Unbalanced Activation of Glutathione Metabolic Pathways Suggests Potential Involvement in Plant Defense against the Gall Midge Mayetiola destructor in Wheat

PubMed Central

Liu, Xuming; Zhang, Shize; Whitworth, R. Jeff; Stuart, Jeffrey J.; Chen, Ming-Shun

2015-01-01

Glutathione, γ-glutamylcysteinylglycine, exists abundantly in nearly all organisms. Glutathione participates in various physiological processes involved in redox reactions by serving as an electron donor/acceptor. We found that the abundance of total glutathione increased up to 60% in resistant wheat plants within 72 hours following attack by the gall midge Mayetiola destructor, the Hessian fly. The increase in total glutathione abundance, however, is coupled with an unbalanced activation of glutathione metabolic pathways. The activity and transcript abundance of glutathione peroxidases, which convert reduced glutathione (GSH) to oxidized glutathione (GSSG), increased in infested resistant plants. However, the enzymatic activity and transcript abundance of glutathione reductases, which convert GSSG back to GSH, did not change. This unbalanced regulation of the glutathione oxidation/reduction cycle indicates the existence of an alternative pathway to regenerate GSH from GSSG to maintain a stable GSSG/GSH ratio. Our data suggest the possibility that GSSG is transported from cytosol to apoplast to serve as an oxidant for class III peroxidases to generate reactive oxygen species for plant defense against Hessian fly larvae. Our results provide a foundation for elucidating the molecular processes involved in glutathione-mediated plant resistance to Hessian fly and potentially other pests as well. PMID:25627558

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

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

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

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.

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.

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.

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

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

[Effects of melaxen and valdoxan on the activity of glutathione antioxidant system and NADPH-producing enzymes in rat heart under experimental hyperthyroidism conditions].

PubMed

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

2013-01-01

The effects of melaxen and valdoxan on the activity of glutathione antioxidant system and some NADPH-producing enzymes have been studied under conditions of experimental hyperthyroidism in rat heart. Under the action of these drugs, reduced glutathione (GSH) content increased as compared to values observed under the conditions of pathology. It has been established that the activities of glutathione reductase (GR), glutathione peroxidase (GP), glucose-6-phosphate dehydrogenase, and NADP isocitrate dehydrogenase (increased under pathological conditions) change toward the intact control values upon the introduction of both drugs. The influence of melaxen and valdoxan, capable of producing antioxidant effect, leads apparently to the inhibition of free-radical oxidation processes and, as a consequence, the reduction of mobilization degree of the glutathione antioxidant system.

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.

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.

[GLUTATHIONE SYSTEM ACTIVITY IN RAT TISSUES UNDER PHENYLETHYL BIGUANIDE ACTION ON THE BACKGROUND OF EXPERIMENTAL BRAIN ISCHEMIA/REPERFUSION DEVELOPMENT].

PubMed

Safonova, O A; Popova, T N; Kryl'skii, D V

2016-01-01

It was studied the total antioxidant activity, content of primary lipid peroxidation (LPO) products and reduced glutathione, and the activity of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and NADP-isocitrate dehydrogenase in rat tissues under phenylethyl biguanide (phenfor- min) action on the background of experimental brain ischemia/reperfusion development. It is stablished the analyzed parameters, increasing under ischemia/reperfusion conditions in the brain and blood serum of animals, exhibit a decrease upon the introduction of this biguanide derivative. The obtained data can be explained by a decrease in degree of mobilization of the antioxidant system--in particular, of its glutathione chain--in the pathologic state. Hence, there is a need in NADPH supply for the system functioning compared with the pathology. Thus, phenylethyl biguanide demonstrates its antioxidant and protective properties under oxidative stress development that is accompanied by accumulation of the products of free radical oxidation of biomolecules during the ischemic brain injury.

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.

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.

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

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.

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.

Blood selenium concentrations and enzyme activities related to glutathione metabolism in wild emperor geese

USGS Publications Warehouse

Franson, J. Christian; Hoffman, David J.; Schmutz, Joel A.

2002-01-01

In 1998, we collected blood samples from 63 emperor geese (Chen canagica) on their breeding grounds on the Yukon-Kuskokwim Delta (YKD) in western Alaska, USA. We studied the relationship between selenium concentrations in whole blood and the activities of glutathione peroxidase and glutathione reductase in plasma. Experimental studies have shown that plasma activities of these enzymes are useful biomarkers of selenium-induced oxidative stress, but little information is available on their relationship to selenium in the blood of wild birds. Adult female emperor geese incubating their eggs in mid-June had a higher mean concentration of selenium in their blood and a greater activity of glutathione peroxidase in their plasma than adult geese or goslings that were sampled during the adult flight feathermolting period in late July and early August. Glutathione peroxidase activity was positively correlated with the concentration of selenium in the blood of emperor geese, and the rate of increase relative to selenium was greater in goslings than in adults. The activity of glutathione reductase was greatest in the plasma of goslings and was greater in molting adults than incubating females but was not significantly correlated with selenium in the blood of adults or goslings. Incubating female emperor geese had high selenium concentrations in their blood, accompanied by increased glutathione peroxidase activity consistent with early oxidative stress. These findings indicate that further study of the effects of selenium exposure, particularly on reproductive success, is warranted in this species.

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.

Impact of glutathione supplementation of parenteral nutrition on hepatic methionine adenosyltransferase activity.

PubMed

Elremaly, Wesam; Mohamed, Ibrahim; Rouleau, Thérèse; Lavoie, Jean-Claude

2016-08-01

The oxidation of the methionine adenosyltransferase (MAT) by the combined impact of peroxides contaminating parenteral nutrition (PN) and oxidized redox potential of glutathione is suspected to explain its inhibition observed in animals. A modification of MAT activity is suspected to be at origin of the PN-associated liver disease as observed in newborns. We hypothesized that the correction of redox potential of glutathione by adding glutathione in PN protects the MAT activity. To investigate whether the addition of glutathione to PN can reverse the inhibition of MAT observed in animal on PN. Three days old guinea pigs received through a jugular vein catheter 2 series of solutions. First with methionine supplement, (1) Sham (no infusion); (2) PN: amino acids, dextrose, lipids and vitamins; (3) PN-GSSG: PN+10μM GSSG. Second without methionine, (4) D: dextrose; (5) D+180μM ascorbylperoxide; (6) D+350μM H2O2. Four days later, liver was sampled for determination of redox potential of glutathione and MAT activity in the presence or absence of 1mM DTT. Data were compared by ANOVA, p<0.05. MAT activity was 45±4% lower in animal infused with PN and 23±7% with peroxides generated in PN. The inhibition by peroxides was associated with oxidized redox potential and was reversible by DTT. Correction of redox potential (PN+GSSG) or DTT was without effect on the inhibition of MAT by PN. The slope of the linear relation between MAT activity and redox potential was two fold lower in animal infused with PN than in others groups. The present study suggests that prevention of peroxide generation in PN and/or correction of the redox potential by adding glutathione in PN are not sufficient, at least in newborn guinea pigs, to restore normal MAT activity. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

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.

Glutathione-dependent extracellular ferric reductase activities in dimorphic zoopathogenic fungi

PubMed Central

Zarnowski, Robert; Woods, Jon P.

2009-01-01

In this study, extracellular glutathione-dependent ferric reductase (GSH-FeR) activities in different dimorphic zoopathogenic fungal species were characterized. Supernatants from Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis and Sporothrix schenckii strains grown in their yeast form were able to reduce iron enzymically with glutathione as a cofactor. Some variations in the level of reduction were noted amongst the strains. This activity was stable in acidic, neutral and slightly alkaline environments and was inhibited when trivalent aluminium and gallium ions were present. Using zymography, single bands of GSH-FeRs with apparent molecular masses varying from 430 to 460 kDa were identified in all strains. The same molecular mass range was determined by size exclusion chromatography. These data demonstrate that dimorphic zoopathogenic fungi produce and secrete a family of similar GSH-FeRs that may be involved in the acquisition and utilization of iron. Siderophore production by these and other fungi has sometimes been considered to provide a full explanation of iron acquisition in these organisms. Our work reveals an additional common mechanism that may be biologically and pathogenically important. Furthermore, while some characteristics of these enzymes such as extracellular location, cofactor utilization and large size are not individually unique, when considered together and shared across a range of fungi, they represent an important novel physiological feature. PMID:16000713

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.

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.

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

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.

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

Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases.

PubMed Central

Danielson, U H; Esterbauer, H; Mannervik, B

1987-01-01

The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism. PMID:3426557

Selenium concentrations and enzyme activities of glutathione metabolism in wild long-tailed ducks and common eiders

USGS Publications Warehouse

Franson, J. Christian; Hoffman, David J.; Flint, Paul L.

2011-01-01

The relationships of selenium (Se) concentrations in whole blood with plasma activities of total glutathione peroxidase, Se-dependent glutathione peroxidase, and glutathione reductase were studied in long-tailed ducks (Clangula hyemalis) and common eiders (Somateria mollissima) sampled along the Beaufort Sea coast of Alaska, USA. Blood Se concentrations were >8 μg/g wet weight in both species. Linear regression revealed that the activities of total and Se-dependent glutathione peroxidase were significantly related to Se concentrations only in long-tailed ducks, raising the possibility that these birds were experiencing early oxidative stress.

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.

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.

Cadmium-Induced Hydrogen Accumulation Is Involved in Cadmium Tolerance in Brassica campestris by Reestablishment of Reduced Glutathione Homeostasis.

PubMed

Wu, Qi; Su, Nana; Chen, Qin; Shen, Wenbiao; Shen, Zhenguo; Xia, Yan; Cui, Jin

2015-01-01

Hydrogen gas (H2) was recently proposed as a therapeutic antioxidant and signaling molecule in clinical trials. However, the underlying physiological roles of H2 in plants remain unclear. In the present study, hydrogen-rich water (HRW) was used to characterize the physiological roles of H2 in enhancing the tolerance of Brassica campestris against cadmium (Cd). The results showed that both 50 μM CdCl2 and 50%-saturated HRW induced an increase of endogenous H2 in Brassica campestris seedlings, and HRW alleviated Cd toxicity related to growth inhibition and oxidative damage. Seedlings supplied with HRW exhibited increased root length and reduced lipid peroxidation, similar to plants receiving GSH post-treatment. Additionally, seedlings post-treated with HRW accumulated higher levels of reduced glutathione (GSH) and ascorbic acid (AsA) and showed increased GST and GPX activities in roots. Molecular evidence illustrated that the expression of genes such as GS, GR1 and GR2, which were down-regulated following the addition of Cd, GSH or BSO, could be reversed to varying degrees by the addition of HRW. Based on these results, it could be proposed that H2 might be an important regulator for enhancing the tolerance of Brassica campestris seedlings against Cd, mainly by governing reduced glutathione homeostasis.

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.

Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

PubMed

Pettersson, Par L; Johansson, Ann-Sofie; Mannervik, Bengt

2002-08-16

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

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.

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.

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.

Atypical Thioredoxins in Poplar: The Glutathione-Dependent Thioredoxin-Like 2.1 Supports the Activity of Target Enzymes Possessing a Single Redox Active Cysteine1[W

PubMed Central

Chibani, Kamel; Tarrago, Lionel; Gualberto, José Manuel; Wingsle, Gunnar; Rey, Pascal; Jacquot, Jean-Pierre; Rouhier, Nicolas

2012-01-01

Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways. PMID:22523226

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.

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

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.

Pharmacogenetics of azathioprine in inflammatory bowel disease: A role for glutathione-S-transferase?

PubMed Central

Stocco, Gabriele; Pelin, Marco; Franca, Raffaella; De Iudicibus, Sara; Cuzzoni, Eva; Favretto, Diego; Martelossi, Stefano; Ventura, Alessandro; Decorti, Giuliana

2014-01-01

Azathioprine is a purine antimetabolite drug commonly used to treat inflammatory bowel disease (IBD). In vivo it is active after reaction with reduced glutathione (GSH) and conversion to mercaptopurine. Although this reaction may occur spontaneously, the presence of isoforms M and A of the enzyme glutathione-S-transferase (GST) may increase its speed. Indeed, in pediatric patients with IBD, deletion of GST-M1, which determines reduced enzymatic activity, was recently associated with reduced sensitivity to azathioprine and reduced production of azathioprine active metabolites. In addition to increase the activation of azathioprine to mercaptopurine, GSTs may contribute to azathioprine effects even by modulating GSH consumption, oxidative stress and apoptosis. Therefore, genetic polymorphisms in genes for GSTs may be useful to predict response to azathioprine even if more in vitro and clinical validation studies are needed. PMID:24707136

N-acetyl cysteine, L-cysteine, and beta-mercaptoethanol augment selenium-glutathione peroxidase activity in glucose-6-phosphate dehydrogenase-deficient human erythrocytes.

