurodegenerative and neurodevelopmental conditions In summary: Healthy human brain glutathione concentration is ∼1-2 mM, but it varies by brain region, with evidence of gender differences and age effects; in neurological disease glutathione appears reduced in multiple sclerosis, motor neurone disease and epilepsy, while being increased in meningiomas; in psychiatric disease the picture is complex and confounded by methodological differences, regional effects, length of disease and drug-treatment. Both increases and decreases in glutathione have been reported in depression and schizophrenia. In Alzheimer's disease and mild cognitive impairment there is evidence for a decrease in glutathione compared to age-matched healthy controls. Improved methods to measure glutathione in vivo will provide better precision in glutathione determination and help resolve the complex biochemistry of this molecule in health and disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Effect of Glutathione on the Taste and Texture of Type I Sourdough Bread.

    PubMed

    Tang, Kai Xing; Zhao, Cindy J; Gänzle, Michael G

    2017-05-31

    Type I sourdough fermentations with Lactobacillus sanfranciscensis as predominant organism accumulate reduced glutathione through glutathione reductase (GshR) activity of L. sanfranciscensis. Reduced glutathione acts as chain terminator for gluten polymerization but is also kokumi-active and may thus enhance bread taste. This study implemented a type I model sourdough fermentations to quantitate glutathione accumulation sourdough, bread dough, and bread and to assess the effect of L. sanfranciscensis GshR on bread volume by comparison of L. sanfranciscensis and an isogenic strain devoid of GshR. L. sanfranciscensis sourdough accumulated the highest amount of reduced glutathione during proofing. Bread produced with the wild type strain had a lower volume when compared to the gshR deficient mutant. The accumulation of γ-glutamyl-cysteine was also higher in L. sanfranciscensis sourdoughs when compared to doughs fermented with the gshR mutant strain. The accumulation of reduced glutathione in L. sanfranciscensis bread did not enhance the saltiness of bread.

  2. Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.

    PubMed

    Pettersson, Eva U; Ljunggren, Erland L; Morrison, David A; Mattsson, Jens G

    2005-01-01

    The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.

  3. Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model.

    PubMed

    Zareba, Ilona; Surazynski, Arkadiusz; Chrusciel, Marcin; Miltyk, Wojciech; Doroszko, Milena; Rahman, Nafis; Palka, Jerzy

    2017-01-01

    The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways. © 2017 The Author(s). Published by S. Karger AG, Basel.

  4. Identification of Glutathione S-Transferase Pi as a Protein Involved in Parkinson Disease Progression

    PubMed Central

    Shi, Min; Bradner, Joshua; Bammler, Theo K.; Eaton, David L.; Zhang, JianPeng; Ye, ZuCheng; Wilson, Angela M.; Montine, Thomas J.; Pan, Catherine; Zhang, Jing

    2009-01-01

    Parkinson disease (PD) typically affects the cortical regions during the later stages of disease, with neuronal loss, gliosis, and formation of diffuse cortical Lewy bodies in a significant portion of patients with dementia. To identify novel proteins involved in PD progression, we prepared synaptosomal fractions from the frontal cortices of pathologically verified PD patients at different stages along with age-matched controls. Protein expression profiles were compared using a robust quantitative proteomic technique. Approximately 100 proteins displayed significant differences in their relative abundances between PD patients at various stages and controls; three of these proteins were validated using independent techniques. One of the confirmed proteins, glutathione S-transferase Pi, was further investigated in cellular models of PD, demonstrating that its level was intimately associated with several critical cellular processes that are directly related to neurodegeneration in PD. These results have, for the first time, suggested that the levels of glutathione S-transferase Pi may play an important role in modulating the progression of PD. PMID:19498008

  5. One-pot synthesis of glutathione by a two-enzyme cascade using a thermophilic ATP regeneration system.

    PubMed

    Zhang, Xing; Wu, Hui; Huang, Bing; Li, Zhimin; Ye, Qin

    2017-01-10

    In vitro cascade catalysis using enzyme-based system is becoming a promising biomanufacturing platform for biofuels and biochemicals production. Glutathione is a pivotal non-protein thiol compound and widely applied in food and pharmaceutical industries. In this study, glutathione was synthesized by a bifunctional glutathione synthetase together with a thermophilic ATP regeneration system through a two-enzyme cascade in vitro. Four bifunctional glutathione synthetases from Streptococcus sanguinis, S. gordonii, S. uberis and Bacillus cereus were applied for glutathione synthesis. The bifunctional glutathione synthetase from S. sanguinis was selected and coupled with the polyphosphate kinase from Thermosynechococcus elongatus BP-1 for regenerating ATP to produce glutathione in one pot. In the optimized system, 28.5mM glutathione was produced within 5h due to efficient ATP regeneration from low-cost polyphosphate. The yield based on added l-cysteine reached 81.4% and the productivity of glutathione achieved 5.7mM/h. The one-pot system indicated a potential biotransformation platform for industrial production of glutathione. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. 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.

  7. Reactivity of 9-aminoacridine drug quinacrine with glutathione limits its antiprion activity.

    PubMed

    Šafařík, Martin; Moško, Tibor; Zawada, Zbigniew; Šafaříková, Eva; Dračínský, Martin; Holada, Karel; Šebestík, Jaroslav

    2017-06-01

    Quinacrine-the drug based on 9-aminoacridine-failed in clinical trials for prion diseases, whereas it was active in in vitro studies. We hypothesize that aromatic nucleophilic substitution at C9 could be contributing factor responsible for this failure because of the transfer of acridine moiety from quinacrine to abundant glutathione. Here, we described the semi-large-scale synthesis of the acridinylated glutathione and the consequences of its formation on biological and biophysical activities. The acridinylated glutathione is one order of magnitude weaker prion protein binder than the parent quinacrine. Moreover, according to log D pH 7.4 , the glutathione conjugate is two orders of magnitude more hydrophilic than quinacrine. Its higher hydrophilicity and higher dsDNA binding potency will significantly decrease its bioavailability in membrane-like environment. The glutathione deactivates quinacrine not only directly but also decreases its bioavailability. Furthermore, the conjugate can spontaneously decompose to practically insoluble acridone, which is precipitated out from the living systems. © 2016 John Wiley & Sons A/S.

  8. A Western diet induced NAFLD in LDLR−/− mice is associated with reduced hepatic glutathione synthesis

    PubMed Central

    Li, Ling; Zhang, Guo-Fang; Lee, Kwangwon; Lopez, Rocio; Previs, Stephen F.; Willard, Belinda; McCullough, Arthur; Kasumov, Takhar

    2017-01-01

    Oxidative stress plays a key role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Glutathione is the major anti-oxidant involved in cellular oxidative defense, however there are currently no simple non-invasive methods for assessing hepatic glutathione metabolism in patients with NAFLD. As a primary source of plasma glutathione, liver plays an important role in interorgan glutathione homeostasis. In this study, we have tested the hypothesis that measurements of plasma glutathione turnover could be used to assess the hepatic glutathione metabolism in LDLR−/− mice, a mouse model of diet-induced NAFLD. Mice were fed a standard low fat diet (LFD) or a high fat diet containing cholesterol (a Western type diet (WD)). The kinetics of hepatic and plasma glutathione were quantified using the 2H2O metabolic labeling approach. Our results show that a WD leads to reduced fractional synthesis rates (FSR) of hepatic (25%/h in LFD vs. 18%/h in WD, P < 0.05) and plasma glutathione (43%/h in LFD vs. 21%/h in WD, P <0.05), without any significant effect on their absolute production rates (PRs). WD-induced concordant changes in both hepatic and plasma glutathione turnover suggest that the plasma glutathione turnover measurements could be used to assess hepatic glutathione metabolism. The safety, simplicity, and low cost of the 2H2O-based glutathione turnover approach suggest that this method has the potential for non-invasive probing of hepatic glutathione metabolism in patients with NAFLD and other diseases. PMID:27036364

  9. Effects of glutathione on sperm quality during liquid storage in boars.

    PubMed

    Zhang, Xiao-Gang; Liu, Qi; Wang, Li-Qiang; Yang, Gong-She; Hu, Jian-Hong

    2016-10-01

    The aim of this study was to investigate the effects of different concentrations of glutathione in Modena on boar sperm quality during liquid storage at 17°C. Boar semen samples were collected and diluted with Modena containing different concentrations (0, 1, 5, 10, 15 mmol/L) of glutathione. Sperm motility, effective survival period, plasma membrane integrity, acrosome integrity, total antioxidant capacity (T-AOC) activity, malondialdehyde (MDA) content and hydrogen peroxide (H 2 O 2 ) content were measured and analyzed. The results showed that Modena supplemented with 1, 5 and 10 mmol/L glutathione improved sperm motility, effective survival period, plasma membrane integrity and T-AOC, and decreased MDA content and H 2 O 2 content. Meanwhile, the semen sample diluted with Modena containing 1 mmol/L glutathione achieved optimum effect, and effective survival period was 6.1 days. After 5 days preservation, sperm motility, plasma membrane integrity and T-AOC of the group treated with 1 mmol/L glutathione were all higher than that of other groups. Meanwhile, MDA content and H 2 O 2 content were lower than that of other groups. In conclusion, Modena supplemented with glutathione decreased the oxidative stress and improved the quality of boar semen during liquid storage at 17°C, and 1 mmol/L concentration was the optimum concentration. © 2016 Japanese Society of Animal Science. © 2016 Japanese Society of Animal Science.

  10. 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.

  11. 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.

  12. 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.

  13. Formation of 5-Oxoproline from Glutathione in Erythrocytes by the γ-Glutamyltranspeptidase-Cyclotransferase Pathway

    PubMed Central

    Palekar, Anil G.; Tate, Suresh S.; Meister, Alton

    1974-01-01

    γ-Glutamyltranspeptidase activity was demonstrated in the membrane fraction of rabbit erythrocytes. The activity observed (with glutathione and various amino-acid acceptors) was similar in magnitude to that of the γ-glutamylcyclotransferase and γ-glutamylcysteine synthetase activities found in the soluble fraction of the cell. No transpeptidase activity was observed with either γ-glutamyl p-nitroanilide or oxidized glutathione in contrast to the rabbit-kidney enzyme for which these compounds and glutathione serve as substrates. Erythrocyte suspensions and hemolysates formed 5-oxoproline (pyroglutamate; pyrrolidone carboxylate); the rate of 5-oxoproline formation from glutathione by hemolysates was increased by addition of methionine. The findings indicate that 5-oxoproline is an end-product of glutathione metabolism in erythrocytes, and that 5-oxoproline passes out of the erythrocyte and is metabolized in other tissues. The observed rate of 5-oxoproline formation is consistent with the conclusion that the γ-glutamyltranspeptidase-cyclotransferase pathway, together with the synthesis of glutathione from glycine, cysteine, and glutamate, account for a large fraction of the observed amino-acid turnover of erythrocyte glutathione. PMID:4150022

  14. Oxidation promotes insertion of the CLIC1 chloride intracellular channel into the membrane.

    PubMed

    Goodchild, Sophia C; Howell, Michael W; Cordina, Nicole M; Littler, Dene R; Breit, Samuel N; Curmi, Paul M G; Brown, Louise Jennifer

    2009-12-01

    Members of the chloride intracellular channel (CLIC) family exist primarily as soluble proteins but can also auto-insert into cellular membranes to form ion channels. While little is known about the process of CLIC membrane insertion, a unique feature of mammalian CLIC1 is its ability to undergo a dramatic structural metamorphosis between a monomeric glutathione-S-transferase homolog and an all-helical dimer upon oxidation in solution. Whether this oxidation-induced metamorphosis facilitates CLIC1 membrane insertion is unclear. In this work, we have sought to characterise the role of oxidation in the process of CLIC1 membrane insertion. We examined how redox conditions modify the ability of CLIC1 to associate with and insert into the membrane using fluorescence quenching studies and a sucrose-loaded vesicle sedimentation assay to measure membrane binding. Our results suggest that oxidation of monomeric CLIC1, in the presence of membranes, promotes insertion into the bilayer more effectively than the oxidised CLIC1 dimer.

  15. Changes in glutathione redox cycle during diapause determination and termination in the bivoltine silkworm, Bombyx mori.