PubMed

Alicigüzel, Y; Aslan, M

2004-09-01

In glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes, failure to maintain normal levels of reduced glutathione (GSH) due to decreased NADPH regeneration in the hexose monophosphate pathway results in acute hemolytic anemia following exposure to oxidative insults, such as ingestion of Vicia fava beans or use of certain drugs. GSH is a source of protection against oxidative attack, used by the selenium-dependent glutathione peroxidase (Se-GSH-Px)/reductase (GR) system to detoxify hydrogen peroxide and organic peroxides, provided that sufficient GSH is made available. In this study, Se-GSH-Px activity was analyzed in G6PD-deficient patients in the presence of reducing agents such as N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol. Se-GSH-Px activity was decreased in G6PD-deficient red blood cells (RBCs). N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol increased Se-GSH-Px activity in G6PD-deficient human erythrocytes, indicating that other reducing agents can be utilized to complement Se-GSH-Px activity in G6PD deficiency. Based on the increased susceptibility of G6PD-deficient patients to oxidative stress, the reported increase in Se-GSH-Px activity can facilitate the detoxification of reactive oxygen species.

Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase

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

Fang, Ti; Li, De-Feng; Zhou, Ning-Yi, E-mail: n.zhou@pentium.whiov.ac.cn

2011-07-08

Highlights: {yields} Application of site-directed mutagenesis to probe the active site residues of glutathione-dependent maleylpyruvate isomerase. {yields} Two conserved residues, Arg8 and Arg176, in zeta class glutathione S-transferases are critical for maleylpyruvate orientation and enolization. {yields} Arg109, found exclusively in NagL, participates in k{sub cat} regulation. {yields} The T11A mutant exhibited a significantly decreased K{sub m} value for glutathione with little impact on maleylpyruvate kinetics. {yields} The Thr11 residue appears to have significance in the evolution of glutathione S-transferase classes. -- Abstract: The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerizationmore » of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k{sub cat} regulation. Surprisingly, the T11A mutant

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

[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.

Dietary fish oil replacement with palm or poultry oil increases fillet oxidative stability and decreases liver glutathione peroxidase activity in barramundi (Lates calcarifer).

PubMed

Wan Ahmad, Wan A R; Stone, David A J; Schuller, Kathryn A

2013-12-01

Complete dietary fish oil replacement with palm or poultry oil in barramundi (Lates calcarifer) had no detrimental effects on growth or hepatosomatic index of juvenile fish up to an average size of ~50 g. However, it significantly decreased the omega-3 (n-3) long-chain polyunsaturated fatty acid content of the fish muscle (fillet) lipids. This was particularly true for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) which are recognised for their health beneficial effects in the human diet. As a result of their decreased EPA and DHA content, the peroxidation index of the muscle lipids was also decreased. This was associated with increased simulated retail storage shelf life as indicated by decreased thiobarbituric acid reactive substances in muscle samples from fish fed the palm or poultry oil-based diets. Concomitantly, glutathione peroxidase (GPx) activity, but not glutathione S-transferase (GST) activity or reduced glutathione concentration, was significantly reduced in the liver of barramundi fed the palm or poultry oil-based diets as compared with the fish fed the fish oil-based diet. Furthermore, GPx and GST activity were very low in muscle, much lower than in gastrointestinal tract, liver or swim bladder. Therefore, we propose that liver GPx activity may be a good predictor of fillet shelf life in barramundi and other fish species.

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.

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

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

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

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

[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.

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

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

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.

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

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.

Imposed glutathione-mediated redox switch modulates the tobacco wound-induced protein kinase and salicylic acid-induced protein kinase activation state and impacts on defence against Pseudomonas syringae

PubMed Central

Matern, Sanja; Peskan-Berghoefer, Tatjana; Gromes, Roland; Kiesel, Rebecca Vazquez; Rausch, Thomas

2015-01-01

The role of the redox-active tripeptide glutathione in plant defence against pathogens has been studied extensively; however, the impact of changes in cellular glutathione redox potential on signalling processes during defence reactions has remained elusive. This study explored the impact of elevated glutathione content on the cytosolic redox potential and on early defence signalling at the level of mitogen-activated protein kinases (MAPKs), as well as on subsequent defence reactions, including changes in salicylic acid (SA) content, pathogenesis-related gene expression, callose depositions, and the hypersensitive response. Wild-type (WT) Nicotiana tabacum L. and transgenic high-glutathione lines (HGL) were transformed with the cytosol-targeted sensor GRX1-roGFP2 to monitor the cytosolic redox state. Surprisingly, HGLs displayed an oxidative shift in their cytosolic redox potential and an activation of the tobacco MAPKs wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK). This activation occurred in the absence of any change in free SA content, but was accompanied by constitutively increased expression of several defence genes. Similarly, rapid activation of MAPKs could be induced in WT tobacco by exposure to either reduced or oxidized glutathione. When HGL plants were challenged with adapted or non-adapted Pseudomonas syringae pathovars, the cytosolic redox shift was further amplified and the defence response was markedly increased, showing a priming effect for SA and callose; however, the initial and transient hyperactivation of MAPK signalling was attenuated in HGLs. The results suggest that, in tobacco, MAPK and SA signalling may operate independently, both possibly being modulated by the glutathione redox potential. Possible mechanisms for redox-mediated MAPK activation are discussed. PMID:25628332

Exogenous sodium nitroprusside and glutathione alleviate copper toxicity by reducing copper uptake and oxidative damage in rice (Oryza sativa L.) seedlings.

PubMed

Mostofa, Mohammad Golam; Seraj, Zeba Islam; Fujita, Masayuki

2014-11-01

Nitric oxide (NO) and glutathione (GSH) regulate a variety of physiological processes and stress responses; however, their involvement in mitigating Cu toxicity in plants has not been extensively studied. This study investigated the interactive effect of exogenous sodium nitroprusside (SNP) and GSH on Cu homeostasis and Cu-induced oxidative damage in rice seedlings. Hydroponically grown 12-day-old seedlings were subjected to 100 μM CuSO4 alone and in combination with 200 μM SNP (an NO donor) and 200 μM GSH. Cu exposure for 48 h resulted in toxicity symptoms such as stunted growth, chlorosis, and rolling in leaves. Cu toxicity was also manifested by a sharp increase in lipoxygenase (LOX) activity, lipid peroxidation (MDA), hydrogen peroxide (H2O2), proline (Pro) content, and rapid reductions in biomass, chlorophyll (Chl), and relative water content (RWC). Cu-caused oxidative stress was evident by overaccumulation of reactive oxygen species (ROS; superoxide (O2 (•-)) and H2O2). Ascorbate (AsA) content decreased while GSH and phytochelatin (PC) content increased significantly in Cu-stressed seedlings. Exogenous SNP, GSH, or SNP + GSH decreased toxicity symptoms and diminished a Cu-induced increase in LOX activity, O2 (•-), H2O2, MDA, and Pro content. They also counteracted a Cu-induced increase in superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and glyoxalase I and glyoxalase II activities, which paralleled changes in ROS and MDA levels. These seedlings also showed a significant increase in catalase (CAT), glutathione peroxidase (GPX), dehydroascorbate reductase (DHAR), glutathione S-transferase (GST) activities, and AsA and PC content compared with the seedlings stressed with Cu alone. Cu analysis revealed that SNP and GSH restricted the accumulation of Cu in the roots and leaves of Cu-stressed seedlings. Our results suggest that Cu exposure provoked an oxidative burden while

Glutathione affects the transport activity of Rhizobium leguminosarum 3841 and is essential for efficient nodulation.

PubMed

Cheng, Guojun; Karunakaran, Ramakrishnan; East, Alison K; Munoz-Azcarate, Olaya; Poole, Philip S

2017-04-01

As glutathione (GSH) plays an essential role in growth and symbiotic capacity of rhizobia, a glutathione synthetase (gshB) mutant of Rhizobium leguminosarum biovar viciae 3841 (Rlv3841) was characterised. It fails to efficiently utilise various compounds as a sole carbon source, including glucose, succinate, glutamine and histidine, and shows 60%-69% reduction in uptake rates of glucose, succinate and the non-metabolisable substrate α-amino isobutyric acid. The defect in glucose uptake can be overcome by addition of exogenous GSH, indicating GSH, but not its bacterial synthesis, is required for efficient transport. GSH is not involved in the regulation of the activity of Rlv3841's transporters via the global regulator of transport, PtsNTR. Although lack of GSH reduces transcription of the branched amino acid transporter, this was not the case for all uptake transport systems, for example, the amino acid permease. This suggests GSH alters activity and/or assembly of transport systems by an unknown mechanism. In interaction with plants, the gshB mutant is not only severely impaired in rhizosphere colonisation, but also shows a 50% reduction in dry weight of plants and nitrogen-fixation ability. This reveals that changes in GSH metabolism affect the bacterial-plant interactions required for symbiosis. © FEMS 2017.

In vitro interactions of thallium with components of the glutathione-dependent antioxidant defence system.

PubMed

Villaverde, Marcela S; Hanzel, Cecilia E; Verstraeten, Sandra V

2004-09-01

We investigated the hypothesis that thallium (Tl) interactions with the glutathione-dependent antioxidant defence system could contribute to the oxidative stress associated with Tl toxicity. Working in vitro with reduced glutathione (GSH), glutathione reductase (GR) or glutathione peroxidase (GPx) in solution, we studied the effects of Tl+ and Tl3+ (1-25 microM) on: (a) the amount of free GSH, investigating whether the metal binds to GSH and/or oxidizes it; (b) the activity of the enzyme GR, that catalyzes GSH regeneration; and (c) the enzyme GPx, that reduces hydroperoxide at expense of GSH oxidation. We found that, while Tl+ had no effect on GSH concentration, Tl3+ oxidized it. Both cations inhibited the reduction of GSSG by GR and the diaphorase activity of this enzyme. In addition, Tl3+ per se oxidized NADPH, the cofactor of GR. The effects of Tl on GPx activity depended on the metal charge: Tl+ inhibited GPx when cumene hydroperoxide (CuOOH) was the substrate, while Tl(3+)-mediated GPx inhibition occurred with both substrates. The present results show that Tl interacts with all the components of GSH/GSSG antioxidant defence system. Alterations of this protective pathway could be partially responsible for the oxidative stress associated with Tl toxicity.

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.

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.

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

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.

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

α-Lipoic acid protects against the cytotoxicity and oxidative stress induced by cadmium in HepG2 cells through regeneration of glutathione by glutathione reductase via Nrf2/ARE signaling pathway.

PubMed

Shi, Chunli; Zhou, Xue; Zhang, Jiayu; Wang, Jiachun; Xie, Hong; Wu, Zhigang

2016-07-01

α-Lipoic acid (α-LA) is a potent natural antioxidant, which is capable of regenerating glutathione (GSH). However, the mechanisms by which α-LA regenerates reduced glutathione (rGSH) via the reduction of oxidized glutathione (GSSG) by glutathione reductase (GR) are still not well understood. In the present study, we investigated if α-LA replenished rGSH by GR via Nrf2/ARE signaling pathway in cadmium-treated HepG2 cells. We found that α-LA antagonized the oxidative damage and alleviated the cytotoxicity in cadmium-induced HepG2 cells by regeneration of rGSH. α-LA regenerated rGSH by activating Nrf2 signaling pathway via promoting the nuclear translocation of Nrf2, which upregulates the transcription of GR, and thus increased the activity of GR. Our results indicated that α-LA was an effective agent to antagonize the oxidative stress and alleviate the cytotoxicity in cadmium-treated HepG2 cells by regenerating rGSH through activating Nrf2 signaling pathway. Copyright © 2016. Published by Elsevier B.V.

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

Imposed glutathione-mediated redox switch modulates the tobacco wound-induced protein kinase and salicylic acid-induced protein kinase activation state and impacts on defence against Pseudomonas syringae.

PubMed

Matern, Sanja; Peskan-Berghoefer, Tatjana; Gromes, Roland; Kiesel, Rebecca Vazquez; Rausch, Thomas

2015-04-01

The role of the redox-active tripeptide glutathione in plant defence against pathogens has been studied extensively; however, the impact of changes in cellular glutathione redox potential on signalling processes during defence reactions has remained elusive. This study explored the impact of elevated glutathione content on the cytosolic redox potential and on early defence signalling at the level of mitogen-activated protein kinases (MAPKs), as well as on subsequent defence reactions, including changes in salicylic acid (SA) content, pathogenesis-related gene expression, callose depositions, and the hypersensitive response. Wild-type (WT) Nicotiana tabacum L. and transgenic high-glutathione lines (HGL) were transformed with the cytosol-targeted sensor GRX1-roGFP2 to monitor the cytosolic redox state. Surprisingly, HGLs displayed an oxidative shift in their cytosolic redox potential and an activation of the tobacco MAPKs wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK). This activation occurred in the absence of any change in free SA content, but was accompanied by constitutively increased expression of several defence genes. Similarly, rapid activation of MAPKs could be induced in WT tobacco by exposure to either reduced or oxidized glutathione. When HGL plants were challenged with adapted or non-adapted Pseudomonas syringae pathovars, the cytosolic redox shift was further amplified and the defence response was markedly increased, showing a priming effect for SA and callose; however, the initial and transient hyperactivation of MAPK signalling was attenuated in HGLs. The results suggest that, in tobacco, MAPK and SA signalling may operate independently, both possibly being modulated by the glutathione redox potential. Possible mechanisms for redox-mediated MAPK activation are discussed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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.

Glutathione Peroxidase Enzyme Activity in Aging

PubMed Central

Espinoza, Sara E.; Guo, Hongfei; Fedarko, Neal; DeZern, Amy; Fried, Linda P.; Xue, Qian-Li; Leng, Sean; Beamer, Brock; Walston, Jeremy D.

2010-01-01

Background It is hypothesized that free radical damage contributes to aging. Age-related decline in activity of the antioxidant enzyme glutathione peroxidase (GPx) may contribute to increased free radicals. We hypothesized that GPx activity decreases with age in a population of older women with disability. Methods Whole blood GPx activity was measured in baseline stored samples from participants in the Women's Health and Aging Study I, a cohort of disabled community-dwelling older women. Linear regression was used to determine cross-sectional associations between GPx activity and age, adjusting for hemoglobin, coronary disease, diabetes, selenium, and body mass index. Results Six hundred one participants had complete demographic, disease, and laboratory information. An inverse association was observed between GPx and age (regression coefficient = −2.9, p < .001), indicating that for each 1-year increase in age, GPx activity decreased by 2.9 μmol/min/L. This finding remained significant after adjustment for hemoglobin, coronary disease, diabetes, and selenium, but not after adjustment for body mass index and weight loss. Conclusion This is the first study to examine the association between age and GPx activity in an older adult cohort with disability and chronic disease. These findings suggest that, after age 65, GPx activity declines with age in older women with disability. This decline does not appear to be related to diseases that have been previously reported to alter GPx activity. Longitudinal examination of GPx activity and other antioxidant enzymes in diverse populations of older adults will provide additional insight into age- and disease-related changes in these systems. PMID:18511755

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.

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.

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.

Glutathione depletion activates the yeast vacuolar transient receptor potential channel, Yvc1p, by reversible glutathionylation of specific cysteines

PubMed Central

Chandel, Avinash; Das, Krishna K.; Bachhawat, Anand K.

2016-01-01

Glutathione depletion and calcium influx into the cytoplasm are two hallmarks of apoptosis. We have been investigating how glutathione depletion leads to apoptosis in yeast. We show here that glutathione depletion in yeast leads to the activation of two cytoplasmically inward-facing channels: the plasma membrane, Cch1p, and the vacuolar calcium channel, Yvc1p. Deletion of these channels partially rescues cells from glutathione depletion–induced cell death. Subsequent investigations on the Yvc1p channel, a homologue of the mammalian TRP channels, revealed that the channel is activated by glutathionylation. Yvc1p has nine cysteine residues, of which eight are located in the cytoplasmic regions and one on the transmembrane domain. We show that three of these cysteines, Cys-17, Cys-79, and Cys-191, are specifically glutathionylated. Mutation of these cysteines to alanine leads to a loss in glutathionylation and a concomitant loss in calcium channel activity. We further investigated the mechanism of glutathionylation and demonstrate a role for the yeast glutathione S-transferase Gtt1p in glutathionylation. Yvc1p is also deglutathionylated, and this was found to be mediated by the yeast thioredoxin, Trx2p. A model for redox activation and deactivation of the yeast Yvc1p channel is presented. PMID:27708136

JS-K, a glutathione S-transferase-activated nitric oxide donor with antineoplastic activity in malignant gliomas

PubMed Central

Weyerbrock, Astrid; Osterberg, Nadja; Psarras, Nikolaos; Baumer, Brunhilde; Kogias, Evangelos; Werres, Anna; Bette, Stefanie; Saavedra, Joseph E.; Keefer, Larry K.; Papazoglou, Anna

2011-01-01

Background 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. Objective To evaluate the role of NO-based antitumor therapy with JS-K in U87 gliomas in vitro and in vivo. Methods 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, PCR and Western blot and NO release from JS-K using a NO assay. The growth-inhibitory effect of JS-K was studied in a U87 xenograft model in vivo. Results 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. GST-inhibition by sulfasalazine, cGMP inhibition by ODQ and MEK 1/2 inhibition by UO126 attenuated the antiproliferative effect of JS-K, suggesting the involvement of various intracellular death signalling 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 significantly reduced, with immunohistochemical evidence for increased necrosis, apoptosis and reduced proliferation. Conclusion Our data for the first time show 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. PMID:21849924

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.

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.

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.

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.

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.

Evidence for the Presence of the Ascorbate-Glutathione Cycle in Mitochondria and Peroxisomes of Pea Leaves.

PubMed Central

Jimenez, A.; Hernandez, J. A.; Del Rio, L. A.; Sevilla, F.

1997-01-01

The presence of the enzymes of the ascorbate-glutathione cycle was investigated in mitochondria and peroxisomes purified from pea (Pisum sativum L.) leaves. All four enzymes, ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), were present in mitochondria and peroxisomes, as well as in the antioxidants ascorbate and glutathione. The activity of the ascorbate-glutathione cycle enzymes was higher in mitochondria than in peroxisomes, except for APX, which was more active in peroxisomes than in mitochondria. Intact mitochondria and peroxisomes had no latent APX activity, and this remained in the membrane fraction after solubilization assays with 0.2 M KCl. Monodehydroascorbate reductase was highly latent in intact mitochondria and peroxisomes and was membrane-bound, suggesting that the electron acceptor and donor sites of this redox protein are not on the external side of the mitochondrial and peroxisomal membranes. Dehydroascorbate reductase was found mainly in the soluble peroxisomal and mitochondrial fractions. Glutathione reductase had a high latency in mitochondria and peroxisomes and was present in the soluble fractions of both organelles. In intact peroxisomes and mitochondria, the presence of reduced ascorbate and glutathione and the oxidized forms of ascorbate and glutathione were demonstrated by high-performance liquid chromatography analysis. The ascorbate-glutathione cycle of mitochondria and peroxisomes could represent an important antioxidant protection system against H2O2 generated in both plant organelles. PMID:12223704

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

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.

[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

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

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.

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.

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.

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

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.

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.

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.

Correlation between Glutathione Peroxidase Activity and Anthropometrical Parameters in Adolescents with Down Syndrome

ERIC Educational Resources Information Center

Ordonez, F. J.; Rosety-Rodriguez, M.

2007-01-01

Since we have recently found that regular exercise increased erythrocyte antioxidant enzyme activities such as glutathione peroxidase (GPX) in adolescents with Down syndrome, these programs may be recommended. This study was designed to assess the role of anthropometrical parameters as easy, economic and non-invasive biomarkers of GPX. Thirty-one…

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.

[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.

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.

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.

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.

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

Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

PubMed Central

Hu, L.; Borleske, B. L.; Colman, R. F.

1997-01-01

Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes. PMID:9007975

Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

PubMed

Hu, L; Borleske, B L; Colman, R F

1997-01-01

Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes.

Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S-transferase, 1-1.

PubMed Central

Wang, J.; Barycki, J. J.; Colman, R. F.

1996-01-01

Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a KI of 0.78 mM and kmax of 0.011 min-1. S-Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S-methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pKa, which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pKa of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that

Epigallocatechin-3-gallate enhances key enzymatic activities of hepatic thioredoxin and glutathione systems in selenium-optimal mice but activates hepatic Nrf2 responses in selenium-deficient mice.

PubMed

Dong, Ruixia; Wang, Dongxu; Wang, Xiaoxiao; Zhang, Ke; Chen, Pingping; Yang, Chung S; Zhang, Jinsong

2016-12-01

Selenium participates in the antioxidant defense mainly through a class of selenoproteins, including thioredoxin reductase. Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechin in green tea. Depending upon the dose and biological systems, EGCG may function either as an antioxidant or as an inducer of antioxidant defense via its pro-oxidant action or other unidentified mechanisms. By manipulating the selenium status, the present study investigated the interactions of EGCG with antioxidant defense systems including the thioredoxin system comprising of thioredoxin and thioredoxin reductase, the glutathione system comprising of glutathione and glutathione reductase coupled with glutaredoxin, and the Nrf2 system. In selenium-optimal mice, EGCG increased hepatic activities of thioredoxin reductase, glutathione reductase and glutaredoxin. These effects of EGCG appeared to be not due to overt pro-oxidant action because melatonin, a powerful antioxidant, did not influence the increase. However, in selenium-deficient mice, with low basal levels of thioredoxin reductase 1, the same dose of EGCG did not elevate the above-mentioned enzymes; intriguingly EGCG in turn activated hepatic Nrf2 response, leading to increased heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 protein levels and thioredoxin activity. Overall, the present work reveals that EGCG is a robust inducer of the Nrf2 system only in selenium-deficient conditions. Under normal physiological conditions, in selenium-optimal mice, thioredoxin and glutathione systems serve as the first line defense systems against the stress induced by high doses of EGCG, sparing the activation of the Nrf2 system. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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

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.

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.

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.

Association between ETFA genotype and activity of superoxide dismutase, catalase and glutathione peroxidase in cryopreserved sperm of Holstein-Friesian bulls.

PubMed

Hering, D M; Lecewicz, M; Kordan, W; Kamiński, S

2015-02-01

The aim of this study was to determine whether C/T missense mutation within the ETFA gene is associated with sperm antioxidant enzymatic activity. One hundred and twenty Holstein-Friesian bulls were genotyped by the PCR-RFLP technique (MwoI). Commercial straws of frozen-thawed semen were used to evaluate the activity of three antioxidant enzymes: superoxide dismutase, catalase and glutathione peroxidase. Among all bulls investigated, genotype CT was the most frequent (44.2%), in comparison with CC (42.5%) and TT (13.3%). Significant differences in glutathione peroxidase activity were observed between homozygous individuals (CC vs TT) with heterozygous CT having intermediate values. Dismutase activity was significantly associated with ETFA genotype, although only bulls with the CT genotype were significantly different from bulls carrying the CC genotype. The activity of catalase showed a similar trend (but was not statistically significant). In conclusion, we found that bulls with the ETFA TT genotype produce sperm with the highest glutathione peroxidase activity and can therefore be more efficiently protected from reactive oxygen. The mechanism of this interaction needs to be elucidated in future research. © 2014 Blackwell Verlag GmbH.

Modulation of nitric oxide-mediated metal release from metallothionein by the redox state of glutathione in vitro.

PubMed

Khatai, Leila; Goessler, Walter; Lorencova, Helena; Zangger, Klaus

2004-06-01

Metallothioneins (MTs) release bound metals when exposed to nitric oxide. At inflammatory sites, both metallothionein and inducible nitric oxide synthase (iNOS) are induced by the same factors and the zinc released from metallothionein by NO suppresses both the induction and activity of iNOS. In a search for a possible modulatory mechanism of this coexpression of counteracting proteins, we investigated the role of the glutathione redox state in vitro because the oxidation state of thiols is involved in the metal binding in Cd-S or Zn-S clusters found in metallothioneins, and NO also binds to reduced glutathione via S-nitrosation. Using a variety of techniques, we found that NO and also ONOO(-)-mediated metal release from purified MTs is suppressed by reduced glutathione (GSH), but not by oxidized glutathione. Considering the millimolar concentrations of GSH present in mammalian cells, the metal release from MTs by NO should play no role in living systems. Therefore, the fact that it has been observed in vivo points to a hitherto unknown mechanism or additional compound(s) being involved in this physiologically relevant reaction and as long as this additional factor is not found experimental results on the MT-NO interaction should be treated with caution. Contrary to the peroxynitrite-induced activation of guanylyl cyclase, where GSH is needed, we found that the metal release from metallothionein by peroxynitrite is not enhanced, but also suppressed by reduced glutathione. In addition, we show that zinc, the major natural metal ligand in mammalian MTs and suppressor of iNOS, is released more readily under the influence of NO than cadmium, but in contrast to the MT isoform 1, the amount of metal released from the beta-domain of MT-2 is comparable to that from the alpha-domain.

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.

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.

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

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.

Expression Patterns of Genes Involved in Ascorbate-Glutathione Cycle in Aphid-Infested Maize (Zea mays L.) Seedlings

PubMed Central

Sytykiewicz, Hubert

2016-01-01

Reduced forms of ascorbate (AsA) and glutathione (GSH) are among the most important non-enzymatic foliar antioxidants in maize (Zea mays L.). The survey was aimed to evaluate impact of bird cherry-oat aphid (Rhopalosiphum padi L.) or grain aphid (Sitobion avenae F.) herbivory on expression of genes related to ascorbate-glutathione (AsA-GSH) cycle in seedlings of six maize varieties (Ambrozja, Nana, Tasty Sweet, Touran, Waza, Złota Karłowa), differing in resistance to the cereal aphids. Relative expression of sixteen maize genes encoding isoenzymes of ascorbate peroxidase (APX1, APX2, APX3, APX4, APX5, APX6, APX7), monodehydroascorbate reductase (MDHAR1, MDHAR2, MDHAR3, MDHAR4), dehydroascorbate reductase (DHAR1, DHAR2, DHAR3) and glutathione reductase (GR1, GR2) was quantified. Furthermore, effect of hemipterans’ attack on activity of APX, MDHAR, DHAR and GR enzymes, and the content of reduced and oxidized ascorbate and glutathione in maize plants were assessed. Seedling leaves of more resistant Z. mays varieties responded higher elevations in abundance of target transcripts. In addition, earlier and stronger aphid-triggered changes in activity of APX, MDHAR, DHAR and GR enzymes, and greater modulations in amount of the analyzed antioxidative metabolites were detected in foliar tissues of highly resistant Ambrozja genotype in relation to susceptible Tasty Sweet plants. PMID:26907270

Expression Patterns of Genes Involved in Ascorbate-Glutathione Cycle in Aphid-Infested Maize (Zea mays L.) Seedlings.