    PubMed

    Zhao, Lin-Chuan; Hou, Yi-Sheng; Sima, Yang-Hu

    2014-02-01

    To explore whether glutathione regulates diapause determination and termination in the bivoltine silkworm Bombyx mori, we monitored the changes in glutathione redox cycle in the ovary of both diapause- and nondiapause-egg producers, as well as those in diapause eggs incubated at different temperatures. The activity of thioredoxin reductase (TrxR) was detected in ovaries but not in eggs, while neither ovaries nor eggs showed activity of glutathione peroxidase. A lower reduced glutathione/oxidized glutathione (GSH/GSSG) ratio was observed in the ovary of diapause-egg producers, due to weaker reduction of oxidized glutathione (GSSG) to the reduced glutathione (GSH) catalyzed by glutathione reductase (GR) and TrxR. This indicates an oxidative shift in the glutathione redox cycle during diapause determination. Compared with the 25°C-treated diapause eggs, the 5°C-treated diapause eggs showed lower GSH/GSSG ratio, a result of stronger oxidation of GSH catalyzed by thioredoxin peroxidase and weaker reduction of GSSG catalyzed by GR. Our study demonstrated the important regulatory role of glutathione in diapause determination and termination of the bivoltine silkworm. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  16. Characterization of thiol-conjugated metabolites of ginger components shogaols in mouse and human rrine and modulation of the glutathione levels in cancer cells by [6]-shogaol

    PubMed Central

    Chen, Huadong; Soroka, Dominique N.; Hu, Yuhui; Chen, Xiaoxin; Sang, Shengmin

    2013-01-01

    Scope Shogaols, a series of major constituents in dried ginger with the most abundant being [6]-, [8]-, and [10]-shogaols, show much higher anti-cancer potencies than gingerols. Previously, we reported the mercapturic acid pathway as a major metabolic route for [6]-shogaol in mice. However, it is still unclear how the side chain length affects the metabolism of shogaols and how shogaols are metabolized in humans. Methods and results We first investigate the metabolism of [10]-shogaol in mouse urine, and then investigate the biotransformation of shogaols in human urine. Our results show that eight major thiol-conjugated metabolites of [10]-shogaol were detected in mouse urine, while six major thiol-conjugated metabolites of [6]-shogaol, two thiol-conjugated metabolites of [8]-shogaol, and two thiol-conjugated metabolites of [10]-shogaol were detected in urine collected from human after drinking ginger tea, using liquid chromatography/electrospray ionization tandem mass spectrometry. Our results clearly indicate the mercapturic acid pathway is a major metabolic route for [10]-shogaol in mice and for shogaols in human. Furthermore, we also investigated the regulation of glutathione (GSH) by [6]-shogaol in human colon cancer cells HCT-116. Our results show [6]-shogaol, after initially depleting glutathione levels, can subsequently restore and increase GSH levels over time. Conclusion Shogaols are metabolized extensively in mouse and human to form thiol-conjugated metabolites and GSH might play an important role in the cancer preventative activity of ginger. PMID:23322393

  17. Characterization of thiol-conjugated metabolites of ginger components shogaols in mouse and human urine and modulation of the glutathione levels in cancer cells by [6]-shogaol.

    PubMed

    Chen, Huadong; Soroka, Dominique N; Hu, Yuhui; Chen, Xiaoxin; Sang, Shengmin

    2013-03-01

    Shogaols, a series of major constituents in dried ginger with the most abundant being [6]-, [8]-, and [10]-shogaols, show much higher anticancer potencies than gingerols. Previously, we reported the mercapturic acid pathway as a major metabolic route for [6]-shogaol in mice. However, it is still unclear how the side chain length affects the metabolism of shogaols and how shogaols are metabolized in humans. We first investigate the metabolism of [10]-shogaol in mouse urine, and then investigate the biotransformation of shogaols in human urine. Our results show that eight major thiol-conjugated metabolites of [10]-shogaol were detected in mouse urine, while six major thiol-conjugated metabolites of [6]-shogaol, two thiol-conjugated metabolites of [8]-shogaol, and two thiol-conjugated metabolites of [10]-shogaol were detected in urine collected from human after drinking ginger tea, using LC/ESI-MS/MS. Our results clearly indicate the mercapturic acid pathway is a major metabolic route for [10]-shogaol in mice and for shogaols in human. Furthermore, we also investigated the regulation of glutathione (GSH) by [6]-shogaol in human colon cancer cells HCT-116. Our results show [6]-shogaol, after initially depleting glutathione levels, can subsequently restore and increase GSH levels over time. Shogaols are metabolized extensively in mouse and human to form thiol-conjugated metabolites and GSH might play an important role in the cancer-preventive activity of ginger. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

    PubMed

    Feary, Marc; Racher, Andrew J; Young, Robert J; Smales, C Mark

    2017-01-01

    In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for

  19. Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves1

    PubMed Central

    Jiménez, Ana; Hernández, José A.; Pastori, Gabriela; del Río, Luis A.; Sevilla, Francisca

    1998-01-01

    We investigated the relationship between H2O2 metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H2O2 content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O2·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria. PMID:9847106

  20. High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila.

    PubMed

    Chiaraviglio, Lucius; Kirby, James E

    2015-12-01

    Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. The photochemical thiol–ene reaction as a versatile method for the synthesis of glutathione S-conjugates targeting the bacterial potassium efflux system Kef† †Electronic supplementary information (ESI) available: Further experimental details and NMR spectra. See DOI: 10.1039/c5qo00436e Click here for additional data file.

    PubMed Central

    Rasmussen, Tim; Miller, Samantha; Booth, Ian R.

    2016-01-01

    The thiol–ene coupling reaction is emerging as an important conjugation reaction that is suitable for use in a biological setting. Here, we explore the utility of this reaction for the synthesis of glutathione-S-conjugates (GSX) and present a general, operationally simple, protocol with a wide substrate scope. The GSX afforded are an important class of compounds and provide invaluable molecular tools to study glutathione-binding proteins. In this study we apply the diverse library of GSX synthesised to further our understanding of the structural requirements for binding to the glutathione-binding protein, Kef, a bacterial K+ efflux system, found in many bacterial pathogens. This system is vital to the survival of bacteria upon exposure to electrophiles, and plays an essential role in the maintenance of intracellular pH and K+ homeostasis. Consequently, Kef is an appealing target for the development of novel antibacterial drugs. PMID:27110363

  2. Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation.

    PubMed

    Fonseca, Fernanda; Meneghel, Julie; Cenard, Stéphanie; Passot, Stéphanie; Morris, G John

    2016-01-01

    During cryopreservation ice nucleation and crystal growth may occur within cells or the intracellular compartment may vitrify. Whilst previous literature describes intracellular vitrification in a qualitative manner, here we measure the intracellular vitrification temperature of bacteria and yeasts under conditions relevant to cryopreservation, including the addition of high levels of permeating and nonpermeating additives and the application of rapid rates of cooling. The effects of growth conditions that are known to modify cellular freezing resistance on the intracellular vitrification temperature are also examined. For bacteria a plot of the activity on thawing against intracellular glass transition of the maximally freeze-concentrated matrix (Tg') shows that cells with the lowest value of intracellular Tg' survive the freezing process better than cells with a higher intracellular Tg'. This paper demonstrates the role of the physical state of the intracellular environment in determining the response of microbial cells to preservation and could be a powerful tool to be manipulated to allow the optimization of methods for the preservation of microorganisms.

  3. Elevation of Glutathione as a Therapeutic Strategy in Alzheimer Disease

    PubMed Central

    Pocernich, Chava B.; Butterfield, D. Allan

    2011-01-01

    Oxidative stress has been associated with the onset and progression of mild cognitive impairment (MCI) and Alzheimer disease (AD). AD and MCI brain and plasma display extensive oxidative stress as indexed by protein oxidation, lipid peroxidation, free radical formation, DNA oxidation, and decreased antioxidants. The most abundant endogenous antioxidant, glutathione, plays a significant role in combating oxidative stress. The ratio of oxidized to reduced glutathione is utilized as a measure of intensity of oxidative stress. Antioxidants have long been considered as an approach to slow down AD progression. In this review, we focus on the elevation on glutathione through N-acytl-cysteine (NAC) and γ-glutamylcysteine ethyl ester (GCEE) as a potential therapeutic approach for Alzheimer disease. PMID:22015471

  4. Cysteic Acid in Dietary Keratin is Metabolized to Glutathione and Liver Taurine in a Rat Model of Human Digestion

    PubMed Central

    Wolber, Frances M.; McGrath, Michelle; Jackson, Felicity; Wylie, Kim; Broomfield, Anne

    2016-01-01

    Poultry feathers, consisting largely of keratin, are a low-value product of the poultry industry. The safety and digestibility of a dietary protein produced from keratin (KER) was compared to a cysteine-supplemented casein-based diet in a growing rat model for four weeks. KER proved to be an effective substitute for casein at 50% of the total dietary protein, with no changes in the rats’ food intake, weight gain, organ weight, bone mineral density, white blood cell counts, liver glutathione, or blood glutathione. Inclusion of KER in the diet reduced total protein digestibility from 94% to 86% but significantly increased total dietary cysteine uptake and subsequent liver taurine levels. The KER diet also significantly increased caecum weight and significantly decreased fat digestibility, resulting in a lower proportion of body fat, and induced a significant increase in blood haemoglobin. KER is therefore a safe and suitable protein substitute for casein, and the cysteic acid in keratin is metabolised to maintain normal liver and blood glutathione levels. PMID:26907334

  5. Rat lung glutathione S-transferases. Evidence for two distinct types of 22000-Mr subunits.

    PubMed Central

    Singh, S V; Partridge, C A; Awasthi, Y C

    1984-01-01

    Two immunologically distinct types of 22000-Mr subunits are present in rat lung glutathione S-transferases. One of these subunits is probably similar to Ya subunits of rat liver glutathione S-transferases, whereas the other subunit Ya' is immunologically distinct. Glutathione S-transferase II (pI7.2) of rat lung is a heterodimer (YaYa') of these subunits, and glutathione S-transferase VI (pI4.8) of rat lung is a homodimer of Ya' subunits. On hybridization in vitro of the subunits of glutathione S-transferase II of rat lung three active dimers having pI values 9.4, 7.2 and 4.8 are obtained. Immunological properties and substrate specificities indicate that the hybridized enzymes having pI7.2 and 4.8 correspond to glutathione S-transferases II and VI of rat lung respectively. Images Fig. 1. Fig. 5. PMID:6433888

  6. Regression of Aflatoxin B1-Induced Hepatocellular Carcinomas by Reduced Glutathione

    NASA Astrophysics Data System (ADS)

    Novi, Anna M.

    1981-05-01

    Reduced glutathione administered to rats bearing aflatoxin B1-induced liver tumors caused regression of tumor growth and resulted in survival of the animals. Since glutathione is a harmless natural product, it merits further investigation as a potential antitumor drug for humans.

  7. Strategic formulation of apigenin-loaded PLGA nanoparticles for intracellular trafficking, DNA targeting and improved therapeutic effects in skin melanoma in vitro.

    PubMed

    Das, Sreemanti; Das, Jayeeta; Samadder, Asmita; Paul, Avijit; Khuda-Bukhsh, Anisur Rahman

    2013-11-25

    The aim of the present study was the evaluation of anti-proliferative potentials of apigenin (Ap), (a dietary flavonoid) loaded in poly (lactic-co-glycolide) nanoparticles (NAp) in A375 cells in vitro. NAp was characterized for particle size, morphology, zeta potential, drug release and encapsulation. Cellular entry and intracellular localization of NAp were assessed by transmission electron and confocal microscopies. Circular dichroic spectral analysis and stability curve for Gibb's free energy of dsDNA of A375 cells were also analyzed. DNA fragmentation, intracellular ROS accumulation, superoxide-dismutase activity, intracellular glutathione-reductase content and mitochondrial functioning through relevant markers like mitochondrial transmembrane potential, ATPase activity, ATP/ADP ratio, volume changes/swelling, cytochrome-c release, expressions of Apaf-1, bax, bcl-2, caspase-9, 3, and PARP cleavage were analyzed. NAp produced better effects due to their smaller size, faster mobility and site-specific action. Photostability studies revealed that PLGA encapsulations were efficient at preserving apigenin ultraviolet-light mediated photodegradation. NAp readily entered cancer cells, could intercalate with dsDNA, inducing conformational change. Corresponding increase in ROS accumulation and depletion of the antioxidant enzyme activities exacerbated DNA damage, mediating apoptosis through mitochondrial dysfunction. Overall results indicate that therapeutic efficacy of NAp may be enhanced by PLGA nanoparticle formulations to have better ameliorative potentials in combating skin melanoma. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  8. 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.

  9. [Effect of bemethyl on the glutathione system in the rat liver in acute hypoxia].

    PubMed

    Zarubina, I V; Mironova, O P

    2002-01-01

    The effect of bemithyl on the state of liver glutathione system was studied in rats under acute hypoxic hypoxia conditions modeled by "elevating" animals in a pressure chamber up to an altitude of 8000-11,000 m for 30 min. Bemithyl (25 mg/kg, i.p.) administered 30 min before the hypoxia onset, prevents a decrease in the content of reduced glutathione and SH groups and impedes a drop in the activity of glutathione reductase and glutathione peroxidase. By means of the inhibition analysis using actinomycin D (a protein synthesis inhibitor), it was established that the protective action of bemithyl is related to the ability of enhancing the synthesis of antioxidant enzymes in the liver glutathione system.

  10. Iodinated chlorin p6 copper complex induces anti-proliferative effect in oral cancer cells through elevation of intracellular reactive oxygen species.