PubMed

Sytykiewicz, Hubert

2016-02-23

Reduced forms of ascorbate (AsA) and glutathione (GSH) are among the most important non-enzymatic foliar antioxidants in maize (Zea mays L.). The survey was aimed to evaluate impact of bird cherry-oat aphid (Rhopalosiphum padi L.) or grain aphid (Sitobion avenae F.) herbivory on expression of genes related to ascorbate-glutathione (AsA-GSH) cycle in seedlings of six maize varieties (Ambrozja, Nana, Tasty Sweet, Touran, Waza, Złota Karłowa), differing in resistance to the cereal aphids. Relative expression of sixteen maize genes encoding isoenzymes of ascorbate peroxidase (APX1, APX2, APX3, APX4, APX5, APX6, APX7), monodehydroascorbate reductase (MDHAR1, MDHAR2, MDHAR3, MDHAR4), dehydroascorbate reductase (DHAR1, DHAR2, DHAR3) and glutathione reductase (GR1, GR2) was quantified. Furthermore, effect of hemipterans' attack on activity of APX, MDHAR, DHAR and GR enzymes, and the content of reduced and oxidized ascorbate and glutathione in maize plants were assessed. Seedling leaves of more resistant Z. mays varieties responded higher elevations in abundance of target transcripts. In addition, earlier and stronger aphid-triggered changes in activity of APX, MDHAR, DHAR and GR enzymes, and greater modulations in amount of the analyzed antioxidative metabolites were detected in foliar tissues of highly resistant Ambrozja genotype in relation to susceptible Tasty Sweet plants.

Effects of GSH1 and GSH2 Gene Mutation on Glutathione Synthetases Activity of Saccharomyces cerevisiae.

PubMed

Xu, Wen; Jia, Haiyan; Zhang, Longmei; Wang, Haiyan; Tang, Hui; Zhang, Liping

2017-08-01

In this paper, three mutants from wild Saccharomyces cerevisiae HBU2.558, called U2.558, UN2.558, and UNA2.558, were screened by UV, sodium nitrite, Atmospheric and room temperature plasma, respectively. Glutathione production of the three mutants increased by 41.86, 72.09 and 56.76%, respectively. We detected the activity of glutathione synthetases and found that its activity was improved. Amino acid sequences of three mutant colonies were compared with HBU2.558. Four mutants: Leu51→Pro51 (L51P), Glu62→Val62 (E62V), Ala332→Glu332 (A332E) and Ser653→Gly653 (S653G) were found in the analysis of γ-glutamylcysteine ligase. L51 is located adjacently to the two active sites of GCL/E/Mg 2+ /ADP complex in the overall GCL structure. L51P mutant spread distortion on the β-sheet due to the fact that the φ was changed from -50.4° to -40.2°. A mutant Leu54→Pro54 (L54P) was found in the analysis of glutathione synthetase, and L54 was an amino acid located between an α-helix and a β-sheet. The results confirm that introduction of proline located at the middle of the β-sheet or at the N- or C-terminal between α-helix and β-sheet or, i.e., L51P and L54P, changed the φ, rigidity, hydrophobicity and conformational entropy, thus increased protein stability and improved the enzyme activity.

A model to environmental monitoring based on glutathione-S-transferase activity and branchial lesions in catfish

NASA Astrophysics Data System (ADS)

Neta, Raimunda Nonata Fortes Carvalho; Torres, Audalio Rebelo

2017-11-01

In this work, we validate the glutathione-S-transferase and branchial lesions as biomarkers in catfish Sciades herzbergii to obtain a predictive model of the environmental impact effects in a harbor of Brazil. The catfish were sampled from a port known to be contaminated with heavy metals and organic compounds and from a natural reserve in São Marcos Bay, Maranhão. Two biomarkers, hepatic glutathione S-transferase (GST) activity and branchial lesions were analyzed. The values for GST activity were modeled with the occurrence of branchial lesions by fitting a third order polynomial. Results from the mathematical model indicate that GST activity has a strong polynomial relationship with the occurrence of branchial lesions in both the wet and the dry seasons, but only at the polluted port site. Our mathematic model indicates that when the GST ceases to act, serious branchial lesions are observed in the catfish of the contaminated port area.

Subchronic glucocorticoids, glutathione depletion and a postpartum model elevate monoamine oxidase a activity in the prefrontal cortex of rats.

PubMed

Raitsin, Sofia; Tong, Junchao; Kish, Stephen; Xu, Xin; Magomedova, Lilia; Cummins, Carolyn; Andreazza, Ana C; Scola, Gustavo; Baker, Glen; Meyer, Jeffrey H

2017-07-01

Recent human brain imaging studies implicate dysregulation of monoamine oxidase-A (MAO-A), in particular in the prefrontal cortex (PFC) and anterior cingulate cortex (ACC), in the development of major depressive disorder (MDD). This study investigates the influence of four alterations underlying important pathologies of MDD, namely, chronic elevation of glucocorticoid levels, glutathione depletion, changes in female gonadal sex hormones and serotonin concentration fluctuation, on MAO-A and MAO-B activities in rats. Young adult rats exposed chronically to the synthetic glucocorticoid dexamethasone at 0, 0.05, 0.5, and 2.0mg/kg/day (osmotic minipumps) for eight days showed significant dose-dependent increases in activities of MAO-A in PFC (+17%, p<0.001) and ACC (+9%, p<0.01) and MAO-B in PFC (+14%, p<0.001) and increased serotonin turnover in the PFC (+31%, p<0.01), not accounted for by dexamethasone-induced changes in serotonin levels, since neither serotonin depletion nor supplementation affected MAO-A activity. Sub-acute depletion of the major antioxidant glutathione by diethyl maleate (5mmol/kg, i.p.) for three days, which resulted in a 36% loss of glutathione in PFC (p=0.0005), modestly, but significantly, elevated activities of MAO-A in PFC and MAO-B in PFC, ACC and hippocampus (+6-9%, p<0.05). Changes in estrogen and progesterone representing pseudopregnancy were associated with significantly elevated MAO-A activity in the ACC day 4-7 postpartum (10-18%, p<0.05 to p<0.0001) but not the PFC or hippocampus. Hence, our study provides data in support of strategies targeting glucocorticoid and glutathione systems, as well as changes in female sex hormones for normalization of MAO-A activities and thus treatment of mood disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of mercury and selenium on glutathione metabolism and oxidative stress in mallard ducks

USGS Publications Warehouse

Hoffman, D.J.; Heinz, G.H.

1998-01-01

Earlier studies reported on the toxicity and related oxidative stress of different forms of Se, including seleno-D,L-methionine, in mallards (Anas platyrhynchos). This study compares the effects of Se (seleno-D,L-methionine) and Hg (methylmercury chloride) separately and in combination. Mallard drakes received one of the following diets: untreated feed (controls), or feed containing 10 ppm Se, 10 ppm Hg, or 10 ppm Se in combination with 10 ppm Hg. After 10 weeks, blood, liver, and brain samples were collected for biochemical assays. The following clinical and biochemical alterations occurred in response to mercury exposure: hematocrit and hemoglobin concentrations decreased; activities of the enzymes glutathione (GSH) peroxidase (plasma and liver), glutathione-S-transferase (liver), and glucose-6-phosphate dehydrogenase (G-6-PDH) (liver and brain) decreased; hepatic oxidized glutathione (GSSG) concentration increased relative to reduced glutathione (GSH); and lipid peroxidation in the brain was evident as detected by increased thiobarbituric reactive substances (TBARS). Effects of Se alone included increased hepatic GSSG reductase activity and brain TBARS concentration. Se in combination with Hg partially or totally alleviated effects of Hg on GSH peroxidase, G-6-PDH, and GSSG. These findings are compared in relation to field observations for diving ducks and other aquatic birds. It is concluded that since both Hg and excess Se can affect thiol status, measurement of associated enzymes in conjunction with thiol status may be a useful bioindicator to discriminate between Hg and Se effects. The ability of Se to restore the activities of G-6-PDH, GSH peroxidase, and glutathione status involved in antioxidative defense mechanisms may be crucial to biological protection from the toxic effects of methyl mercury.

Efficacy of Jian'ganle () versus Hugan Pian (), glucuronolactone and reduced glutathione in prevention of antituberculosis drug-induced liver injury.

PubMed

Zhang, Quan; Zhong, Fang-ying; Wu, Meng; Zhang, Xin-ping

2014-06-01

Evidence-based medicine is advocated by WHO and adopted by developed countries for many years. In China, however, the selection of essential medicine and various medical insurance reimbursement schemes medicine is usually based on experts' experience of prescription practice which is under heavy critics resulting from the lack of related comparative efficacy and evidence-based research. The efficacy of Jian'ganle in prevention of drug-induced liver injury (DILI) caused by antituberculotics was evaluated in this study by comparison with Hugan Pian, glucuronolactone and reduced glutathione. Evidence was provided for relevant sectors such as Ministry for Human Resources and Social Security of the People's Republic of China and National Health and Family Planning Commission of the People's Republic of China to select and renew the Essential Medicine List (EML), the new rural cooperative medical scheme in China (NRCMS) list or the reimbursement list of industrial injury insurance. A total of 189 patients with initial pulmonary tuberculosis were divided into four groups who took antituberculotics combined with Jian'ganle, Hugan Pian, glucuronolactone and reduced glutathione respectively. Their liver function profile including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), total protein (TP), albumin (A) and globulin (G) were detected at admission as baseline and after treatment. The Jian'ganle group was compared with the three others by chi-square tests. In an aspect of maintaining bilirubin indexes normal, Jian'ganle was more efficacious than glucuronolactone. And Jian'ganle had a little more efficacy than reduced glutathione to maintain protein indexes normal as well. And the therapeutic regimen of antituberculotics combined with Jian'ganle was the best in treating tuberculosis and preventing DILI at the same time. The study showed that among the four hepatinicas which demonstrated similar prevention

Effects of commercial selenium products on glutathione peroxidase activity and semen quality in stud boars

USDA-ARS?s Scientific Manuscript database

The aim of this study was to determine how dietary supplementation of inorganic and organic selenium affects selenium concentration and glutathione peroxidase activity in blood and sperm of sexually mature stud boars. Twenty-four boars of the Large White, Landrace, Pietrain, and Duroc breeds of opt...

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

Diagnosis of glutathione synthetase deficiency in newborn screening.

PubMed

Simon, E; Vogel, M; Fingerhut, R; Ristoff, E; Mayatepek, E; Spiekerkötter, U

2009-12-01

Glutathione synthetase (GSS) deficiency is a rare disorder of glutathione metabolism with varying clinical severity. Patients may present with haemolytic anaemia 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 GSS activity in erythrocytes or cultured skin fibroblasts. Diagnosis can be confirmed by mutational analysis. Treatment consists of the correction of acidosis, blood transfusion, and supplementation with antioxidants. The most important determinants for outcome and survival in patients with GSS deficiency are early diagnosis and early initiation of treatment. The case of a newborn with GSS deficiency diagnosed by tandem mass spectrometry (MS/MS)-based newborn screening is described. After onset of clinical symptoms on the 2nd day of life, expanded newborn screening revealed normal results for all disorders included in the German screening programme; however, selective MS/MS screening revealed a >10-fold elevation of 5-oxoproline in dried blood, leading to the presumptive diagnosis of GSS deficiency by the 5th day of life. Diagnosis was later confirmed by detection of markedly reduced glutathione concentration in erythrocytes and mutational analysis of the GSS gene. Presently, GSS deficiency is not included in newborn screening programmes in Europe. As outcome depends significantly on early start of treatment, routine inclusion of this disorder in newborn screening panels should be considered.

Role of vitamin B6 status on antioxidant defenses, glutathione, and related enzyme activities in mice with homocysteine-induced oxidative stress.

PubMed

Hsu, Cheng-Chin; Cheng, Chien-Hsiang; Hsu, Chin-Lin; Lee, Wan-Ju; Huang, Shih-Chien; Huang, Yi-Chia

2015-01-01

Vitamin B6 may directly or indirectly play a role in oxidative stress and the antioxidant defense system. The purpose of this study was to examine the associations of vitamin B6 status with cysteine, glutathione, and its related enzyme activities in mice with homocysteine-induced oxidative stress. Four-week-old male BALB/c mice were weighed and divided into one of four dietary treatment groups fed either a normal diet (as a control group and a homocysteine group), a vitamin B6-deficient diet (as a B6-deficient group), or a B6-supplemented diet (a pyridoxine-HCl-free diet supplemented with 14 mg/kg of pyridoxine-HCl, as a B6 supplement group) for 28 days. Homocysteine thiolactone was then added to drinking water in three groups for 21 days to induce oxidative stress. At the end of the study, mice were sacrificed by decapitation and blood and liver samples were obtained. Mice with vitamin B6-deficient diet had the highest homocysteine concentration in plasma and liver among groups. Significantly increased hepatic malondialdehyde levels were observed in the vitamin B6-deficient group. Among homocysteine-treated groups, mice with vitamin B6-deficient diet had the highest plasma glutathione concentration and relatively lower hepatic glutathione concentration. The glutathione peroxidase activities remained relatively stable in plasma and liver whether vitamin B6 was adequate, deficient, or supplemented. Mice with deficient vitamin B6 intakes had an aggravate effect under homocysteine-induced oxidative stress. The vitamin B6-deficient status seems to mediate the oxidative stress in connection with the redistribution of glutathione from liver to plasma, but not further affect glutathione-related enzyme activities in mice with homocysteine-induced oxidative stress.