    PubMed

    Sarbadhikary, Paromita; Dube, Alok

    2017-11-01

    We investigated the anticancer chemotoxicity of previously reported iodinated chlorin p 6 copper complex (ICp 6 -Cu), a novel chlorophyll derivative in which copper is attached to the side chain carboxylate groups via coordination. Human oral carcinoma cells NT8e, 4451 and the non-cancerous keratinocyte HaCaT cells were treated with ICp 6 -Cu for 48 h in dark and cell viability, proliferation and morphological alterations were examined. ICp 6 -Cu showed pronounced cytotoxicity in cancer cells with IC 50 ∼40 μM, whereas, the viability of HaCaT cells was not affected. Cell proliferation assay revealed that ICp 6 -Cu at IC 50 concentration led to complete inhibition of cell proliferation in both the cell lines. Cell morphology studied by confocal microscopy showed absence of cell death via necrosis or apoptosis. Instead, the treated cells displayed distinct features of non-apoptotic death such as highly vacuolated cytoplasm, lysosomal membrane permeabilization and damage to cytoskeleton F-actin filaments. In addition, ICp 6 -Cu treatment led to time dependent increase in the intracellular level of reactive oxygen species (ROS) and the cytotoxicity of ICp 6 -Cu was significantly inhibited by pre-treatment of cells with antioxidants (glutathione and trolox). These findings revealed that ICp 6 -Cu is a potent chemotoxic agent which can induce cytotoxic effect in cancer cells through elevation of intracellular ROS. It is suggested that ICp 6 -Cu may provide tumor selective chemotoxicity by exploiting difference of redox environment in normal and cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Crystallization and preliminary X-ray crystallographic analysis of Escherichia coli glutaredoxin 2 in complex with glutathione and of a cysteine-less variant without glutathione

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

    Sheng, Ju; Ye, Jun; Rosen, Barry P., E-mail: brosen@med.wayne.edu

    2007-04-01

    Glutaredoxin 2 from E. coli was cocrystallized with glutathione and data were collected to 1.60 Å. A mutant with the active-site residues Cys9 and Cys12 changed to serine was crystallized in the absence of glutathione and data were collected to 2.4 Å. Glutaredoxin 2 (Grx2) from Escherichia coli is larger in size than classical glutaredoxins. It is extremely efficient in the catalysis of reduced glutathione-dependent disulfide reduction. A complex of Grx2 with reduced glutathione (GSH) has been crystallized. Data were collected to 1.60 Å. The crystals belong to space group P3{sub 2}21, with one Grx2–GSH complex in the asymmetric unit.more » The unit-cell parameters are a = b = 50.10, c = 152.47 Å. A Grx2 mutant, C9S/C12S, which cannot form a disulfide bond with GSH was also crystallized. The crystals diffracted to 2.40 Å and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with one molecule in the asymmetric unit. The unit-cell parameters are a = 28.16, b = 78.65, c = 89.16 Å.« less

  12. The Chilean wild raspberry (Rubus geoides Sm.) increases intracellular GSH content and protects against H2O2 and methylglyoxal-induced damage in AGS cells.

    PubMed

    Jiménez-Aspee, Felipe; Theoduloz, Cristina; Ávila, Felipe; Thomas-Valdés, Samanta; Mardones, Claudia; von Baer, Dietrich; Schmeda-Hirschmann, Guillermo

    2016-03-01

    The Chilean raspberry Rubus geoides Sm. (Rosaceae) is a native species occurring in the Patagonia. Five R. geoides samples were assessed for phenolic content and composition, antioxidant activity, effect on total reduced glutathione (GSH) synthesis and protective effect against H2O2 and methylglyoxal (MGO)-induced stress in epithelial gastric AGS cells. The HPLC-DAD/ESI-MS profiles allowed the tentative identification of 39 phenolics including flavonol glycosides and tannins. R. geoides presented higher total phenolic and flavonoid content than Rubus idaeus. Two out of the five phenolic enriched R. geoides extracts (PEEs) exhibited better antioxidant activity than R. idaeus in the DPPH, FRAP and TEAC assays. A significant cytoprotective activity was observed when AGS cells were pre-incubated with extracts and subsequently challenged with H2O2 or MGO. Treatment with the PEEs increased the intracellular GSH content. R. geoides fruit extracts may induce the activation of intracellular protection mechanisms against oxidative and dicarbonyl-induced stress. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Genetics Home Reference: glutathione synthetase deficiency

    MedlinePlus

    ... Ristoff E, Larsson A. Inborn errors in the metabolism of glutathione. Orphanet J Rare Dis. 2007 Mar 30;2:16. Review. Citation on PubMed or Free article on PubMed Central Wu G, Fang YZ, Yang S, ...

  14. Long-term dietary supplementation with cystathionine improves tissue glutathione in ageing rats.

    PubMed

    Pouget, Mélanie; Perrot, Marie; Denis, Philippe; Vuichoud, Jacques; Dardevet, Dominique; Vidal, Karine; Breuillé, Denis; Papet, Isabelle

    2016-08-01

    Ageing is associated with decrease in tissue glutathione that can be reduced by food fortification with the amino acid cysteine. However, cysteine is not stable in solution and generates bad taste. Cystathionine, the direct precursor of cysteine, could be a valuable alternative. This study aimed to determine whether long-term dietary supplementation with cystathionine induces an increase in glutathione pools. Aged rats (20.5-month-old) were fed ad libitum during 29 weeks with either a cystathionine-supplemented diet (7.3 g/kg, n = 90 rats) or a control iso-nitrogenous alanine-supplemented diet (2.9 g/kg, n = 90 rats). Cystathionine was detected in the plasma of the cystathionine-supplemented rats but not in the control alanine-supplemented rats. Cystathionine increased glutathione concentrations in liver, small intestine and gastrocnemius muscle (P < 0.03). No adverse effect was observed. Cystathionine supplementation being able to increase moderately glutathione in healthy old rats could be considered as a candidate for nutritional supports aiming to revert the stronger glutathione depletions occurring in unhealthy elderly.

  15. Enhancement of Combined Umami and Salty Taste by Glutathione in the Human Tongue and Brain.

    PubMed

    Goto, Tazuko K; Yeung, Andy Wai Kan; Tanabe, Hiroki C; Ito, Yuki; Jung, Han-Sung; Ninomiya, Yuzo

    2016-09-01

    Glutathione, a natural substance, acts on calcium receptors on the tongue and is known to enhance basic taste sensations. However, the effects of glutathione on brain activity associated with taste sensation on the tongue have not been determined under standardized taste delivery conditions. In this study, we investigated the sensory effect of glutathione on taste with no effect of the smell when glutathione added to a combined umami and salty taste stimulus. Twenty-six volunteers (12 women and 14 men; age 19-27 years) performed a sensory evaluation of taste of a solution of monosodium L-glutamate and sodium chloride, with and without glutathione. The addition of glutathione changed taste qualities and significantly increased taste intensity ratings under standardized taste delivery conditions (P < 0.001). Functional magnetic resonance imaging showed that glutathione itself elicited significant activation in the left ventral insula. These results are the first to demonstrate the enhancing effect of glutathione as reflected by brain data while tasting an umami and salty mixture. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Zn2+-dependent redox switch in the intracellular T1-T1 interface of a Kv channel.

    PubMed

    Wang, Guangyu; Strang, Candace; Pfaffinger, Paul J; Covarrubias, Manuel

    2007-05-04

    The thiol-based redox regulation of proteins plays a central role in cellular signaling. Here, we investigated the redox regulation at the Zn(2+) binding site (HX(5)CX(20)CC) in the intracellular T1-T1 inter-subunit interface of a Kv4 channel. This site undergoes conformational changes coupled to voltage-dependent gating, which may be sensitive to oxidative stress. The main results show that internally applied nitric oxide (NO) inhibits channel activity profoundly. This inhibition is reversed by reduced glutathione and suppressed by intracellular Zn(2+), and at least two Zn(2+) site cysteines are required to observe the NO-induced inhibition (Cys-110 from one subunit and Cys-132 from the neighboring subunit). Biochemical evidence suggests strongly that NO induces a disulfide bridge between Cys-110 and Cys-132 in intact cells. Finally, further mutational studies suggest that intra-subunit Zn(2+) coordination involving His-104, Cys-131, and Cys-132 protects against the formation of the inhibitory disulfide bond. We propose that the interfacial T1 Zn(2+) site of Kv4 channels acts as a Zn(2+)-dependent redox switch that may regulate the activity of neuronal and cardiac A-type K(+) currents under physiological and pathological conditions.

  17. Glutathione system participation in thoracic aneurysms from patients with Marfan syndrome.

    PubMed

    Zúñiga-Muñoz, Alejandra María; Pérez-Torres, Israel; Guarner-Lans, Verónica; Núñez-Garrido, Elías; Velázquez Espejel, Rodrigo; Huesca-Gómez, Claudia; Gamboa-Ávila, Ricardo; Soto, María Elena

    2017-05-01

    Aortic dilatation in Marfan syndrome (MFS) is progressive. It is associated with oxidative stress and endothelial dysfunction that contribute to the early acute dissection of the vessel and can result in rupture of the aorta and sudden death. We evaluated the participation of the glutathione (GSH) system, which could be involved in the mechanisms that promote the formation and progression of the aortic aneurysms in MFS patients. Aortic aneurysm tissue was obtained during chest surgery from eight control subjects and 14 MFS patients. Spectrophotometrical determination of activity of glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), lipid peroxidation (LPO) index, carbonylation, total antioxidant capacity (TAC), and concentration of reduced and oxidized glutathione (GSH and GSSG respectively), was performed in the homogenate from aortic aneurysm tissue. LPO index, carbonylation, TGF-β1, and GR activity were increased in MFS patients (p < 0.04), while TAC, GSH/GSSG ratio, GPx, and GST activity were significantly decreased (p < 0.04). The depletion of GSH, in spite of the elevated activity of GR, not only diminished the activity of GSH-depend GST and GPx, but increased LPO, carbonylation and decreased TAC. These changes could promote the structural and functional alterations in the thoracic aorta of MFS patients.

  18. 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

  19. 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

  20. Deficient synthesis of glutathione underlies oxidative stress in aging and can be corrected by dietary cysteine and glycine supplementation1234

    PubMed Central

    Patel, Sanjeet G; Guthikonda, Anuradha P; Reid, Marvin; Balasubramanyam, Ashok; Taffet, George E; Jahoor, Farook

    2011-01-01

    Background: Aging is associated with oxidative stress, but underlying mechanisms remain poorly understood. Objective: We tested whether glutathione deficiency occurs because of diminished synthesis and contributes to oxidative stress in aging and whether stimulating glutathione synthesis with its precursors cysteine and glycine could alleviate oxidative stress. Design: Eight elderly and 8 younger subjects received stable-isotope infusions of [2H2]glycine, after which red blood cell (RBC) glutathione synthesis and concentrations, plasma oxidative stress, and markers of oxidant damage (eg, F2-isoprostanes) were measured. Elderly subjects were restudied after 2 wk of glutathione precursor supplementation. Results: Compared with younger control subjects, elderly subjects had markedly lower RBC concentrations of glycine (486.7 ± 28.3 compared with 218.0 ± 23.7 μmol/L; P < 0.01), cysteine (26.2 ± 1.4 compared with 19.8 ± 1.3 μmol/L; P < 0.05), and glutathione (2.08 ± 0.12 compared with 1.12 ± 0.18 mmol/L RBCs; P < 0.05); lower glutathione fractional (83.14 ± 6.43% compared with 45.80 ± 5.69%/d; P < 0.01) and absolute (1.73 ± 0.16 compared with 0.55 ± 0.12 mmol/L RBCs per day; P < 0.01) synthesis rates; and higher plasma oxidative stress (304 ± 16 compared with 346 ± 20 Carratelli units; P < 0.05) and plasma F2-isoprostanes (97.7 ± 8.3 compared with 136.3 ± 11.3 pg/mL; P < 0.05). Precursor supplementation in elderly subjects led to a 94.6% higher glutathione concentration, a 78.8% higher fractional synthesis rate, a 230.9% higher absolute synthesis rate, and significantly lower plasma oxidative stress and F2-isoprostanes. No differences in these measures were observed between younger subjects and supplemented elderly subjects. Conclusions: Glutathione deficiency in elderly humans occurs because of a marked reduction in synthesis. Dietary supplementation with the glutathione precursors cysteine and glycine fully restores glutathione synthesis and

  1. [Effect of extremely low frequency magnetic field on glutathione in rat muscles].