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.

Inhibition of Tapeworm Thioredoxin and Glutathione Pathways by an Oxadiazole N-Oxide Leads to Reduced Mesocestoides vogae Infection Burden in Mice.

PubMed

Pasquet, Vivian; Bisio, Hugo; López, Gloria V; Romanelli-Cedrez, Laura; Bonilla, Mariana; Saldaña, Jenny; Salinas, Gustavo

2015-06-26

Parasitic flatworms cause serious infectious diseases that affect humans and livestock in vast regions of the world, yet there are few effective drugs to treat them. Thioredoxin glutathione reductase (TGR) is an essential enzyme for redox homeostasis in flatworm parasites and a promising pharmacological target. We purified to homogeneity and characterized the TGR from the tapeworm Mesocestoides vogae (syn. M. corti). This purification revealed absence of conventional TR and GR. The glutathione reductase activity of the purified TGR exhibits a hysteretic behavior typical of flatworm TGRs. Consistently, M. vogae genome analysis revealed the presence of a selenocysteine-containing TGR and absence of conventional TR and GR. M. vogae thioredoxin and glutathione reductase activities were inhibited by 3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole N2-oxide (VL16E), an oxadiazole N-oxide previously identified as an inhibitor of fluke and tapeworm TGRs. Finally, we show that mice experimentally infected with M. vogae tetrathyridia and treated with either praziquantel, the reference drug for flatworm infections, or VL16E exhibited a 28% reduction of intraperitoneal larvae numbers compared to vehicle treated mice. Our results show that oxadiazole N-oxide is a promising chemotype in vivo and highlights the convenience of M. vogae as a model for rapid assessment of tapeworm infections in vivo.

Inducible NAD(H)-linked methylglyoxal oxidoreductase regulates cellular methylglyoxal and pyruvate through enhanced activities of alcohol dehydrogenase and methylglyoxal-oxidizing enzymes in glutathione-depleted Candida albicans.

PubMed

Kwak, Min-Kyu; Ku, MyungHee; Kang, Sa-Ouk

2018-01-01

High methylglyoxal content disrupts cell physiology, but mammals have scavengers to prevent glycolytic and mitochondrial dysfunctions. In yeast, methylglyoxal accumulation triggers methylglyoxal-oxidizing alcohol dehydrogenase (Adh1) activity. While methylglyoxal reductases and glyoxalases have been well studied in prokaryotes and eukaryotes, experimental evidence for methylglyoxal dehydrogenase (Mgd) and other catalytic activities of this enzyme affecting glycolysis and the tricarboxylic acid cycle is lacking. A glycine-rich cytoplasmic Mgd protein, designated as Mgd1/Grp2, was isolated from glutathione-depleted Candida albicans. The effects of Mgd1/Grp2 activities on metabolic pathophysiology were investigated using knockout and overexpression mutants. We measured glutathione-(in)dependent metabolite contents and metabolic effects, including viability, oxygen consumption, ADH1 transcripts, and glutathione reductase and α-ketoglutarate dehydrogenase activities in the mutants. Based on the findings, methylglyoxal-oxidizing proteins were monitored to determine effects of MGD1/GRP2 disruption on methylglyoxal-scavenging traits during glutathione deprivation. Methylglyoxal-oxidizing NAD(H)-linked Mgd1/Grp2 was found solely in glutathione auxotrophs, and it catalyzed the reduction of both methylglyoxal and pyruvate. MGD1/GRP2 disruptants showed growth defects, cell-cycle arrest, and methylglyoxal and pyruvate accumulation with mitochondrial impairment, regardless of ADH1 compensation. Other methylglyoxal-oxidizing enzymes were identified as key glycolytic enzymes with enhanced activity and transcription in MGD1/GRP2 disruptants, irrespective of glutathione content. Failure of methylglyoxal and pyruvate dissimilation by Mgd1/Grp2 deficiency leads to poor glutathione-dependent redox regulation despite compensation by Adh1. This is the first report that multifunctional Mgd activities contribute to scavenging methylglyoxal and pyruvate to maintain metabolic homeostasis

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.

Application of Hyperbaric Oxygen Reduce Oxidative Damage of Plasmatic Carbonyl Proteins and 8-OHdG by Activating Glutathion Peroxidase.

PubMed

Matzi, Veronika; Greilberger, Joachim F; Lindenmann, Joerg; Neuboeck, Nicole; Nuhsbaumer, Stephan; Zelzer, Sieglinde; Tafeit, Erwin; Maier, Alfred; Smolle-Juettner, Maria-Frey

2015-01-01

It is postulated that application of hyperbaric oxygenation may induce the production of radicals after HBO. Higher oxygenation and transport of oxygen increase the mitochondrial energy turnover, whereas inner mitochondrial radical formation decreases. Several markers of oxidative stress in healthy volunteers (n = 21), including plasma carbonyl proteins (CP), malondialdehyde (MDA), oxidized LDL (oxLDL), 8-hydroxy-deoxyguanosine (8-OHdG), and erythrocyte glutathione peroxidase (GPx) activity are measured before, during, and after HBO. Median plasma concentrations of CP decreased significantly during HBO compared to CP levels before HBO (from 77.1 to 61.7 pmol/mg; p < 0.001) and increased again after HBO (to 78.1 pmol/mg; p = 0.035). 8-OHdG decreased significantly during HBO (8.1 ng/mL; p < 0.001) and remained constant after HBO (8.1 ng/mL) compared to "before HBO" (9.4 ng/mL). MDA increased significantly from 0.92 μM (before HBO) to 1.26 μM (during HBO, p < 0.01) and decreased again to 1.00 μM (after HBO, p = 0.023). Erythrocyte GPx activity also increased significantly during HBO (26.5 ± 14.7; p = 0.005), but not after HBO (25.6 ± 17.2 IU/mg). A negative correlation was observed between GPx and MDA only during HBO (r = -0.518; p = 0.016). We assume that higher oxygen consumption decreases, on the one hand, the inner mitochondrial generation of free radicals and, on the other, RONS by activation of detoxifying enzymes like GPx.

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

The 28-day exposure to fenpropathrin decreases locomotor activity and reduces activity of antioxidant enzymes in mice brains.

PubMed

Nieradko-Iwanicka, Barbara; Borzęcki, Andrzej

2016-04-01

Fenpropathrin (Fen) is a pyrethroid (Pyr) insecticide. Pyrs are used in veterinary medicine, in agriculture and for domestic purposes. As their use increases, new questions about their side effects and mode of action in non-target organisms arise. The objective of this work was to characterize dose-response relationship for in vivo motor function and memory in mice exposed to Fen for 28 days and to assess its influence on activity of antioxidant enzymes in mice brains. The experiment was performed using 64 female mice. Fen at the dose of 11.9mg/kg of body mass, 5.95mg/kg or 2.38mg/kg was administered ip to the mice for 28 consecutive days. Motor function and spatial working memory were tested on days 7, 14 and 28. On day 29, the animals were sacrificed and brains were used to determine activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Fen significantly decreased locomotor activity in mice receiving the highest dose at every stage of the experiment. Lower doses reduced locomotion on days 7 and 14. Fen did not produce memory impairment. A decrease in activities of SOD and GPx was recorded in mice brains. The decrease of SOD activity in mice brains results from direct inhibition of the enzyme by Fen and/or increased utilization due to excessive free radical formation in conditions of Fen-induced oxidative stress. The reduction in GPx activity is probably due to limited glutathione availability. The reduced locomotor activity is a behavioral demonstration of Fen-induced damage in the dopaminergic system. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Inactivation of thioredoxin reductases reveals a complex interplay between thioredoxin and glutathione pathways in Arabidopsis development.

PubMed

Reichheld, Jean-Philippe; Khafif, Mehdi; Riondet, Christophe; Droux, Michel; Bonnard, Géraldine; Meyer, Yves

2007-06-01

NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem.

Influence of galactose cataract on erythrocytic and lenticular glutathione metabolism in albino rats.

PubMed

Jyothi, M; Sanil, R; Shashidhar, S

2011-01-01

Glutathione depletion has been postulated to be the prime reason for galactose cataract. The current research seeks the prospect of targeting erythrocytes to pursue the lens metabolism by studying the glutathione system. To study the activity of the glutathione-linked scavenger enzyme system in the erythrocyte and lens of rats with cataract. Experiments were conducted in 36 male albino rats weighing 80 ± 20 g of 28 days of age. The rats were divided into two major groups, viz. experimental and control. Six rats in each group were sacrificed every 10 days, for 30 days. Cataract was induced in the experimental group by feeding the rats 30% galactose (w/w). The involvement of reduced glutathione (GSH) and the linked enzymes was studied in the erythrocytes and lens of cataractous as well as control rats. Parametric tests like one-way ANOVA and Student's 't' test were used for comparison. Correlation linear plot was used to compare the erythrocyte and lens metabolism. The concentration of GSH and the activity of linked enzymes were found decreased with the progression of cataract, and also in comparison to the control. The same linear fashion was also observed in the erythrocytes. Depletion of GSH was the prime factor for initiating galactose cataract in the rat model. This depletion may in turn result in enzyme inactivation leading to cross-linking of protein and glycation. The correlation analysis specifies that the biochemical mechanism in the erythrocytes and lens is similar in the rat model.

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.

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.

Assessment of human 4-hydroxynonenal, 8-isoprostane concentrations and glutathione reductase activity after synbiotics administration.

PubMed

Kleniewska, Paulina; Pawliczak, Rafał

2018-05-30

Probiotics and prebiotics have become an object of intense research, to identify methods of mitigating oxidative stress. Over the past few years, the number of in vitro and in vivo studies, related to antioxidant properties of probiotics/prebiotics has significantly increased. The aim of the present study was to assess whether probiotic in combination with prebiotic influences the level of human 4-hydroxynonenal, 8-isoprostane and glutathione reductase activity. Experiments were carried out on healthy volunteers (male and female). All oxidative stress markers were measured in blood plasma pre- and post-administration of synbiotic. The administration of synbiotic resulted in a significant decrease in 4-hydroxynonenal in the female-synbiotic group (p < 0.05), 8-isoprostanes in the female-synbiotic group and male-synbiotic group (p < 0.05) and non-significant increase in the activity of glutathione reductase (p > 0.05) vs. control. The present results show that supplementation of synbiotics contributed to the decrease in oxidative stress parameters in the female patients. Copyright © 2018 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.

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

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

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

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

Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

PubMed

Drozd, Ewa; Krzysztoń-Russjan, Jolanta; Marczewska, Jadwiga; Drozd, Janina; Bubko, Irena; Bielak, Magda; Lubelska, Katarzyna; Wiktorska, Katarzyna; Chilmonczyk, Zdzisław; Anuszewska, Elżbieta; Gruber-Bzura, Beata

2016-10-01

Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely

Glutathione reductase gsr-1 is an essential gene required for Caenorhabditis elegans early embryonic development.

PubMed

Mora-Lorca, José Antonio; Sáenz-Narciso, Beatriz; Gaffney, Christopher J; Naranjo-Galindo, Francisco José; Pedrajas, José Rafael; Guerrero-Gómez, David; Dobrzynska, Agnieszka; Askjaer, Peter; Szewczyk, Nathaniel J; Cabello, Juan; Miranda-Vizuete, Antonio

2016-07-01

Glutathione is the most abundant thiol in the vast majority of organisms and is maintained in its reduced form by the flavoenzyme glutathione reductase. In this work, we describe the genetic and functional analysis of the Caenorhabditis elegans gsr-1 gene that encodes the only glutathione reductase protein in this model organism. By using green fluorescent protein reporters we demonstrate that gsr-1 produces two GSR-1 isoforms, one located in the cytoplasm and one in the mitochondria. gsr-1 loss of function mutants display a fully penetrant embryonic lethal phenotype characterized by a progressive and robust cell division delay accompanied by an aberrant distribution of interphasic chromatin in the periphery of the cell nucleus. Maternally expressed GSR-1 is sufficient to support embryonic development but these animals are short-lived, sensitized to chemical stress, have increased mitochondrial fragmentation and lower mitochondrial DNA content. Furthermore, the embryonic lethality of gsr-1 worms is prevented by restoring GSR-1 activity in the cytoplasm but not in mitochondria. Given the fact that the thioredoxin redox systems are dispensable in C. elegans, our data support a prominent role of the glutathione reductase/glutathione pathway in maintaining redox homeostasis in the nematode. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Antioxidative defence mechanisms against reactive oxygen species in bovine retained and not-retained placenta: activity of glutathione peroxidase, glutathione transferase, catalase and superoxide dismutase.