    PubMed

    Ciejka, Elzbieta; Jakubowska, Ewa; Zelechowska, Paulina; Huk-Kolega, Halina; Kowalczyk, Agata; Goraca, Anna

    2014-01-01

    Free radicals (FR) are atoms, molecules or their fragments. Their excess leads to the development of oxidizing stress, the cause of many neoplastic, neurodegenerative and inflammatory diseases, and aging of the organism. Industrial pollution, tobacco smoke, ionizing radiation, ultrasound and magnetic field are the major FR exogenous sources. The low frequency magnetic field is still more commonly applied in the physical therapy. The aim of the presented study was to evaluate the effect of extremely low frequency magnetic field used in the magnetotherapy on the level of total glutathione, oxidized and reduced, and the redox state of the skeletal muscle cells, depending on the duration of exposure to magnetic field. The male rats, weight of 280-300 g, were randomly devided into 3 experimental groups: controls (group I) and treatment groups exposed to extremely low frequency magnetic field (ELF-MF) (group II exposed to 40 Hz, 7 mT for 0.5 h/day for 14 days and group III exposed to 40 Hz, 7 mT for 1 h/day for 14 days). Control rats were kept in a separate room not exposed to extremely low frequency magnetic field. Immediately after the last exposure, part of muscles was taken under pentobarbital anesthesia. Total glutathione, oxidized and reduced, and the redox state in the muscle tissue of animals were determined after exposure to magnetic fields. Exposure to low magnetic field: 40 Hz, 7 mT for 30 min/day and 60 min/day for 2 weeks significantly increased the total glutathione levels in the skeletal muscle compared to the control group (p < 0.001). Exposure to magnetic fields used in the magnetic therapy plays an important role in the development of adaptive mechanisms responsible for maintaining the oxidation-reduction balance in the body and depends on exposure duration.

  2. Subcellular characteristics of functional intracellular renin–angiotensin systems☆

    PubMed Central

    Abadir, Peter M.; Walston, Jeremy D.; Carey, Robert M.

    2013-01-01

    The renin–angio tensin system (RAS) is now regarded as an integral component in not only the development of hypertension, but also in physiologic and pathophysiologic mechanisms in multiple tissues and chronic disease states. While many of the endocrine (circulating), paracrine (cell-to-different cell) and autacrine (cell-to-same cell) effects of the RAS are believed to be mediated through the canonical extracellular RAS, a complete, independent and differentially regulated intracellular RAS (iRAS) has also been proposed. Angiotensinogen, the enzymes renin and angiotensin-converting enzyme (ACE) and the angiotensin peptides can all be synthesized and retained intracellularly. Angiotensin receptors (types I and 2) are also abundant intracellularly mainly at the nuclear and mitochondrial levels. The aim of this review is to focus on the most recent information concerning the subcellular localization, distribution and functions of the iRAS and to discuss the potential consequences of activation of the subcellular RAS on different organ systems. PMID:23032352

  3. Identification of Small-Molecule Frequent Hitters of Glutathione S-Transferase-Glutathione Interaction.

    PubMed

    Brenke, Jara K; Salmina, Elena S; Ringelstetter, Larissa; Dornauer, Scarlett; Kuzikov, Maria; Rothenaigner, Ina; Schorpp, Kenji; Giehler, Fabian; Gopalakrishnan, Jay; Kieser, Arnd; Gul, Sheraz; Tetko, Igor V; Hadian, Kamyar

    2016-07-01

    In high-throughput screening (HTS) campaigns, the binding of glutathione S-transferase (GST) to glutathione (GSH) is used for detection of GST-tagged proteins in protein-protein interactions or enzyme assays. However, many false-positives, so-called frequent hitters (FH), arise that either prevent GST/GSH interaction or interfere with assay signal generation or detection. To identify GST-FH compounds, we analyzed the data of five independent AlphaScreen-based screening campaigns to classify compounds that inhibit the GST/GSH interaction. We identified 53 compounds affecting GST/GSH binding but not influencing His-tag/Ni(2+)-NTA interaction and general AlphaScreen signals. The structures of these 53 experimentally identified GST-FHs were analyzed in chemoinformatic studies to categorize substructural features that promote interference with GST/GSH binding. Here, we confirmed several existing chemoinformatic filters and more importantly extended them as well as added novel filters that specify compounds with anti-GST/GSH activity. Selected compounds were also tested using different antibody-based GST detection technologies and exhibited no interference clearly demonstrating specificity toward their GST/GSH interaction. Thus, these newly described GST-FH will further contribute to the identification of FH compounds containing promiscuous substructures. The developed filters were uploaded to the OCHEM website (http://ochem.eu) and are publicly accessible for analysis of future HTS results. © 2016 Society for Laboratory Automation and Screening.

  4. Glutathione reduces cytotoxicity of polyethyleneimine coated magnetic nanoparticles in CHO cells.

    PubMed

    Strojan, Klemen; Lojk, Jasna; Bregar, Vladimir B; Veranič, Peter; Pavlin, Mojca

    2017-06-01

    Polyethyleneimine (PEI) is a polycationic compound frequently used as a transfection agent. However, cytotoxicity of PEI and PEI-coated nanoparticles (PEI NPs) is still a major obstacle in its use. In this study we report a method for reducing cytotoxicity of PEI NPs by addition of glutathione in NPs synthesis. Glutathione reduced cytotoxic effects for at least 30% and decreased observed oxidative stress response compared to standard formulation. Results showed that the effect was partially due to reduced zeta potential and partially due to protective antioxidant properties of glutathione. Addition of glutathione to cell culture media with concurrent exposure to PEI NPs proved to be insufficient for cytotoxicity reduction. Additionally, we compared internalization pathways of both PEI NPs and GSH NPs. NPs were only found in endosomes and no NPs were found free in the cytosol, as would be expected according to so called proton sponge hypothesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. 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

  6. Arabidopsis dehydroascorbate reductase 1 and 2 modulate redox states of ascorbate-glutathione cycle in the cytosol in response to photooxidative stress.

    PubMed

    Noshi, Masahiro; Yamada, Hiroki; Hatanaka, Risa; Tanabe, Noriaki; Tamoi, Masahiro; Shigeoka, Shigeru

    2017-03-01

    Ascorbate and glutathione are indispensable cellular redox buffers and allow plants to acclimate stressful conditions. Arabidopsis contains three functional dehydroascorbate reductases (DHAR1-3), which catalyzes the conversion of dehydroascorbate into its reduced form using glutathione as a reductant. We herein attempted to elucidate the physiological role in DHAR1 and DHAR2 in stress responses. The total DHAR activities in DHAR knockout Arabidopsis plants, dhar1 and dhar2, were 22 and 92%, respectively, that in wild-type leaves. Under high light (HL), the levels of total ascorbate and dehydroascorbate were only reduced and increased, respectively, in dhar1. The oxidation of glutathione under HL was significantly inhibited in both dhar1 and dhar2, while glutathione contents were only enhanced in dhar1. The dhar1 showed stronger visible symptoms than the dhar2 under photooxidative stress conditions. Our results demonstrated a pivotal role of DHAR1 in the modulation of cellular redox states under photooxidative stress.

  7. Silver ions increase plasma membrane permeability through modulation of intracellular calcium levels in tobacco BY-2 cells.

    PubMed

    Klíma, Petr; Laňková, Martina; Vandenbussche, Filip; Van Der Straeten, Dominique; Petrášek, Jan

    2018-05-01

    Silver ions increase plasma membrane permeability for water and small organic compounds through their stimulatory effect on plasma membrane calcium channels, with subsequent modulation of intracellular calcium levels and ion homeostasis. The action of silver ions at the plant plasma membrane is largely connected with the inhibition of ethylene signalling thanks to the ability of silver ion to replace the copper cofactor in the ethylene receptor. A link coupling the action of silver ions and cellular auxin efflux has been suggested earlier by their possible direct interaction with auxin efflux carriers or by influencing plasma membrane permeability. Using tobacco BY-2 cells, we demonstrate here that besides a dramatic increase of efflux of synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthalene acetic acid (NAA), treatment with AgNO 3 resulted in enhanced efflux of the cytokinin trans-zeatin (tZ) as well as the auxin structural analogues tryptophan (Trp) and benzoic acid (BA). The application of AgNO 3 was accompanied by gradual water loss and plasmolysis. The observed effects were dependent on the availability of extracellular calcium ions (Ca 2+ ) as shown by comparison of transport assays in Ca 2+ -rich and Ca 2+ -free buffers and upon treatment with inhibitors of plasma membrane Ca 2+ -permeable channels Al 3+ and ruthenium red, both abolishing the effect of AgNO 3 . Confocal microscopy of Ca 2+ -sensitive fluorescence indicator Fluo-4FF, acetoxymethyl (AM) ester suggested that the extracellular Ca 2+ availability is necessary to trigger the response to silver ions and that the intracellular Ca 2+ pool alone is not sufficient for this effect. Altogether, our data suggest that in plant cells the effects of silver ions originate from the primal modification of the internal calcium levels, possibly by their interaction with Ca 2+ -permeable channels at the plasma membrane.

  8. Quantifying intracellular hydrogen peroxide perturbations in terms of concentration

    PubMed Central

    Huang, Beijing K.; Sikes, Hadley D.

    2014-01-01

    Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS) in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies. PMID:25460730

  9. Choroid plexus glutathione peroxidases are instrumental in protecting the brain fluid environment from hydroperoxides during postnatal development.

    PubMed

    Saudrais, Elodie; Strazielle, Nathalie; Ghersi-Egea, Jean-Francois

    2018-06-27

    Hydrogen peroxide, released at low physiological concentration, is involved in different cell signaling pathways during brain development. When released at supraphysiological concentrations in brain fluids following an inflammatory, hypoxic or toxic stress, it can initiate lipid peroxidation, protein and nucleic acid damage and contribute to long-term neurological impairment associated with perinatal diseases. We found high glutathione peroxidase and glutathione reductase enzymatic activities in both lateral and fourth ventricle choroid plexus tissue isolated from developing rats, in comparison to the cerebral cortex and liver. Consistent with these, a high protein expression of glutathione peroxidases 1 and 4 was observed in choroid plexus epithelial cells, which form the blood-cerebrospinal fluid barrier. Live choroid plexuses isolated from newborn rats were highly efficient in detoxifying H2O2 from mock cerebrospinal fluid, illustrating the capacity of the choroid plexuses to control H2O2 concentration in the ventricular system of the brain. We used a differentiated cellular model of the blood-cerebrospinal fluid barrier coupled to kinetic and inhibition analyses to show that glutathione peroxidases are more potent than catalase to detoxify extracellular H2O2 at concentrations up to 250 µM. The choroidal cells also formed an enzymatic barrier preventing blood-borne hydroperoxides to reach the cerebrospinal fluid. These data point out the choroid plexuses as key structures in the control of hydroperoxide levels in the cerebral fluid environment during development, at a time when the protective glial cell network is still immature. Glutathione peroxidases are the main effectors of this choroidal hydroperoxide inactivation.

  10. High Intracellular Chloride Slows the Decay of Glycinergic Currents

    PubMed Central

    Pitt, Samantha J.; Sivilotti, Lucia G.; Beato, Marco

    2009-01-01

    The time course of currents mediated by native and recombinant glycine receptors was examined with a combination of rapid agonist applications to outside-out patches and single-channel recording. The deactivation time constant of currents evoked by brief, saturating pulses of glycine is profoundly affected by the chloride concentration on the intracellular side of the cell membrane. Deactivation was threefold slower when intracellular chloride was increased from a low level (10 mm), similar to that observed in living mature neurons, to 131 mm (“symmetrical” chloride, often used in pipette internal solutions). Single-channel analysis revealed that high chloride has its greatest effect on the channel closing rate, slowing it by a factor of 2 compared with the value we estimated in the cell-attached mode (in which the channels are at physiological intracellular chloride concentrations). The same effect of chloride was observed when glycinergic evoked synaptic currents were recorded from juvenile rat spinal motoneurons in vitro, because the decay time constant was reduced from ∼7ms to ∼3 ms when cells were dialyzed with 10 mm chloride intracellular recording solution. Our results indicate that the time course of glycinergic synaptic inhibition in intact neurons is much faster than is estimated by measurements in symmetrical chloride and can be modulated by changes in intracellular chloride concentration in the range that can occur in physiological or pathological conditions. PMID:18987182

  11. Improvements in the bread-making quality of gluten-free rice batter by glutathione.

    PubMed

    Yano, Hiroyuki

    2010-07-14

    The wide prevalence of celiac disease and wheat allergy has led to a growing demand for gluten-free foods. Rice proteins do not possess the viscoelastic properties typically found in gluten, thus making rice flour unsuitable for the production of yeast-leavened products. In the present study, we found that the addition of glutathione to rice batter improves its gas-retaining properties. Glutathione was found to prevent the formation of the disulfide-linked macromolecular protein barrier, which is reported to confer resistance to the deformation of rice batter in the baking process. Also, glutathione appeared to gelatinize rice starch at lower temperatures. Microstructure analyses of glutathione-added rice bread revealed it to have a perforated structure like wheat bread but with a smoother-looking surface. These data collectively suggest that glutathione facilitates the deformation of rice batter, thus increasing its elasticity in the early stages of bread baking and the volume of the resulting bread.

  12. 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.

  13. 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.