PubMed

Kankofer, M

2001-05-01

Glutathione peroxidase (GSH-Px), glutathione transferase (GSH-Tr), catalase (CAT) and superoxide dismutase (SOD)-the members of enzymatic antioxidative defence mechanisms against reactive oxygen species-may play an important role in proper or improper release of bovine fetal membranes. The aim of the following study was the determination of GSH-Px, GSH-Tr, CAT and SOD activity in order to define antioxidative status of bovine placenta during retention of fetal membranes (RFM) in cows. Placental samples were collected immediately after spontaneous parturition or during caesarean section before term and at term and divided into six groups as follows: A: caesarean section before term without RFM; B: caesarean section before term with RFM; C: caesarean section at term without RFM; D: caesarean section at term with RFM; E: spontaneous delivery at term without RFM; F: spontaneous delivery at term with RFM. The enzyme activities in placental homogenates were measured spectrophotometrically. GSH-Px activity was statistically significantly higher in fetal than in maternal placenta in all examined groups, increased towards parturition and was higher in caesarean section groups than spontaneous delivery groups. Statistically significantly higher activities were noticed in retained than not-retained placentae. GSH-Tr activity was significantly lower in fetal than in maternal placenta. In preterm groups, the activity was statistically significantly higher in retained than not retained placenta. In term groups, the opposite relationship was observed, higher values in caesarean section groups than spontaneous delivery were noticed. CAT activity was statistically significantly higher in fetal than in maternal part of placenta in all groups examined. The highest values in C and D groups and the differences between retained and not-retained placenta were observed. SOD exhibited the highest values in preterm placenta and alterations between retained and not-retained fetal membranes

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.

Inactivation of Thioredoxin Reductases Reveals a Complex Interplay between Thioredoxin and Glutathione Pathways in Arabidopsis Development[W

PubMed Central

Reichheld, Jean-Philippe; Khafif, Mehdi; Riondet, Christophe; Droux, Michel; Bonnard, Géraldine; Meyer, Yves

2007-01-01

NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem. PMID:17586656

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

Marked differences in drug-induced methemoglobinemia in sheep are not due to RBC glucose-6-phosphate dehydrogenase, reduced glutathione, or methemoglobin reductase activity

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

Martin, D.G.; Guertler, A.T.; Lagutchik, M.S.

1993-05-13

Benzocaine is a commonly used topical anesthetic that is structurally similar to current candidates for cyanide prophylaxis. Benzocaine induces profound methemoglobinemia in some sheep but not others. After topical benzocaine administration certain sheep respond to form MHb (elevated MHb 16-50% after a 56-280 mg dose, a 2-10 second spray with benzocine), while other phenotypically similar sheep fail to significantly form MHb (less than a 2% increase from baseline). Deficiencies in Glucose-6-phosphate dehydrogenase (G-6-PD), reduced glutathione (GSH), and MHb reductase increase the susceptibility to methemoglobinemia in man and animals. Sheep are used as a model for G-6-PD deficiency in man, andmore » differences in this enzyme level could cause the variable response seen in these sheep. Similarly, differences in GSH and MHb reductase could be responsible for the observed differences in MHb formation.« less

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

The insect repellent DEET (N,N-diethyl-3-methylbenzamide) increases the synthesis of glutathione S-transferase in cultured mosquito cells.

PubMed

Hellestad, Vanessa J; Witthuhn, Bruce A; Fallon, Ann M

2011-04-01

DEET (N,N-diethyl-3-methylbenzamide) is the active ingredient used in many commonly used insect repellents, but its mode of action remains poorly understood. Efforts to identify properties that could lead to the development of more effective active ingredients have distinguished among DEET's repellent, deterrent, and insecticidal activities. We used an Aedes albopictus mosquito cell line to evaluate DEET's toxicological properties in the absence of sensory input mediated by the olfactory system. When cells were treated with DEET and labeled with [(35)S]methionine/cysteine, a single 25-kDa protein was induced, relative to other proteins, on SDS-polyacrylamide gels. The 25-kDa band from DEET-treated cells was enriched in peptides corresponding to glutathione S-transferase D10 and/or theta in the Aedes aegypti genome. Consistent with the increased expression of the labeled protein, DEET-treated cells had increased glutathione S-transferase activity, and the radiolabeled band bound to Sepharose 4B containing reduced glutathione. By analyzing partial tryptic digests, we established that DEET induces the homolog of A. aegypti glutathione S-transferase, class theta, corresponding to protein XP_001658009.1 in the NCBI database. This specific effect of DEET at the subcellular level suggests that DEET induces physiological responses that extend beyond recognition by the peripheral olfactory system.

Simultaneous determination of the impurity and radial tensile strength of reduced glutathione tablets by a high selective NIR-PLS method.

PubMed

Li, Juan; Jiang, Yue; Fan, Qi; Chen, Yang; Wu, Ruanqi

2014-05-05

This paper establishes a high-throughput and high selective method to determine the impurity named oxidized glutathione (GSSG) and radial tensile strength (RTS) of reduced glutathione (GSH) tablets based on near infrared (NIR) spectroscopy and partial least squares (PLS). In order to build and evaluate the calibration models, the NIR diffuse reflectance spectra (DRS) and transmittance spectra (TS) for 330 GSH tablets were accurately measured by using the optimized parameter values. For analyzing GSSG or RTS of GSH tablets, the NIR-DRS or NIR-TS were selected, subdivided reasonably into calibration and prediction sets, and processed appropriately with chemometric techniques. After selecting spectral sub-ranges and neglecting spectrum outliers, the PLS calibration models were built and the factor numbers were optimized. Then, the PLS models were evaluated by the root mean square errors of calibration (RMSEC), cross-validation (RMSECV) and prediction (RMSEP), and by the correlation coefficients of calibration (R(c)) and prediction (R(p)). The results indicate that the proposed models have good performances. It is thus clear that the NIR-PLS can simultaneously, selectively, nondestructively and rapidly analyze the GSSG and RTS of GSH tablets, although the contents of GSSG impurity were quite low while those of GSH active pharmaceutical ingredient (API) quite high. This strategy can be an important complement to the common NIR methods used in the on-line analysis of API in pharmaceutical preparations. And this work expands the NIR applications in the high-throughput and extraordinarily selective analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

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.

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

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.

[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.

Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders.

PubMed

Gadea, Joaquín; Sellés, Elena; Marco, Marco Antonio; Coy, Pilar; Matás, Carmen; Romar, Raquel; Ruiz, Salvador

2004-08-01

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen-thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84 +/- 0.21 nM GSH/10(8) sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P < 0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P = 0.0102 ). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5 mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the

Regulative roles of glutathione reductase and four glutaredoxins in glutathione redox, antioxidant activity, and iron homeostasis of Beauveria bassiana.

PubMed

Zhang, Long-Bin; Tang, Li; Ying, Sheng-Hua; Feng, Ming-Guang

2016-07-01

Multiple glutaredoxins (Grx) and glutathione reductase (Glr) are vital for the thiol-disulfide redox system in budding yeast but generally unexplored in filamentous fungi. Here we characterized the Beauveria bassiana redox system comprising dithiol Grx1, monothiol Grx2-4, Grx-like Grx5, and Glr orthologue. Each grx or glr deletion was compensated by increased transcripts of some other grx genes in normal cultures. Particularly, grx3 compensated the absence of grx1, grx2, grx5, or glr under oxidative stress while its absence was compensated only by undeletable grx4 under normal conditions but by most of other undeleted grx and glr genes in response to menadione. Consequently, the redox state was disturbed in Δglr more than in Δgrx3 but not in Δgrx1/2/5. Superoxide dismutases were more active in normal Δgrx1-3 cultures but less in Δgrx5 or Δglr response to menadione. Total catalase activity increased differentially in all the mutant cultures stressed with or without H2O2 while total peroxidase activity decreased more in the normal or H2O2-stressed culture of Δglr than of Δgrx3. Among the mutants, Δgrx3 showed slightly increased sensitivity to menadione or H2O2; Δglr exhibited greater sensitivity to thiol-oxidizing diamide than thiol-reducing 1-chloro-2,4-dinitrobenzene as well as increased sensitivity to the two oxidants. Intriguingly, all the mutants grew slower in a Fe(3+)-inclusive medium perhaps due to elevated transcripts of two Fe(3+) transporter genes. More or fewer phenotypes linked with biocontrol potential were altered in four deletion mutants excluding Δgrx5. All the changes were restored by targeted gene complementation. Overall, Grx3 played more critical role than other Grx homologues in the Glr-dependent redox system of the fungal entomopathogen.

Intercellular Distribution of Glutathione Synthesis in Maize Leaves and Its Response to Short-Term Chilling1

PubMed Central

Gómez, Leonardo D.; Vanacker, Hélène; Buchner, Peter; Noctor, Graham; Foyer, Christine H.

2004-01-01

To investigate the intercellular control of glutathione synthesis and its influence on leaf redox state in response to short-term chilling, genes encoding γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GSH-S) were cloned from maize (Zea mays) and specific antibodies produced. These tools were used to provide the first information on the intercellular distribution of γ-ECS and GSH-S transcript and protein in maize leaves, in both optimal conditions and chilling stress. A 2-d exposure to low growth temperatures (chill) had no effect on leaf phenotype, whereas return to optimal temperatures (recovery) caused extensive leaf bleaching. The chill did not affect total leaf GSH-S transcripts but strongly induced γ-ECS mRNA, an effect reversed during recovery. The chilling-induced increase in γ-ECS transcripts was not accompanied by enhanced total leaf γ-ECS protein or extractable activity. In situ hybridization and immunolocalization of leaf sections showed that γ-ECS and GSH-S transcripts and proteins were found in both the bundle sheath (BS) and the mesophyll cells under optimal conditions. Chilling increased γ-ECS transcript and protein in the BS but not in the mesophyll cells. Increased BS γ-ECS was correlated with a 2-fold increase in both leaf Cys and γ-glutamylcysteine, but leaf total glutathione significantly increased only in the recovery period, when the reduced glutathione to glutathione disulfide ratio decreased 3-fold. Thus, while there was a specific increase in the potential contribution of the BS cells to glutathione synthesis during chilling, it did not result in enhanced leaf glutathione accumulation at low temperatures. Return to optimal temperatures allowed glutathione to increase, particularly glutathione disulfide, and this was associated with leaf chlorosis. PMID:15047902

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.

Enzymatic improvement of mitochondrial thiol oxidase Erv1 for oxidized glutathione fermentation by Saccharomyces cerevisiae.

PubMed

Kobayashi, Jyumpei; Sasaki, Daisuke; Hara, Kiyotaka Y; Hasunuma, Tomohisa; Kondo, Akihiko

2017-03-15

Oxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our previous study, over-expression of the mitochondrial thiol oxidase ERV1 gene was the most effective for high GSSG production in Saccharomyces cerevisiae cells among three types of different thiol oxidase genes. We improved Erv1 enzyme activity for oxidation of GSH and revealed that S32 and N34 residues are critical for the oxidation. Five engineered Erv1 variant proteins containing S32 and/or N34 replacements exhibited 1.7- to 2.4-fold higher in vitro GSH oxidation activity than that of parental Erv1, whereas the oxidation activities of these variants for γ-glutamylcysteine were comparable. According to three-dimensional structures of Erv1 and protein stability assays, S32 and N34 residues interact with nearby residues through hydrogen bonding and greatly contribute to protein stability. These results suggest that increased flexibility by amino acid replacements around the active center decrease inhibitory effects on GSH oxidation. Over-expressions of mutant genes coding these Erv1 variants also increased GSSG and consequently total glutathione production in S. cerevisiae cells. Over-expression of the ERV1 S32A gene was the most effective for GSSG production in S. cerevisiae cells among the parent and other mutant genes, and it increased GSSG production about 1.5-fold compared to that of the parental ERV1 gene. This is the first study demonstrating the pivotal effects of S32 and N34 residues to high GSH oxidation activity of Erv1. Furthermore, in vivo validity of Erv1 variants containing these S32 and N34 replacements were also demonstrated. This study indicates potentials of Erv1 for high GSSG production.

Glutathione S-Transferases Interact with AMP-Activated Protein Kinase: Evidence for S-Glutathionylation and Activation In Vitro

PubMed Central

Polge, Cécile; Ramirez, Sacnicte; Michelland, Sylvie; Sève, Michel; Vertommen, Didier; Rider, Mark; Lentze, Nicolas; Auerbach, Daniel; Schlattner, Uwe

2013-01-01

AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions. PMID:23741294

N-ACETYLCYSTEINE REDUCES DISEASE ACTIVITY BY BLOCKING MTOR IN T CELLS OF LUPUS PATIENTS

PubMed Central

Lai, Zhi-Wei; Hanczko, Robert; Bonilla, Eduardo; Caza, Tiffany N.; Clair, Brandon; Bartos, Adam; Miklossy, Gabriella; Jimah, John; Doherty, Edward; Tily, Hajra; Francis, Lisa; Garcia, Ricardo; Dawood, Maha; Yu, Jianghong; Ramos, Irene; Coman, Ioana; Faraone, Stephen V.; Phillips, Paul E.; Perl, Andras

2012-01-01

Background Systemic lupus erythematosus (SLE) patients exhibit T-cell dysfunction which can be regulated through the mitochondrial transmembrane potential (Δψm) and mammalian target of rapamycin (mTOR) by glutathione. Therefore, the safety, tolerance, and efficacy of glutathione-precursor N-acetylcysteine (NAC) were examined in this randomized double-blind placebo-controlled study. Methods 36 SLE patients received daily placebo or 1.2 g, 2.4 g or 4.8 g of NAC. Disease activity was monthly evaluated by BILAG, SLEDAI and fatigue assessment scale (FAS) before, during, and after 3-month treatment. Δψm and mTOR were assessed by flow cytometry. 42 healthy subjects matched for patients’ age, gender, and ethnicity were studied as controls. Results NAC was tolerated by all patients up to 2.4 g/day while 33% of those receiving 4.8 g/day had reversible nausea. Placebo or 1.2 g/day NAC did not influence disease activity. Considered together, 2.4 g and 4.8 g NAC reduced: 1) SLEDAI after 1 month (p=0.0007), 2 months (p=0.0009), 3 months (p=0.0030) and 4 months (p=0.0046); 2) BILAG after 1 month (p=0.029) and 3 months (p=0.0009); and 3) FAS after 2 months (p=0.002) and 3 months (p=0.004). NAC increased Δψm (p=0.0001) in all T cells, it profoundly reduced mTOR activity (p=0.0001), enhanced apoptosis (p=0.0004) and reversed expansion of CD4−/CD8− T cells (1.35 ± 0.12-fold; p=0.008), stimulated Foxp3 expression in CD4+/CD25+ T cells (p=0.045), and reduced anti-DNA production (p=0.049). Conclusions This pilot study suggests that NAC safely improves lupus disease activity by blocking mTOR in T lymphocytes. PMID:22549432

Yeast mitochondrial glutathione is an essential antioxidant with mitochondrial thioredoxin providing a back-up system

PubMed Central

Gostimskaya, Irina; Grant, Chris M.

2016-01-01

Glutathione is an abundant, low-molecular-weight tripeptide whose biological importance is dependent upon its redox-active free sulphydryl moiety. Its role as the main determinant of thiol-redox control has been challenged such that it has been proposed to play a crucial role in iron–sulphur clusters maturation, and only a minor role in thiol redox regulation, predominantly as a back-up system for the cytoplasmic thioredoxin system. Here, we have tested the importance of mitochondrial glutathione in thiol-redox regulation. Glutathione reductase (Glr1) is an oxidoreductase which converts oxidized glutathione to its reduced form. Yeast Glr1 localizes to both the cytosol and mitochondria and we have used a Glr1M1L mutant that is constitutively localized to the cytosol to test the requirement for mitochondrial Glr1. We show that the loss of mitochondrial Glr1 specifically accounts for oxidant sensitivity of a glr1 mutant. Loss of mitochondrial Glr1 does not influence iron–sulphur cluster maturation and we have used targeted roGFP2 fluorescent probes to show that oxidant sensitivity is linked to an altered redox environment. Our data indicate mitochondrial glutathione is crucial for mitochondrial thiol-redox regulation, and the mitochondrial thioredoxin system provides a back-up system, but cannot bear the redox load of the mitochondria on its own. PMID:26898146

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

Glutathione and thioredoxin systems contribute to recombinant monoclonal antibody interchain disulfide bond reduction during bioprocessing.

PubMed

Handlogten, Michael W; Zhu, Min; Ahuja, Sanjeev

2017-07-01

Antibody interchain disulfide bond reduction during biopharmaceutical manufacturing has received increased attention since it was first reported in 2010. Antibody reduction leads to loss of product and reduced product stability. It is therefore critical to understand the underlying mechanisms of reduction. To date, the thioredoxin system has been reported as the sole contributor to antibody reduction during bioprocessing. In this work, we show that the glutathione system, in addition to the thioredoxin system, is involved in reducing antibody molecules and the contributions of the two systems can vary depending upon the cell culture process. The roles of the glutathione and thioredoxin systems were evaluated for three molecules with different IgG subclass where reduction was observed during manufacturing: mAb A, mAb B, and mAb C representing an IgG 1 , IgG 2 , and IgG 4, respectively. The expression of enzymes for both the thioredoxin and glutathione systems were confirmed in all three cell lines. Inhibitors were evaluated using purified mammalian reductases to evaluate their specificity. The optimized experimental conditions enabled both the determination of reductase activity contributed from as well as the amount of antibody reduced by each enzymatic system. Our results demonstrate that the underlying enzymatic mechanisms are different depending upon the cell culture process; one of the two systems may be the dominant mechanism, or both enzymatic systems may be involved. Specifically, the glutathione system was found to be the major contributor to mAb A reduction while the thioredoxin system was the major contributor to mAb C reduction. Intriguingly, mAb B experienced significant reduction from both enzymatic systems. In summary, we have demonstrated that in addition to the thioredoxin pathway, the glutathione system is a second major pathway contributing to antibody reduction and this knowledge can be leveraged to develop more specific antibody reduction

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.

Association of mercury and selenium with altered glutathione metabolism and oxidative stress in diving ducks from the San Francisco Bay region

USGS Publications Warehouse

Hoffman, D.J.; Ohlendorf, H.M.; Marn, C.M.; Pendleton, G.W.

1998-01-01

Adult male greater scaup (Aythya marila) (GS), surf scoters (Melanitta perspicillata)(SS), and ruddy ducks (Oxyura jamaicensis) (RD) were collected from Suisun Bay and coastal Tomales Bay in the greater San Francisco Bay area to assess exposure to inorganic contaminants. Hepatic selenium (Se) concentrations were highest in GS (geometric mean = 67 ppm, dw) and SS (119 ppm) in Suisun Bay, whereas hepatic mercury (Hg) was highest (19 ppm) in GS and SS from Tomales Bay. Hepatic Se and Hg were lower in RD and did not differ between locations. Hepatic supernatants were assayed for enzymes related to glutathione metabolism and antioxidant activity including: glucose-6-phosphate dehydrogenase (G-6-PDH), glutathione peroxidase (GSH-peroxidase), glutathione reductase (GSSG-reductase), and glutathione-S-transferase (GSH-transferase). GSH-peroxidase activity was higher in SS and RD, and G-6-PDH higher in GS and SS from Suisun Bay than Tomales Bay. GSSG-reductase was higher in SS from Suisun Bay. The ratio of oxidized glutathione (GSSG) to reduced glutathione (GSH) was greater in all species from Tomales Bay. The following significant relationships were found in one or more species with increasing hepatic Hg concentration: lower body, liver and heart weights; decreased hepatic GSH concentration, G-6-PDH and GSH-peroxidase activities; increased ratio of GSSG to GSH, and increased GSSG-reductase activity. With increasing hepatic Se concentration, GSH-peroxidase increased but GSH decreased. It is concluded that measurement of associated enzymes in conjunction with thiol status may be a useful bioindicator to discriminate between Hg and Se effects. Concentrations of mercury and selenium and variable affected have been associated with adverse effects on reproduction and neurological function in experimental studies with mallards.

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

The structure of a zeta class glutathione S-transferase from Arabidopsis thaliana: characterisation of a GST with novel active-site architecture and a putative role in tyrosine catabolism.

PubMed

Thom, R; Dixon, D P; Edwards, R; Cole, D J; Lapthorn, A J

2001-05-18

The cis-trans isomerisation of maleylacetoacetate to fumarylacetoacetate is the penultimate step in the tyrosine/phenylalanine catabolic pathway and has recently been shown to be catalysed by glutathione S-transferase enzymes belonging to the zeta class. Given this primary metabolic role it is unsurprising that zeta class glutathione S-transferases are well conserved over a considerable period of evolution, being found in vertebrates, plants, insects and fungi. The structure of this glutathione S-transferase, cloned from Arabidopsis thaliana, has been solved by single isomorphous replacement with anomalous scattering and refined to a final crystallographic R-factor of 19.6% using data from 25.0 A to 1.65 A. The zeta class enzyme adopts the canonical glutathione S-transferase fold and forms a homodimer with each subunit consisting of 221 residues. In agreement with structures of glutathione S-transferases from the theta and phi classes, a serine residue (Ser17) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione. Site-directed mutagenesis of this residue confirms its importance in catalysis. In addition, the role of a highly conserved cysteine residue (Cys19) present in the active site of the zeta class glutathione S-transferase enzymes is discussed. Copyright 2001 Academic Press.

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).

Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines

PubMed Central

Sappington, Daniel R.; Siegel, Eric R.; Hiatt, Gloria; Desai, Abhishek; Penney, Rosalind B.; Jamshidi-Parsian, Azemat; Griffin, Robert J.; Boysen, Gunnar

2016-01-01

Background Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Glutamine provides additional carbon and nitrogen sources for cell growth. The first step in glutamine utilization is its conversion to glutamate by glutaminase (GLS). Glutamate is a precursor for glutathione synthesis, and we investigated the hypothesis that glutamine drives glutathione synthesis and thereby contributes to cellular defense systems. Methods The importance of glutamine for glutathione synthesis was studied in H460 and A549 lung cancer cell lines using glutamine-free medium and Bis-2-(5-phenyl-acetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) a GLS inhibitor. Metabolic activities were determined by targeted mass spectrometry. Results A significant correlation between glutamine consumption and glutathione excretion was demonstrated in H460 and A549 tumor cells. Culturing in the presence of [13C5]glutamine demonstrated that by 12 hrs >50% of excreted glutathione is derived from glutamine. Culturing in glutamine-free medium or treatment with BPTES, a glutaminase (GLS)-specific inhibitor, reduced cell proliferation and viability, and abolished glutathione excretion. Treatment with glutathione-ester prevented BPTES induced cytotoxicity. Inhibition of GLS markedly radiosensitized the lung tumor cell lines, suggesting an important role of glutamine-derived glutathione in determining radiation sensitivity. Conclusions We demonstrate here for the first time that a significant amount of extracellular glutathione is directly derived from glutamine. This finding adds yet another important function to the already known glutamine dependence of tumor cells and probably tumors as well. General significance Glutamine is essential for synthesis and excretion of glutathione to promote cell growth and viability. PMID:26825773

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

Selenium reduces the proapoptotic signaling associated to NF-kappaB pathway and stimulates glutathione peroxidase activity during excitotoxic damage produced by quinolinate in rat corpus striatum.

PubMed

Santamaría, Abel; Vázquez-Román, Beatriz; La Cruz, Verónica Pérez-De; González-Cortés, Carolina; Trejo-Solís, Ma Cristina; Galván-Arzate, Sonia; Jara-Prado, Aurelio; Guevara-Fonseca, Jorge; Ali, Syed F

2005-12-15

Quinolinate (QUIN) neurotoxicity has been attributed to degenerative events in nerve tissue produced by sustained activation of N-methyl-D-aspartate receptor (NMDAr) and oxidative stress. We have recently described the protective effects that selenium (Se), an antioxidant, produces on different markers of QUIN-induced neurotoxicity (Santamaría et al., 2003, J Neurochem 86:479-488.). However, the mechanisms by which Se exerts its protective actions remain unclear. Since some of these events are thought to be related with inhibition of deadly molecular cascades through the activation of antioxidant selenoproteins, in this study we investigated the effects of Se on QUIN-induced cell damage elicited by the nuclear factor kappaB (NF-kappaB) pathway, as well as the time-course response of striatal glutathione peroxidase (GPx) activity. Se (sodium selenite, 0.625 mg/kg/day, i.p.) was administered to rats for 5 days, and 120 min after the last administration, animals received a single striatal injection of QUIN (240 nmol/mul). Twenty-four hours later, their striata were tested for the expression of IkappaB-alpha (the NF-kappaB cytosolic binding protein), the immunohistochemical expression of NF-kappaB (evidenced as nuclear expression of P65), caspase-3-like activation, and DNA fragmentation. Additional groups were killed at 2, 6, and 24 h for measurement of GPx activity. Se reduced the QUIN-induced decrease in IkappaB-alpha expression, evidencing a reduction in its cytosolic degradation. Se also prevented the QUIN-induced increase in P65-immunoreactive cells, suggesting a reduction of NF-kappaB nuclear translocation. Caspase-3-like activation and DNA fragmentation produced by QUIN were also inhibited by Se. Striatal GPx activity was stimulated by Se at 2 and 6 h, but not at 24 h postlesion. Altogether, these data suggest that the protective effects exerted by Se on QUIN-induced neurotoxicity are partially mediated by the inhibition of proapoptotic events underlying Ikappa

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.

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.

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

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.

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

Dietary supplementation with the microalga Galdieria sulphuraria (Rhodophyta) reduces prolonged exercise-induced oxidative stress in rat tissues.