  14. Comparison of the biological effects of {sup 18}F at different intracellular levels

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

    Kashino, Genro, E-mail: kashino@oita-u.ac.jp; Hayashi, Kazutaka; Douhara, Kazumasa

    Highlights: • We estimated the inductions of DNA DSB in cell treated with {sup 18}F-FDG. • We found that inductions of DNA DSB are dependent on accumulation of {sup 18}F in cell. • Accumulation of {sup 18}F in cell may be indispensable for risk estimation of PET. - Abstract: We herein examined the biological effects of cells treated with {sup 18}F labeled drugs for positron emission tomography (PET). The relationship between the intracellular distribution of {sup 18}F and levels of damaged DNA has yet to be clarified in detail. We used culture cells (Chinese Hamster Ovary cells) treated with twomore » types of {sup 18}F labeled drugs, fluorodeoxyglucose (FDG) and fluorine ion (HF). FDG efficiently accumulated in cells, whereas HF did not. To examine the induction of DNA double strand breaks (DSB), we measured the number of foci for 53BP1 that formed at the site of DNA DSB. The results revealed that although radioactivity levels were the same, the induction of 53BP1 foci was stronger in cells treated with {sup 18}F-FDG than in those treated with {sup 18}F-HF. The clonogenic survival of cells was significantly lower with {sup 18}F-FDG than with {sup 18}F-HF. We concluded that the efficient accumulation of {sup 18}F in cells led to stronger biological effects due to more severe cellular lethality via the induction of DNA DSB.« less

  15. Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and Viability

    PubMed Central

    Das, Theerthankar; Simone, Martin; Ibugo, Amaye I.; Witting, Paul K.; Manefield, Mike; Manos, Jim

    2017-01-01

    Pyocyanin secreted by Pseudomonas aeruginosa is a virulence factor that damages epithelial cells during infection through the action of reactive oxygen species, however, little is known about its direct effect on biofilms. We demonstrated that pyocyanin-producing P. aeruginosa strains (PA14WT, DKN370, AES-1R, and AES-2) formed robust biofilms in contrast to the poorly formed biofilms of the pyocyanin mutant PA14ΔphzA-G and the low pyocyanin producer AES-1M. Addition of DNase I and reduced glutathione (GSH) significantly reduced biofilm biomass of pyocyanin-producing strains (P < 0.05) compared to non-pyocyanin producers. Subsequently we showed that a combined treatment comprising: GSH + DNase I + antibiotic, disrupted and reduced biofilm biomass up to 90% in cystic fibrosis isolates AES-1R, AES-2, LESB58, and LES431 and promoted lung epithelial cell (A549) recovery and growth. We also showed that exogenously added GSH restored A549 epithelial cell glutathione reductase activity in the presence of pyocyanin through recycling of GSSG to GSH and consequently increased total intracellular GSH levels, inhibiting oxidative stress, and facilitating cell growth and confluence. These outcomes indicate that GSH has multiple roles in facilitating a return to normal epithelial cell growth after insult by pyocyanin. With increased antibiotic resistance in many bacterial species, there is an urgency to establish novel antimicrobial agents. GSH is able to rapidly and comprehensively destroy P. aeruginosa associated biofilms while at a same time assisting in the recovery of host cells and re-growth of damaged tissue. PMID:29312161

  16. Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants

    PubMed Central

    Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Mendes-Silva, Leonardo; Pinho Melo, Eduardo; Harding, Heather P; Ron, David

    2014-01-01

    Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER. DOI: http://dx.doi.org/10.7554/eLife.03421.001 PMID:25073928

  17. Mercury(II) Complex Formation With Glutathione in Alkaline Aqueous Solution

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

    Mah, V.; Jalilehvand, F.

    2009-05-19

    The structure and speciation of the complexes formed between mercury(II) ions and glutathione (GSH = L-glutamyl-L-cysteinyl-glycine) have been studied for a series of alkaline aqueous solutions (C{sub Hg{sup 2+}} {approx} 18 mmol dm{sup -3} and C{sub GSH} = 40-200 mmol dm{sup -3} at pH {approx} 10.5) by means of extended X-ray absorption fine structure (EXAFS) and {sup 199}Hg NMR spectroscopy at ambient temperature. The dominant complexes are [Hg(GS){sub 2}]{sup 4-} and [Hg(GS){sub 3}]{sup 7-}, with mean Hg-S bond distances of 2.32(1) and 2.42(2) {angstrom} observed in digonal and trigonal Hg-S coordination, respectively. The proportions of the Hg{sup 2+}-glutathione complexes weremore » evaluated by fitting linear combinations of model EXAFS oscillations representing each species to the experimental EXAFS spectra. The [Hg(GS){sub 4}]{sup 10-} complex, with four sulfur atoms coordinated at a mean Hg-S bond distance of 2.52(2) {angstrom}, is present in minor amounts (<30%) in solutions containing a large excess of glutathione (C{sub GSH} {ge} 160 mmol dm{sup -3}). Comparable alkaline mercury(II) cysteine (H{sub 2}Cys) solutions were also investigated and a reduced tendency to form higher complexes was observed, because the deprotonated amino group of Cys{sup 2-} allows the stable [Hg(S,N-Cys){sub 2}]{sup 2-} chelate to form. The effect of temperature on the distribution of the Hg{sup 2+}-glutathione complexes was studied by comparing the EXAFS spectra at ambient temperature and at 25 K of a series of glycerol/water (33/67, v/v) frozen glasses with and C{sub Hg{sup 2+}} {approx} 7 mmol dm{sup -3} and C{sub GSH} = 16-81 mmol dm{sup -3}. Complexes with high Hg-S coordination numbers, [Hg(GS){sub 3}]{sup 7-} and [Hg(GS){sub 4}]{sup 10-}, became strongly favored when just a moderate excess of glutathione (C{sub GSH} {ge} 28 mmol dm{sup -3}) was used in the glassy samples, as expected for a stepwise exothermic bond formation. Addition of glycerol had no effect on the Hg(II)-glutathione

  18. JS-K, a glutathione S-transferase-activated nitric oxide donor with antineoplastic activity in malignant gliomas.

    PubMed

    Weyerbrock, Astrid; Osterberg, Nadja; Psarras, Nikolaos; Baumer, Brunhilde; Kogias, Evangelos; Werres, Anna; Bette, Stefanie; Saavedra, Joseph E; Keefer, Larry K; Papazoglou, Anna

    2012-02-01

    Glutathione S-transferases (GSTs) control multidrug resistance and are upregulated in many cancers, including malignant gliomas. The diazeniumdiolate JS-K generates nitric oxide (NO) on enzymatic activation by glutathione and GST, showing promising NO-based anticancer efficacy. To evaluate the role of NO-based antitumor therapy with JS-K in U87 gliomas in vitro and in vivo. U87 glioma cells and primary glioblastoma cell lines were exposed to JS-K and a variety of inhibitors to study cell death by necrosis, apoptosis, and other mechanisms. GST expression was evaluated by immunocytochemistry, polymerase chain reaction, and Western blot, and NO release from JS-K was studied with a NO assay. The growth-inhibitory effect of JS-K was studied in a U87 xenograft model in vivo. Dose-dependent inhibition of cell proliferation was observed in human U87 glioma cells and primary glioblastoma cells in vitro. Cell death was partially induced by caspase-dependent apoptosis, which could be blocked by Z-VAD-FMK and Q-VD-OPH. Inhibition of GST by sulfasalazine, cGMP inhibition by ODQ, and MEK1/2 inhibition by UO126 attenuated the antiproliferative effect of JS-K, suggesting the involvement of various intracellular death signaling pathways. Response to JS-K correlated with mRNA and protein expression of GST and the amount of NO released by the glioma cells. Growth of U87 xenografts was reduced significantly, with immunohistochemical evidence for increased necrosis and apoptosis and reduced proliferation. Our data show for the first time the potent antiproliferative effect of JS-K in gliomas in vitro and in vivo. These findings warrant further investigation of this novel NO-releasing prodrug in gliomas.

  19. Intracellular protein determination using droplet-based immunoassays.

    PubMed

    Martino, Chiara; Zagnoni, Michele; Sandison, Mairi E; Chanasakulniyom, Mayuree; Pitt, Andrew R; Cooper, Jonathan M

    2011-07-01

    This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ~50 pM to 1 μM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells.

  20. [Effects of glutamic acid and glutathione on the secretory function of the stomach].

    PubMed

    Shlygin, G K; Vasilevskaia, L S; Ignatenko, L G

    1988-10-01

    Experiments on dogs with Pavlov isolated pouches and gastric fistulas have shown that the ingested solution of MSG produces a potentiating effect on maximal gastric secretion caused by pentagastrin. This effect is apparently connected with the formation of glutathione in intestine. The glutathione concentration in blood after the intake of MSG is significantly elevated. It has been established that reduced glutathione administered in blood produced the similar potentiating effect on gastric secretion caused by pentagastrin.

  1. PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis.

    PubMed

    Micheva-Viteva, Sofiya N; Shou, Yulin; Ganguly, Kumkum; Wu, Terry H; Hong-Geller, Elizabeth

    2017-01-01

    Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis , we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host

  2. Intracellular mechanisms of solar water disinfection

    NASA Astrophysics Data System (ADS)

    Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

    2016-12-01

    Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection.

  3. Intracellular mechanisms of solar water disinfection

    PubMed Central

    Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

    2016-01-01

    Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection. PMID:27909341

  4. Intracellular mechanisms of solar water disinfection.

    PubMed

    Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

    2016-12-02

    Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection.

  5. 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

  6. Exogenous salicylic acid improves photosynthesis and growth through increase in ascorbate-glutathione metabolism and S assimilation in mustard under salt stress

    PubMed Central

    Nazar, Rahat; Umar, Shahid; Khan, Nafees A.

    2015-01-01

    Ascorbate (AsA)–glutathione (GSH) cycle metabolism has been regarded as the most important defense mechanism for the resistance of plants under stress. In this study the influence of salicylic acid (SA) was studied on ascorbate-glutathione pathway, S-assimilation, photosynthesis and growth of mustard (Brassica juncea L.) plants subjected to 100 mM NaCl. Treatment of SA (0.5 mM) alleviated the negative effects of salt stress and improved photosynthesis and growth through increase in enzymes of ascorbate-glutathione pathway which suggest that SA may participate in the redox balance under salt stress. The increase in leaf sulfur content through higher activity of ATP sulfurylase (ATPS) and serine acetyl transferase (SAT) by SA application was associated with the increased accumulation of glutathione (GSH) and lower levels of oxidative stress. These effects of SA were substantiated by the findings that application of SA-analog, 2,6, dichloro-isonicotinic acid (INA) and 1 mM GSH treatment produced similar results on rubisco, photosynthesis and growth of plants establishing that SA application alleviates the salt-induced decrease in photosynthesis mainly through inducing the enzyme activity of ascorbate-glutathione pathway and increased GSH production. Thus, SA/GSH could be a promising tool for alleviation of salt stress in mustard plants. PMID:25730495

  7. A mitochondria-targeted turn-on fluorescent probe for the detection of glutathione in living cells.

    PubMed

    Zhang, Jian; Bao, Xiaolong; Zhou, Junliang; Peng, Fangfang; Ren, Hang; Dong, Xiaochun; Zhao, Weili

    2016-11-15

    A novel turn-on red fluorescent BODIPY-based probe (Probe 1) for the detection of glutathione was developed. Such a probe carries a para-dinitrophenoxy benzyl pyridinium moiety at the meso position of a BODIPY dye as self-immolative linker. Probe 1 responds selectively to glutathione with the detection limit of 109nM over other amino acids, common metal ions, reactive oxygen species, reactive nitrogen species, and reactive sulfur species. A novel electrostatic interaction to modulate the SNAr attack of glutathione was believed to play significant role for the observed selective response to glutathione. The cleavage of dinitrophenyl ether by glutathione leads to the production of para-hydroxybenzyl moiety which is able to self-immolate through an intramolecular 1,4-elimination reaction to release the fluorescent BODIPY dye. The low toxic probe has been successfully used to detect mitochondrial glutathione in living cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Glutathione and zebrafish: Old assays to address a current issue.

    PubMed

    Massarsky, Andrey; Kozal, Jordan S; Di Giulio, Richard T

    2017-02-01

    Several xenobiotic agents (e.g. metals, polycyclic aromatic hydrocarbons, nanoparticles, etc.) commonly involve the generation of reactive oxygen species (ROS) and oxidative stress as part of their toxic mode of action. Among piscine models, the zebrafish is a popular vertebrate model to study toxicity of various xenobiotic agents. Similarly to other vertebrates, zebrafish possess an extensive antioxidant system, including the reduced form of glutathione (GSH), which is an important antioxidant that acts alone or in conjunction with enzymes, such as glutathione peroxidase (GPx). Upon interaction with ROS, GSH is oxidized, resulting in the formation of glutathione disulfide (GSSG). GSSG is recycled by an auxiliary antioxidant enzyme glutathione reductase (GR). This article outlines detailed methods to measure the concentrations of GSH and GSSG, as well as the activities of GPx and GR in zebrafish larvae as robust and economical means to assess oxidative stress. The studies that have assessed these endpoints in zebrafish and alternative methods are also discussed. We conclude that the availability of these robust and economical methods support the use of zebrafish as a model organism in studies evaluating redox biology, as well as the induction of oxidative stress following exposure to toxic agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Streptozotocin-Induced Autophagy Reduces Intracellular Insulin in Insulinoma INS-1E Cells.

    PubMed

    Yoo, Yeong-Min; Park, Yung Chul

    2018-03-01

    Streptozotocin (STZ), a glucose analog, induces diabetes in experimental animals by inducing preferential cytotoxicity in pancreatic beta cells. We investigated whether STZ reduced the production of intracellular insulin through autophagy in insulinoma INS-1E cells. Typically, 2 mM STZ treatment for 24 h significantly decreased cell survival. STZ treatment led to significant decrease in phospho-AMP-activated protein kinase (p-AMPK) level; reduction in levels of phospho-protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1α (IRE1α); significant reduction in levels of p85α, p110, phospho-serine and threonine kinase/protein kinase B (p-Akt/PKB) (Ser473), phospho-extracellular-regulated kinase (p-ERK), and phospho-mammalian target of rapamycin (p-mTOR); increase in levels of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase; decrease in B-cell lymphoma 2 (Bcl-2) expression; increase in Bcl-2-associated X protein (Bax) expression; increase in levels of microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1; and reduction in production of intracellular insulin. These results suggest that insulin synthesis during STZ treatment involves autophagy in INS-1E cells and, subsequently, results in a decrease in intracellular production of insulin.

  10. An early-branching microbialite cyanobacterium forms intracellular carbonates.

    PubMed

    Couradeau, Estelle; Benzerara, Karim; Gérard, Emmanuelle; Moreira, David; Bernard, Sylvain; Brown, Gordon E; López-García, Purificación

    2012-04-27

    Cyanobacteria have affected major geochemical cycles (carbon, nitrogen, and oxygen) on Earth for billions of years. In particular, they have played a major role in the formation of calcium carbonates (i.e., calcification), which has been considered to be an extracellular process. We identified a cyanobacterium in modern microbialites in Lake Alchichica (Mexico) that forms intracellular amorphous calcium-magnesium-strontium-barium carbonate inclusions about 270 nanometers in average diameter, revealing an unexplored pathway for calcification. Phylogenetic analyses place this cyanobacterium within the deeply divergent order Gloeobacterales. The chemical composition and structure of the intracellular precipitates suggest some level of cellular control on the biomineralization process. This discovery expands the diversity of organisms capable of forming amorphous calcium carbonates.

  11. Glutathione and redox signaling in substance abuse.

    PubMed

    Uys, Joachim D; Mulholland, Patrick J; Townsend, Danyelle M

    2014-07-01

    Throughout the last couple decades, the cause and consequences of substance abuse has expanded to identify the underlying neurobiological signaling mechanisms associated with addictive behavior. Chronic use of drugs, such as cocaine, methamphetamine and alcohol leads to the formation of oxidative or nitrosative stress (ROS/RNS) and changes in glutathione and redox homeostasis. Of importance, redox-sensitive post-translational modifications on cysteine residues, such as S-glutathionylation and S-nitrosylation could impact on the structure and function of addiction related signaling proteins. In this commentary, we evaluate the role of glutathione and redox signaling in cocaine-, methamphetamine- and alcohol addiction and conclude by discussing the possibility of targeting redox pathways for the therapeutic intervention of these substance abuse disorders. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  12. Enzyme activity of α-chymotrypsin: Deactivation by gold nano-cluster and reactivation by glutathione.

    PubMed

    Ghosh, Catherine; Mondal, Tridib; Bhattacharyya, Kankan

    2017-05-15

    Effect of gold nanoclusters (Au-NCs) on the circular dichroism (CD) spectra and enzymatic activity of α-chymotrypsin (ChT) (towards hydrolysis of a substrate, N-succinyl-l-phenylalanine p-nitroanilide) are studied. The CD spectra indicate that on binding to Au-NC, ChT is completely unfolded, resulting in nearly zero ellipticity. α-chymotrypsin (ChT) coated gold nano-clusters exhibit almost no enzymatic activity. Addition of glutathione (GSH) or oxidized glutathione (GSSG) restore the enzyme activity of α-chymotrypsin by 30-45%. ChT coated Au-NC exhibits two emission maxima-one at 480nm (corresponding to Au 10 ) and one at 640nm (Au 25 ). On addition of glutathione (GSH) or oxidized glutathione (GSSG) the emission peak at 640nm vanishes and only one peak at 480nm (Au 10 ) remains. MALDI mass spectrometry studies suggest addition of glutathione (GSH) to α-chymotrypsin capped Au-NCs results in the formation of glutathione-capped Au-NCs and α-chymotrypsin is released from Au-NCs. CD spectroscopy indicates that the conformation of the released α-chymotrypsin is different from that of the native α-chymotrypsin. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Iron storage, lipid peroxidation and glutathione turnover in chronic anti-HCV positive hepatitis.

    PubMed

    Farinati, F; Cardin, R; De Maria, N; Della Libera, G; Marafin, C; Lecis, E; Burra, P; Floreani, A; Cecchetto, A; Naccarato, R

    1995-04-01

    Little is known about the pathogenesis of liver damage related to hepatitis C virus. The presence of steatosis or increased ferritin levels, and preliminary data on the relevance of iron as a prognostic factor prompted us to ascertain whether hepatitis C virus-related liver damage might be mediated by iron accumulation. We evaluated the degree of hepatic inflammation and steatosis, serum ferritin, transferrin saturation and iron levels, tissue iron concentrations and iron index, liver glutathione and malondialdehyde in 33 males and 20 females with chronic hepatitis C virus- or hepatitis B virus-related hepatitis (42 + 11). We also considered six patients with both alcohol abuse and hepatitis C virus, four males with chronic alcoholic liver disease and four males with genetic hemochromatosis, giving a total of 67. All diagnoses were histologically confirmed. Patients with cirrhosis were excluded. Our data show that: 1. Steatosis is more frequent in hepatitis C virus and hepatitis C virus+alcohol abuse patients; 2. In males, serum ferritin and tissue iron are significantly higher in hepatitis C virus- than in hepatitis B virus-positive patients (p < 0.01 and 0.05); transferrin saturation is higher (p < 0.05) in hepatitis C virus-positive than in hepatitis B virus-positive patients only when males and females are considered together; 3. Serum ferritin and transferrin saturation only correlate with liver iron (r = 0.833 and r = 0.695, respectively, p = 0.00001); tissue iron is significantly higher in hepatitis C virus- than in hepatitis B virus-positive patients (p < 0.05); 4. In patients with chronic hepatitis, serum ferritin is a better marker of liver iron storage than transferrin saturation, both in males and in females; 5. Hepatitis C virus-positive patients have higher malondialdehyde levels and activation of turnover of glutathione, probably in response to free-radical-mediated liver damage. Females have lower liver iron levels but similar trends. These findings

  14. CHARACTERIZATION OF DANSYLATED CYSTEINE, CYSTINE, GLUTATHIONE, AND GLUTATHIONE DISULFIDE BY NARROW BORE LIQUID CHROMATOGRAPHY - ELECTROSPRAY IONIZATION MASS SPECTROMETRY

    EPA Science Inventory

    A method using reversed phase high performance liquid chromtography/electrospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to confirm the dientity of dansylated derivatives of cysteine (C) and glutathione (GSH), and their respective dimers, cystine (CSSC) and...

  15. Dietary docosahexaenoic acid-induced generation of liver lipid peroxides is not suppressed further by elevated levels of glutathione in ODS rats.

    PubMed

    Sekine, Seiji; Kubo, Kazuhiro; Tadokoro, Tadahiro; Saito, Morio

    2006-04-01

    We examined the effects of ascorbic acid (AsA) and glutathione (GSH; experiment 1) and of GSH in acetaminophen-fed rats (experiment 2) on dietary docosahexaenoic acid (DHA)-induced tissue lipid peroxidation. In experiment 1, AsA-requiring Osteogenic Disorder Shionogi/Shi-od/od (ODS) rats were fed soybean protein diets containing DHA (10.0% total energy) and AsA at 50 (low) or 300 (normal) mg/kg without (low) or with (normal) methionine at 2 g/kg for 32 d. In experiment 2, ODS rats were fed diets containing DHA (7.8% total energy) and acetaminophen (4 g/kg) with different levels of dietary methionine (low, moderate, high, and excessive at 0, 3, 6, and 9 g/kg, respectively) for 30 d. Tissue lipid peroxides and antioxidant levels were determined. In experiment 1, liver lipid peroxide levels in the low-AsA group were lower than those in the normal-AsA group, but kidney and testis lipid peroxide levels in the low-AsA group were higher than those in the normal-AsA group. Dietary methionine tended to decrease tissue lipid peroxide levels but did not decrease vitamin E (VE) consumption. In experiment 2, a high level of methionine (6 g/kg) decreased liver lipid peroxide levels and VE consumption. However, generation of tissue lipid peroxides and VE consumption were not decreased further by a higher dose of methionine (9 g/kg). Higher than normal levels of dietary methionine are not necessarily associated with decreased dietary DHA-induced generation of tissue lipid peroxides and VE consumption except that the GSH requirement is increased in a condition such as acetaminophen feeding.

  16. 3-Nitrotyrosine and glutathione antioxidant system in patients in the early and late stages of bipolar disorder

    PubMed Central

    Andreazza, Ana Cristina; Kapczinski, Flavio; Kauer-Sant’Anna, Marcia; Walz, Julio C.; Bond, David J.; Gonçalves, Carlos A.; Young, L. Trevor; Yatham, Lakshmi N.

    2009-01-01

    Background There has been an increasing interest in the role of oxidative stress in the pathophysiology of bipolar disorder. To explore this further, we evaluated the activity of glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST), as well as 3-nitrotyrosine levels and carbonyl content in patients in the early (within 3 years of illness onset) and late (a minimum of 10 years of illness) stages of bipolar disorder. Methods We matched 30 patients in the early stage and 30 patients in the late stage of bipolar disorder, diagnosed according to DSM-IV criteria, with 60 healthy controls (30 matched for each group of patients). We measured symptomatic status using the Hamilton Rating Scale for Depression and the Young Mania Rating Scale. Results We found a significant increase in 3-nitrotyrosine levels among patients in the early (p < 0.010) and late (p < 0.010) stages of bipolar disorder. The activity of GR and GST was increased only among patients in the late stage of illness. Glutathione peroxidase activity and carbonyl content did not differ among the groups. Limitations Limitations of our study include its cross-sectional design, which did not allow us to examine direct causative mechanisms or the effects of progression of illness, and the potential environmental bias introduced by comparing patient groups recruited from different regions of the world. Conclusion Our data indicate a possible tyrosine nitration-induced damage in patients with bipolar disorder that is present from the early stage of illness. Our data also indicate that patients in the late stage of illness demonstrate enhanced activity of GR and GST, which could suggest the involvement of a compensatory system in bipolar disorder. PMID:19568477

  17. Nanovehicular Intracellular Delivery Systems

    PubMed Central

    PROKOP, ALES; DAVIDSON, JEFFREY M.

    2013-01-01

    This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood–brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list “elementary” phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach. PMID:18200527

  18. Fluorescence detection of glutathione and oxidized glutathione in blood with a NIR-excitable cyanine probe

    NASA Astrophysics Data System (ADS)

    Liu, Chang-hui; Qi, Feng-pei; Wen, Fu-bin; Long, Li-ping; Liu, Ai-juan; Yang, Rong-hua

    2018-04-01

    Cyanine has been widely utilized as a near infrared (NIR) fluorophore for detection of glutathione (GSH). However, the excitation of most of the reported cyanine-based probes was less than 800 nm, which inevitably induce biological background absorption and lower the sensitivity, limiting their use for detection of GSH in blood samples. To address this issue, here, a heptamethine cyanine probe (DNIR), with a NIR excitation wavelength at 804 nm and a NIR emission wavelength at 832 nm, is employed for the detection of GSH and its oxidized form (GSSG) in blood. The probe displays excellent selectivity for GSH over GSSG and other amino acids, and rapid response to GSH, in particular a good property for indirect detection of GSSG in the presence of enzyme glutathione reductase and the reducing agent nicotinamideadenine dinucleotide phosphate, without further separation prior to fluorescent measurement. To the best of our knowledge, this is the first attempt to explore NIR fluorescent approach for the simultaneous assay of GSH and GSSG in blood. As such, we expect that our fluorescence sensors with both NIR excitation and NIR emission make this strategy suitable for the application in complex physiological systems.

  19. Detection of Glutathione by Glutathione-S-Transferase-Nanoconjugate Ensemble Electrochemical Device.

    PubMed

    Barman, Ujjwol; Mukhopadhyay, Gargi; Goswami, Namami; Ghosh, Siddhartha Sankar; Paily, Roy P

    2017-06-01

    This paper reports a novel electrochemical method for detection of Glutathione (GSH) using Glutathione-S-Transferase (GST) - ZnO composite nanoparticles to investigate the prospects of the method for detection of cancer at an early stage. The purified GST enzyme was bound with ZnO nanoparticles by electrostatic interactions and the nanocomposite was dropcast on a silicon dioxide wafer. The GST functionalized deposited layer was then used as a chemiresistive channel to detect conjugation reaction between GSH and 1-Chloro-2, 4-Dinitrobenzene (CDNB). The zeta potential values of the ZnO nanoparticles and the GST were found to be 13.4 mV and-6.21 mV, respectively. Around 73.8% binding was observed between the enzyme and ZnO nanoparticles. I - V analysis of the chemiresistive channel showed an increase in conductivity of the channel due to conjugation reaction between GSH and CDNB as compared with that of GSH or CDNB alone. I - V characterization of the GST functionalized layer was performed at various concentrations of GSH and a sensitivity and limit of detection of 5.68 nA/ [Formula: see text] and 41.9 nM were obtained, respectively. Thus from I - V analysis of the chemiresistivechannel, the detectionand quantification of GSH could be obtained. The kinetic parameters of both GST and nanoconjugate of ZnO nanoparticles andGSTwere determinedwith respect to its substrates, GSH and CDNB, using Michaelis-Mentenmodel. This novel approach of detection of GSH bymeans of ZnO nanoparticle and GST enzyme composite can be further analyzed for in vitro experiments, which will lead us to a new and efficient way of detecting certain types of cancers at an early stage.

  20. Intracellular Phosphate Dynamics in Muscle Measured by Magnetic Resonance Spectroscopy during Hemodialysis

    PubMed Central

    Fournier, Thomas; Kocevar, Gabriel; Belloi, Amélie; Normand, Gabrielle; Ibarrola, Danielle; Sappey-Marinier, Dominique; Juillard, Laurent

    2016-01-01

    Of the 600–700 mg inorganic phosphate (Pi) removed during a 4-hour hemodialysis session, a maximum of 10% may be extracted from the extracellular space. The origin of the other 90% of removed phosphate is unknown. This study tested the hypothesis that the main source of phosphate removed during hemodialysis is the intracellular compartment. Six binephrectomized pigs each underwent one 3-hour hemodialysis session, during which the extracorporeal circulation blood flow was maintained between 100 and 150 ml/min. To determine in vivo phosphate metabolism, we performed phosphorous (31P) magnetic resonance spectroscopy using a 1.5-Tesla system and a surface coil placed over the gluteal muscle region. 31P magnetic resonance spectra (repetition time =10 s; echo time =0.35 ms) were acquired every 160 seconds before, during, and after dialysis. During the dialysis sessions, plasma phosphate concentrations decreased rapidly (−30.4 %; P=0.003) and then, plateaued before increasing approximately 30 minutes before the end of the sessions; 16 mmol phosphate was removed in each session. When extracellular phosphate levels plateaued, intracellular Pi content increased significantly (11%; P<0.001). Moreover, βATP decreased significantly (P<0.001); however, calcium levels remained balanced. Results of this study show that intracellular Pi is the source of Pi removed during dialysis. The intracellular Pi increase may reflect cellular stress induced by hemodialysis and/or strong intracellular phosphate regulation. PMID:26561642

  1. Triggered-release polymeric conjugate micelles for on-demand intracellular drug delivery

    NASA Astrophysics Data System (ADS)

    Cao, Yanwu; Gao, Min; Chen, Chao; Fan, Aiping; Zhang, Ju; Kong, Deling; Wang, Zheng; Peer, Dan; Zhao, Yanjun

    2015-03-01

    Nanoscale drug delivery platforms have been developed over the past four decades that have shown promising clinical results in several types of cancer and inflammatory disorders. These nanocarriers carrying therapeutic payloads are maximizing the therapeutic outcomes while minimizing adverse effects. Yet one of the major challenges facing drug developers is the dilemma of premature versus on-demand drug release, which influences the therapeutic regiment, efficacy and potential toxicity. Herein, we report on redox-sensitive polymer-drug conjugate micelles for on-demand intracellular delivery of a model active agent, curcumin. Biodegradable methoxy poly(ethylene glycol)-poly(lactic acid) copolymer (mPEG-PLA) was conjugated with curcumin via a disulfide bond or ester bond (control), respectively. The self-assembled redox-sensitive micelles exhibited a hydrodynamic size of 115.6 ± 5.9 (nm) with a zeta potential of -10.6 ± 0.7 (mV). The critical micelle concentration was determined at 6.7 ± 0.4 (μg mL-1). Under sink conditions with a mimicked redox environment (10 mM dithiothreitol), the extent of curcumin release at 48 h from disulfide bond-linked micelles was nearly three times higher compared to the control micelles. Such rapid release led to a lower half maximal inhibitory concentration (IC50) in HeLa cells at 18.5 ± 1.4 (μg mL-1), whereas the IC50 of control micelles was 41.0 ± 2.4 (μg mL-1). The cellular uptake study also revealed higher fluorescence intensity for redox-sensitive micelles. In conclusion, the redox-sensitive polymeric conjugate micelles could enhance curcumin delivery while avoiding premature release, and achieving on-demand release under the high glutathione concentration in the cell cytoplasm. This strategy opens new avenues for on-demand drug release of nanoscale intracellular delivery platforms that ultimately might be translated into pre-clinical and future clinical practice.

  2. Intracellular magnesium detection by fluorescent indicators.

    PubMed

    Trapani, Valentina; Schweigel-Röntgen, Monika; Cittadini, Achille; Wolf, Federica I

    2012-01-01

    Magnesium is essential for a wide variety of biochemical reactions and physiological functions, but its regulatory mechanisms (both at the cellular and at the systemic level) are still poorly characterized. Not least among the reasons for this gap are the technical difficulties in sensing minor changes occurring over a high background concentration. Specific fluorescent indicators are highly sensitive tools for dynamic evaluation of intracellular magnesium concentration. We herein discuss the main criteria to consider when choosing a magnesium-specific fluorescent indicator and provide examples among commercial as well as developmental sensors. We focus on spectrofluorimetric approaches to quantify Mg(2+) concentration in cell or mitochondria suspensions, and on imaging techniques to detect intracellular magnesium distribution and fluxes by live microscopy, reporting a detailed description of standard protocols for each method. The general guidelines we provide should be applicable to specific issues by any researcher in the field. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Toxicity of nickel and silver to Nostoc muscorum: interaction with ascorbic acid, glutathione, and sulfur-containing amino acids

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

    Rai, L.C.; Raizada, M.

    1987-08-01

    Exposure of Nostoc muscorum to different concentrations of Ni and Ag brought about reduction in growth, carbon fixation, heterocyst production, and nitrogenase activity and increase in the loss of ions (K+, Na+). In an attempt to ameliorate the toxicity of test metals by ascorbic acid, glutathione, and sulfur-containing amino acids (L-cysteine and L-methionine), it was found that the level of protection by ascorbic acid and glutathione was more for Ag than Ni. However, metal-induced inhibition of growth and carbon fixation was equally ameliorated by methionine. But the level of protection by cysteine was quite different, i.e., 27% for Ni andmore » 22% for Ag. Protection of metal toxicity in N. muscorum by amino acids lends further support to self-detoxifying ability of cyanobacteria because they are known to synthesize all essential amino acids.« less

  4. The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

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

    Chen, Yanyan; The Hamner Institutes for Health Sciences, Research Triangle Park, NC; Xu, Yuanyuan, E-mail: yyxu@cmu.edu.cn

    Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-doublemore » knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.« less

  5. A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis.

    PubMed

    Gopal, Shubha; Borovok, Ilya; Ofer, Amos; Yanku, Michaela; Cohen, Gerald; Goebel, Werner; Kreft, Jürgen; Aharonowitz, Yair

    2005-06-01

    Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, gamma-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate gamma-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a gamma-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.

  6. 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.

  7. Review: Intracardiac intracellular angiotensin system in diabetes

    PubMed Central

    Kumar, Rajesh; Yong, Qian Chen; Thomas, Candice M.

    2012-01-01

    The renin-angiotensin system (RAS) has mainly been categorized as a circulating and a local tissue RAS. A new component of the local system, known as the intracellular RAS, has recently been described. The intracellular RAS is defined as synthesis and action of ANG II intracellularly. This RAS appears to differ from the circulating and the local RAS, in terms of components and the mechanism of action. These differences may alter treatment strategies that target the RAS in several pathological conditions. Recent work from our laboratory has demonstrated significant upregulation of the cardiac, intracellular RAS in diabetes, which is associated with cardiac dysfunction. Here, we have reviewed evidence supporting an intracellular RAS in different cell types, ANG II's actions in cardiac cells, and its mechanism of action, focusing on the intracellular cardiac RAS in diabetes. We have discussed the significance of an intracellular RAS in cardiac pathophysiology and implications for potential therapies. PMID:22170614

  8. Identification of residues critical for proton-coupled glutathione translocation in the yeast glutathione transporter, Hgt1p.

    PubMed

    Zulkifli, Mohammad; Bachhawat, Anand Kumar

    2017-05-16

    The proton gradient acts as the driving force for the transport of many metabolites across fungal and plant plasma membranes. Identifying the mechanism of proton relay is critical for understanding the mechanism of transport mediated by these transporters. We investigated two strategies for identifying residues critical for proton-dependent substrate transport in the yeast glutathione transporter, Hgt1p, a member of the poorly understood oligopeptide transporter family of transporters. In the first strategy, we tried to identify the pH-independent mutants that could grow at higher pH when dependant on glutathione transport. Screening a library of 269 alanine mutants of the transmembrane domains (TMDs) along with a random mutagenesis strategy yielded two residues (E135K on the cusp of TMD2 and N710S on TMD12) that permitted growth on glutathione at pH 8.0. Further analysis revealed that these residues were not involved in proton symport even though they conferred better transport at a higher pH. The second strategy involved a knowledge-driven approach, targeting 31 potential residues based on charge, conservation and location. Mutation of these residues followed by functional and biochemical characterization revealed E177A, Y193A, D335A, Y374A, H445A and R554A as being defective in proton transport. Further analysis enabled possible roles of these residues to be assigned in proton relay. The implications of these findings in relation to Hgt1p and the suitability of these strategic approaches for identifying such residues are discussed. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  9. Glutathione-S-transferase-oxidative stress relationship in the internal spermatic vein blood of infertile men with varicocele.

    PubMed

    Mostafa, T; Rashed, L A; Zeidan, A S; Hosni, A

    2015-02-01

    This study aimed to assess glutathione-S-transferase (GST) enzyme- oxidative stress (OS) relationship in the internal spermatic vein (ISV) of infertile men associated with varicocele (Vx). Ninety five infertile oligoasthenoteratozoospemic (OAT) men associated with Vx were subjected to history taking, clinical examination and semen analysis. During inguinal varicocelectomy, GST, malondialdehyde (MDA) and glutathione peroxidase (GPx) were estimated in the blood samples drawn from ISV and median cubital veins. The mean levels of GST, GPx were significantly decreased and the mean level of GPx was significantly increased in the ISV compared with the peripheral blood. The mean level of GST and GPx in the ISV was significantly decreased, and the mean level of MDA was significantly increased in Vx grade III compared with Vx grade II cases. There was nonsignificant difference in the mean level of GST in the ISV in unilateral Vx cases compared with bilateral Vx cases. There was significant positive correlation of GST with sperm count, sperm motility, GPx and significant negative correlation with sperm abnormal forms, MDA. It is concluded that ISV of infertile men associated with Vx has decreased levels of GST compared with peripheral venous circulation that is correlated with both OS and Vx grade. © 2014 Blackwell Verlag GmbH.

  10. Insecticide resistance and intracellular proteases.

    PubMed

    Wilkins, Richard M

    2017-12-01

    Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  11. Prolonged fasting increases glutathione biosynthesis in postweaned northern elephant seals.

    PubMed

    Vázquez-Medina, José Pablo; Zenteno-Savín, Tania; Forman, Henry Jay; Crocker, Daniel E; Ortiz, Rudy M

    2011-04-15

    Northern elephant seals experience prolonged periods of absolute food and water deprivation (fasting) while breeding, molting or weaning. The postweaning fast in elephant seals is characterized by increases in the renin-angiotensin system, expression of the oxidant-producing protein Nox4, and NADPH oxidase activity; however, these increases are not correlated with increased oxidative damage or inflammation. Glutathione (GSH) is a potent reductant and a cofactor for glutathione peroxidases (GPx), glutathione-S transferases (GST) and 1-cys peroxiredoxin (PrxVI) and thus contributes to the removal of hydroperoxides, preventing oxidative damage. The effects of prolonged food deprivation on the GSH system are not well described in mammals. To test our hypothesis that GSH biosynthesis increases with fasting in postweaned elephant seals, we measured circulating and muscle GSH content at the early and late phases of the postweaning fast in elephant seals along with the activity/protein content of glutamate-cysteine ligase [GCL; catalytic (GCLc) and modulatory (GCLm) subunits], γ-glutamyl transpeptidase (GGT), glutathione disulphide reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), GST and PrxVI, as well as plasma changes in γ-glutamyl amino acids, glutamate and glutamine. GSH increased two- to four-fold with fasting along with a 40-50% increase in the content of GCLm and GCLc, a 75% increase in GGT activity, a two- to 2.5-fold increase in GR, G6PDH and GST activities and a 30% increase in PrxVI content. Plasma γ-glutamyl glutamine, γ-glutamyl isoleucine and γ-glutamyl methionine also increased with fasting whereas glutamate and glutamine decreased. Results indicate that GSH biosynthesis increases with fasting and that GSH contributes to counteracting hydroperoxide production, preventing oxidative damage in fasting seals.

  12. Prolonged fasting increases glutathione biosynthesis in postweaned northern elephant seals

    PubMed Central

    Vázquez-Medina, José Pablo; Zenteno-Savín, Tania; Forman, Henry Jay; Crocker, Daniel E.; Ortiz, Rudy M.

    2011-01-01

    SUMMARY Northern elephant seals experience prolonged periods of absolute food and water deprivation (fasting) while breeding, molting or weaning. The postweaning fast in elephant seals is characterized by increases in the renin–angiotensin system, expression of the oxidant-producing protein Nox4, and NADPH oxidase activity; however, these increases are not correlated with increased oxidative damage or inflammation. Glutathione (GSH) is a potent reductant and a cofactor for glutathione peroxidases (GPx), glutathione-S transferases (GST) and 1-cys peroxiredoxin (PrxVI) and thus contributes to the removal of hydroperoxides, preventing oxidative damage. The effects of prolonged food deprivation on the GSH system are not well described in mammals. To test our hypothesis that GSH biosynthesis increases with fasting in postweaned elephant seals, we measured circulating and muscle GSH content at the early and late phases of the postweaning fast in elephant seals along with the activity/protein content of glutamate-cysteine ligase [GCL; catalytic (GCLc) and modulatory (GCLm) subunits], γ-glutamyl transpeptidase (GGT), glutathione disulphide reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), GST and PrxVI, as well as plasma changes in γ-glutamyl amino acids, glutamate and glutamine. GSH increased two- to four-fold with fasting along with a 40–50% increase in the content of GCLm and GCLc, a 75% increase in GGT activity, a two- to 2.5-fold increase in GR, G6PDH and GST activities and a 30% increase in PrxVI content. Plasma γ-glutamyl glutamine, γ-glutamyl isoleucine and γ-glutamyl methionine also increased with fasting whereas glutamate and glutamine decreased. Results indicate that GSH biosynthesis increases with fasting and that GSH contributes to counteracting hydroperoxide production, preventing oxidative damage in fasting seals. PMID:21430206

  13. Cofilin-1 levels and intracellular localization are associated with melanoma prognosis in a cohort of patients

    PubMed Central

    Bracalente, Candelaria; Rinflerch, Adriana R.; Ibañez, Irene L.; García, Francisco M.; Volonteri, Victoria; Galimberti, Gastón N.; Klamt, Fabio; Durán, Hebe

    2018-01-01

    Melanoma is an aggressive cancer with highly metastatic ability. We propose cofilin-1, a key protein in the regulation of actin dynamics and migration, as a prognostic marker. We determined cofilin-1 levels in a retrospective cohort of patients with melanomas and benign lesions of melanocytes (nevi) by immunohistochemistry. Higher cofilin-1 levels were found in malignant melanoma (MM) with Breslow Index (BI)>2 vs MM with BI<2, melanoma in situ (MIS) and nevi and also in MM with metastasis vs MM without detected metastasis. Kaplan-Meier survival curves were performed, clustering patients according to either the type of melanocytic lesions or cofilin-1 level. Survival curves demonstrated worse prognosis of patients with high vs low cofilin-1 levels. TCGA database analysis of melanoma also showed low survival in patients with upregulated cofilin-1 mRNA vs patients without alteration in CFL1 mRNA expression. As cofilin-1 has a dual function depending on its intracellular localization, we evaluated nuclear and cytoplasmic levels of cofilin-1 in melanoma and nevi samples by immunofluorescence. MM with high Breslow index and metastatic cells not only presented cytoplasmic cofilin-1, but also showed this protein at the nucleus. An increase in nuclear/cytoplasmic cofilin-1 mean fluorescence ratio was observed in MM with BI>2 vs MM with BI<2, MIS and nevi. In conclusion, an association of cofilin-1 levels with malignant features and an inverse correlation with survival were demonstrated. Moreover, this study suggests that not only the higher levels of cofilin-1, but also its nuclear localization can be proposed as marker of worse outcome of patients with melanoma. PMID:29844875

  14. Cofilin-1 levels and intracellular localization are associated with melanoma prognosis in a cohort of patients.

    PubMed

    Bracalente, Candelaria; Rinflerch, Adriana R; Ibañez, Irene L; García, Francisco M; Volonteri, Victoria; Galimberti, Gastón N; Klamt, Fabio; Durán, Hebe

    2018-05-08

    Melanoma is an aggressive cancer with highly metastatic ability. We propose cofilin-1, a key protein in the regulation of actin dynamics and migration, as a prognostic marker. We determined cofilin-1 levels in a retrospective cohort of patients with melanomas and benign lesions of melanocytes (nevi) by immunohistochemistry. Higher cofilin-1 levels were found in malignant melanoma (MM) with Breslow Index (BI)>2 vs MM with BI<2, melanoma in situ (MIS) and nevi and also in MM with metastasis vs MM without detected metastasis. Kaplan-Meier survival curves were performed, clustering patients according to either the type of melanocytic lesions or cofilin-1 level. Survival curves demonstrated worse prognosis of patients with high vs low cofilin-1 levels. TCGA database analysis of melanoma also showed low survival in patients with upregulated cofilin-1 mRNA vs patients without alteration in CFL1 mRNA expression. As cofilin-1 has a dual function depending on its intracellular localization, we evaluated nuclear and cytoplasmic levels of cofilin-1 in melanoma and nevi samples by immunofluorescence. MM with high Breslow index and metastatic cells not only presented cytoplasmic cofilin-1, but also showed this protein at the nucleus. An increase in nuclear/cytoplasmic cofilin-1 mean fluorescence ratio was observed in MM with BI>2 vs MM with BI<2, MIS and nevi. In conclusion, an association of cofilin-1 levels with malignant features and an inverse correlation with survival were demonstrated. Moreover, this study suggests that not only the higher levels of cofilin-1, but also its nuclear localization can be proposed as marker of worse outcome of patients with melanoma.

  15. The interaction of intracellular Mg2+ and pH on Cl- fluxes associated with intracellular pH regulation in barnacle muscle fibers

    PubMed Central

    1988-01-01

    The intracellular dialysis technique was used to measure unidirectional Cl- fluxes and net acid extrusion by single muscle fibers from the giant barnacle. Decreasing pHi below normal levels of 7.35 stimulated both Cl- efflux and influx. These increases of Cl- fluxes were blocked by disulfonic acid stilbene derivatives such as SITS and DIDS. The SITS- sensitive Cl- efflux was sharply dependent upon pHi, increasing approximately 20-fold as pHi was decreased from 7.35 to 6.7. Under conditions of normal intracellular Mg2+ concentration, the apparent pKa for the activation of Cl- efflux was 7.0. We found that raising [Mg2+]i, but not [Mg2+]o, had a pronounced inhibitory effect on both SITS-sensitive unidirectional Cl- fluxes as well as on SITS-sensitive net acid extrusion. Increasing [Mg2+]i shifted the apparent pKa of Cl- efflux to a more acid value without affecting the maximal flux that could be attained. This relation between pHi and [Mg2+]i on SITS- sensitive Cl- efflux is consistent with a competition between H ions and Mg ions. We conclude that the SITS-inhibitable Cl- fluxes are mediated by the pHi-regulatory transport mechanism and that changes of intracellular Mg2+ levels can modify the activity of the pHi regulator/anion transporter. PMID:3392519

  16. Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells

    PubMed Central

    Mauzeroll, Janine; Bard, Allen J.

    2004-01-01

    The uptake of menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy. The progression of the in vitro reaction between menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV–visible) behavior. By observing the scanning electrochemical microscope tip current of yeast cells suspended in a menadione-containing solution, the export of the conjugate from the cells with time could be measured. Similar experiments were performed on immobilized yeast cell aggregates stressed by a menadione solution. From the export of the menadione-glutathione conjugate detected at a 1-μm-diameter electrode situated 10 μm from the cells, a flux of about 30,000 thiodione molecules per second per cell was extracted. Numerical simulations based on an explicit finite difference method further revealed that the observation of a constant efflux of thiodione from the cells suggested the rate was limited by the uptake of menadione and that the efflux through the glutathione-conjugate pump was at least an order of magnitude faster. PMID:15148374

  17. Formation of the accumulative human metabolite and human-specific glutathione conjugate of diclofenac in TK-NOG chimeric mice with humanized livers.

    PubMed

    Kamimura, Hidetaka; Ito, Satoshi; Nozawa, Kohei; Nakamura, Shota; Chijiwa, Hiroyuki; Nagatsuka, Shin-ichiro; Kuronuma, Miyuki; Ohnishi, Yasuyuki; Suemizu, Hiroshi; Ninomiya, Shin-ichi

    2015-03-01

    3'-Hydroxy-4'-methoxydiclofenac (VI) is a human-specific metabolite known to accumulate in the plasma of patients after repeated administration of diclofenac sodium. Diclofenac also produces glutathione-conjugated metabolites, some of which are human-specific. In the present study, we investigated whether these metabolites could be generated in humanized chimeric mice produced from TK-NOG mice. After a single oral administration of diclofenac to humanized mice, the unchanged drug in plasma peaked at 0.25 hour and then declined with a half-life (t1/2) of 2.4 hours. 4'-Hydroxydiclofenac (II) and 3'-hydroxydiclofenac also peaked at 0.25 hour and were undetectable within 24 hours. However, VI peaked at 8 hours and declined with a t1/2 of 13 hours. When diclofenac was given once per day, peak and trough levels of VI reached plateau within 3 days. Studies with administration of II suggested VI was generated via II as an intermediate. Among six reported glutathione-conjugated metabolites of diclofenac, M1 (5-hydroxy-4-(glutathion-S-yl)diclofenac) to M6 (2'-(glutathion-S-yl)monoclofenac), we found three dichlorinated conjugates [M1, M2 (4'-hydroxy-3'-(glutathion-S-yl)diclofenac), and M3 (5-hydroxy-6-(glutathion-S-yl)diclofenac)], and a single monochlorinated conjugate [M4 (2'-hydroxy-3'-(glutathion-S-yl)monoclofenac) or M5 (4'-hydroxy-2'-(glutathion-S-yl)monoclofenac)], in the bile of humanized chimeric mice. M4 and M5 are positional isomers and have been previously reported as human-specific in vitro metabolites likely generated via arene oxide and quinone imine-type intermediates, respectively. The biliary monochlorinated metabolite exhibited the same mass spectrum as those of M4 and M5, and we discuss whether this conjugate corresponded to M4 or M5. Overall, humanized TK-NOG chimeric mice were considered to be a functional tool for the study of drug metabolism of diclofenac in humans. Copyright © 2015 by The American Society for Pharmacology and Experimental

  18. [35S]-LABELING OF THE SALMONELLA TYPHIMURIUM GLUTATHIONE POOL TO ASSESS GLUTATHIONE-MEDIATED DNA BINDING BY 1,2-DIBROMOETHANE

    EPA Science Inventory

    Biotransformation of drugs and environmental chemicals to reactive intermediates is often studied with the use of radiolabeled compounds that are synthesized by expensive and technically difficult procedures. In general, glutathione (GSH) conjugation serves as a detoxification m...

  19. Reaction of rat liver glutathione S-transferases and bacterial dichloromethane dehalogenase with dihalomethanes.

    PubMed

    Blocki, F A; Logan, M S; Baoli, C; Wackett, L P

    1994-03-25

    Dichloromethane dehalogenase from Methylophilus sp. DM11 is a glutathione S-transferase homolog that is specifically active with dihalomethane substrates. This bacterial enzyme and rat liver glutathione S-transferases were purified to investigate their relative reactivity with CH2Cl2 and related substrates. Rat liver alpha class glutathione transferases were inactive and mu class enzymes showed low activity (7-23 nmol/min/mg of protein) with CH2Cl2. theta class glutathione transferase 5-5 from rat liver and Methylophilus sp. dichloromethane dehalogenase showed specific activities of > or = 1 mumol/min/mg of protein. Apparent Kcat/Km were determined to be 3.3 x 10(4) and 6.0 x 10(4) L M-1 S-1 for the two enzymes, respectively. Dideutero-dichloromethane was processed to dideutereo-formaldehyde, consistent with a nucleophilic halide displacement mechanism. The possibility of a GSCH2X reaction intermediate (GS, glutathione; X, halide) was probed using CH2ClF to generate a more stable halomethylglutathione species (GSCH2F). The reaction of CH2ClF with dichloromethane dehalogenase produced a kinetically identifiable intermediate that decomposed to formaldehyde at a similar rate to synthetic HOCH2CH2SCH2F. 19F-NMR revealed the transient formation of an intermediate identified as GSCH2F by its chemical shift, its triplet resonance, and H-F coupling constant consistent with a fluoromethylthioether. Its decomposition was matched by a stoichiometric formation of fluoride. These studies indicated that the bacterial dichloromethane dehalogenase directs a nucleophilic attack of glutathione on CH2Cl2 to produce a halomethylthioether intermediate. This focuses attention on the mechanism used by theta class glutathione transferases to generate a halomethylthioeter from relatively unreactive dihalomethanes.

  20. 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.