PubMed

Carfagna, Simona; Napolitano, Gaetana; Barone, Daniela; Pinto, Gabriele; Pollio, Antonino; Venditti, Paola

2015-01-01

We studied the effects of ten-day 1% Galdieria sulphuraria dietary supplementation on oxidative damage and metabolic changes elicited by acute exercise (6-hour swimming) determining oxygen consumption, lipid hydroperoxides, protein bound carbonyls in rat tissue (liver, heart, and muscle) homogenates and mitochondria, tissue glutathione peroxidase and glutathione reductase activities, glutathione content, and rates of H2O2 mitochondrial release. Exercise increased oxidative damage in tissues and mitochondria and decreased tissue content of reduced glutathione. Moreover, it increased State 4 and decreased State 3 respiration in tissues and mitochondria. G. sulphuraria supplementation reduced the above exercise-induced variations. Conversely, alga supplementation was not able to modify the exercise-induced increase in mitochondrial release rate of hydrogen peroxide and in liver and heart antioxidant enzyme activities. The alga capacity to reduce lipid oxidative damage without reducing mitochondrial H2O2 release can be due to its high content of C-phycocyanin and glutathione, which are able to scavenge peroxyl radicals and contribute to phospholipid hydroperoxide metabolism, respectively. In conclusion, G. sulphuraria ability to reduce exercise-linked oxidative damage and mitochondrial dysfunction makes it potentially useful even in other conditions leading to oxidative stress, including hyperthyroidism, chronic inflammation, and ischemia/reperfusion.

Dietary Supplementation with the Microalga Galdieria sulphuraria (Rhodophyta) Reduces Prolonged Exercise-Induced Oxidative Stress in Rat Tissues

PubMed Central

Carfagna, Simona; Napolitano, Gaetana; Barone, Daniela; Pinto, Gabriele; Venditti, Paola

2015-01-01

We studied the effects of ten-day 1% Galdieria sulphuraria dietary supplementation on oxidative damage and metabolic changes elicited by acute exercise (6-hour swimming) determining oxygen consumption, lipid hydroperoxides, protein bound carbonyls in rat tissue (liver, heart, and muscle) homogenates and mitochondria, tissue glutathione peroxidase and glutathione reductase activities, glutathione content, and rates of H2O2 mitochondrial release. Exercise increased oxidative damage in tissues and mitochondria and decreased tissue content of reduced glutathione. Moreover, it increased State 4 and decreased State 3 respiration in tissues and mitochondria. G. sulphuraria supplementation reduced the above exercise-induced variations. Conversely, alga supplementation was not able to modify the exercise-induced increase in mitochondrial release rate of hydrogen peroxide and in liver and heart antioxidant enzyme activities. The alga capacity to reduce lipid oxidative damage without reducing mitochondrial H2O2 release can be due to its high content of C-phycocyanin and glutathione, which are able to scavenge peroxyl radicals and contribute to phospholipid hydroperoxide metabolism, respectively. In conclusion, G. sulphuraria ability to reduce exercise-linked oxidative damage and mitochondrial dysfunction makes it potentially useful even in other conditions leading to oxidative stress, including hyperthyroidism, chronic inflammation, and ischemia/reperfusion. PMID:25874021

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.

The Ascorbate-glutathione-α-tocopherol Triad in Abiotic Stress Response

PubMed Central

Szarka, András; Tomasskovics, Bálint; Bánhegyi, Gábor

2012-01-01

The life of any living organism can be defined as a hurdle due to different kind of stresses. As with all living organisms, plants are exposed to various abiotic stresses, such as drought, salinity, extreme temperatures and chemical toxicity. These primary stresses are often interconnected, and lead to the overproduction of reactive oxygen species (ROS) in plants, which are highly reactive and toxic and cause damage to proteins, lipids, carbohydrates and DNA, which ultimately results in oxidative stress. Stress-induced ROS accumulation is counteracted by enzymatic antioxidant systems and non-enzymatic low molecular weight metabolites, such as ascorbate, glutathione and α-tocopherol. The above mentioned low molecular weight antioxidants are also capable of chelating metal ions, reducing thus their catalytic activity to form ROS and also scavenge them. Hence, in plant cells, this triad of low molecular weight antioxidants (ascorbate, glutathione and α-tocopherol) form an important part of abiotic stress response. In this work we are presenting a review of abiotic stress responses connected to these antioxidants. PMID:22605990

Functional characterisation of ganglioside-induced differentiation-associated protein 1 as a glutathione transferase.

PubMed

Shield, Alison J; Murray, Tracy P; Board, Philip G

2006-09-08

Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene have been linked with Charcot-Marie-Tooth (CMT) disease. This protein, and its paralogue GDAP1L1, appear to be structurally related to the cytosolic glutathione S-transferases (GST) including an N-terminal thioredoxin fold domain with conserved active site residues. The specific function, of GDAP1 remains unknown. To further characterise their structure and function we purified recombinant human GDAP1 and GDAP1L1 proteins using bacterial expression and immobilised metal affinity chromatography. Like other cytosolic GSTs, GDAP1 protein has a dimeric structure. Although the full-length proteins were largely insoluble, the deletion of a proposed C-terminal transmembrane domain allowed the preparation of soluble protein. The purified proteins were assayed for glutathione-dependent activity against a library of 'prototypic' GST substrates. No evidence of glutathione-dependent activity or an ability to bind glutathione immobilised on agarose was found.

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.

Glutathione exposes sequential IgE-epitopes in ovomucoid relevant in persistent egg allergy.

PubMed

Roth-Walter, Franziska; Starkl, Philipp; Zuberbier, Torsten; Hummel, Karin; Nöbauer, Karin; Razzazi-Fazeli, Ebrahim; Brunner, Richard; Pali-Schöll, Isabella; Kinkel, Janis; Felix, Ferdinand; Jensen-Jarolim, Erika; Kinaciyan, Tamar

2013-03-01

Patients with persistent egg allergy have more immunoglobulin E (IgE) against sequential than conformational epitopes of ovomucoid (OVO). Here, we aimed to identify compounds capable to render sequential epitopes in egg. Glutathione was used for in vitro reduction of OVO and circular dichroism analyses were performed. Glutathione reduced OVO in a concentration-dependent manner. Egg white was analyzed for reduced proteins with a thiol probe and by MALDI-TOF/TOF. In unprocessed total egg white, several reduced proteins were detected by the thiol probe, among them reduced ovalbumin could be confirmed with MS analyses. Egg-allergics or sensitized controls were tested serologically (n = 19) for IgE against native and reduced OVO and in skin prick tests (n = 9). More patients had IgE against reduced than native OVO in Western blots. In skin prick test, five out of seven persistent egg-allergics and none of the controls reacted with reduced OVO. Reduced egg proteins are present in natural egg white. Glutathione, which is present in egg and furthermore is used as texture-improving additive in processed food, is capable of reducing OVO. Patients with persistent egg allergy reacted rather to reduce the native OVO. Hence, our data indicate that reduction is a novel natural and processing-associated principle, which contributes to the allergenicity of food. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

[Malondialdehyde and reduced glutathione increase in patients with chronic stable ischaemic cardiopathy].

PubMed

Cabrera de Bravo, Mayela Carolina; Bermúdez Pirela, Valmore José; Bermúdez Arias, Fernando; Cano Ponce, Clímaco; Mengual Moreno, Edgardo José; Leal González, Elliuz Marina

2005-03-19

Cardiovascular diseases are associated with the ischemia/reperfusion phenomena and therefore to the oxidation/antioxidation balance. The aim of this study was to determine malondialdehyde, nitric oxide, glutathione, ascorbic and dehydroascorbic acid in patients with chronic ischemic heart disease. 32 male patients, with chronic ischemic heart disease, between 40 and 60 years of age were studied. These individuals were divided in two groups: 16 with hypertension and 16 without hypertension. Both groups were compared with 31 healthy male subjects (control group). Significant differences (p < 0.001) was observed in malondialdehyde between no-hypertension ischemic group: 5.3 (1.5) microM and the hypertension ischemic group: 4.8 (1.3) microM in contrast with the healthy group: 2.2 (0.5) microM. Hypertension ischemic group showed significant greater reduced glutation levels: 286.1 (31.4) microg/ml than control group 262.0 (38.8) microg/ml; p < 0.03 and no-hypertension ischemic group: 256.4 (41.5) microg/ml; p < 0.02. No significant difference in the rest of the parameters for all study groups. Oxidation/antioxidation balance during chronic ischemic heart disease can be considered as a good metabolic ischemia indicator, that used in the monitoring and therapeutic evaluation could detect molecular changes that anticipate installation of tissue damage.

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

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

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.

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.

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.

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

FAD-induced in vitro activation of glutathione reductase in the lens of B2 deficient rats.

PubMed

Ono, S; Hirano, H

1984-04-01

We studied the FAD-induced in vitro stimulation of lenticular glutathione reductase in riboflavin-deficient rats. The stimulatory effect of FAD on lenticular glutathione reductase in rats fed a B2-deficient diet for 4 weeks was remarkably higher than in paired control rats fed a B2-supplemented basal diet and control rats had ad libitum access to a B2-supplemented basal diet. The in vitro FAD stimulation effect on rat lenticular glutathione reductase represents a sensitive indicator of the B2 deficient status.

S-glutathionyl-(chloro)hydroquinone reductases: a new class of glutathione transferases functioning as oxidoreductases

PubMed Central

Belchik, Sara M.; Xun, Luying

2011-01-01

Glutathione transferases (GSTs) are best known for transferring glutathione (GSH) to hydrophobic organic compounds, making the conjugates more soluble. However, the omega-class GSTs of animals and the lambda-class GSTs and dehydroascorbate reductases (DHARs) of plants have little or no activity for GSH transfer. Instead, they catalyze GSH-dependent oxidoreductions. The lambda-class GSTs reduce disulfide bonds, the DHARs reduce the disulfide bonds and dehydroascorbate, and the omega-class GSTs can reduce more substrates, including disulfide bonds, dehydroascorbate, and dimethylarsinate. Glutathionyl-(chloro)hydroquinone reductases (GS-HQRs) are the newest class of GSTs that mainly catalyze oxidoreductions. Besides the activities of the other three classes, GS-HQRs also reduce GS-hydroquinones, including GS-trichloro-p-hydroquinone, GS-dichloro-p-hydroquinone, GS-2-hydroxy-p-hydroquinone, and GS-p-hydroquinone. They are conserved and widely distributed in bacteria, fungi, protozoa, and plants, but not in animals. The four classes are phylogenetically more related to each other than to other GSTs, and they share a Cys-Pro motif at the GSH-binding site. Hydroquinones are metabolic intermediates of certain aromatic compounds. They can be auto-oxidized by O2 to benzoquinones, which spontaneously react with GSH to form GS-hydroquinones via Michael’s addition. GS-HQRs are expected to channel GS-hydroquinones, formed spontaneously or enzymatically, back to hydroquinones. When the released hydroquinones are intermediates of metabolic pathways, GS-HQRs play a maintenance role for the pathways. Further, the common presence of GS-HQRs in plants, green algae, cyanobacteria, and halobacteria suggest a beneficial role in the light-using organisms. PMID:21425927

Effect of Patulin from Penicillium vulpinum on the Activity of Glutathione-S-Transferase and Selected Antioxidative Enzymes in Maize

PubMed Central

Ismaiel, Ahmed A.

2017-01-01

The mycotoxin patulin (PAT) was purified from Penicillium vulpinum CM1 culture that has been isolated from a soil cultivated with maize. The effect of PAT and of a fungal culture filtrate on the activities of glutathione-S-transferase (GST) and some antioxidant enzymes viz. ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) was investigated in roots and shoots of 8-day-old maize seedlings. PAT and culture filtrate caused significant reduction effects in a dose-related manner on the total GST activity. Upon application of the high PAT concentration (25 μg·mL−1) and of the concentrated fungal filtrate (100%, v/v), the reduction in GST activity of roots was 73.8–76.0% and of shoots was 60–61.7%. Conversely, significant increases in the activities of antioxidant enzymes were induced. Application of 25 μg·PAT·mL−1 increased APX, GR, DHAR, and MDHAR activity of root by 2.40-, 2.00-, 1.24-, and 2.16-fold, respectively. In shoots, the enzymatic activity was increased by 1.57-, 1.45-, 1.45-, and 1.61-fold, respectively. Similar induction values of the enzymatic activity were obtained upon application of the concentrated fungal filtrate. This is the first report describing the response of GST and antioxidant enzyme activities of plant cells to PAT toxicity. PMID:28737668

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.

Glutathione-induced drought stress tolerance in mung bean: coordinated roles of the antioxidant defence and methylglyoxal detoxification systems

PubMed Central

Nahar, Kamrun; Hasanuzzaman, Mirza; Alam, Md. Mahabub; Fujita, Masayuki

2015-01-01

Drought is considered one of the most acute environmental stresses presently affecting agriculture. We studied the role of exogenous glutathione (GSH) in conferring drought stress tolerance in mung bean (Vigna radiata L. cv. Binamoog-1) seedlings by examining the antioxidant defence and methylglyoxal (MG) detoxification systems and physiological features. Six-day-old seedlings were exposed to drought stress (−0.7 MPa), induced by polyethylene glycol alone and in combination with GSH (1 mM) for 24 and 48 h. Drought stress decreased seedling dry weight and leaf area; resulted in oxidative stress as evidenced by histochemical detection of hydrogen peroxide (H2O2) and O2⋅− in the leaves; increased lipid peroxidation (malondialdehyde), reactive oxygen species like H2O2 content and O2⋅− generation rate and lipoxygenase activity; and increased the MG level. Drought decreased leaf succulence, leaf chlorophyll and relative water content (RWC); increased proline (Pro); decreased ascorbate (AsA); increased endogenous GSH and glutathione disulfide (GSSG) content; decreased the GSH/GSSG ratio; increased ascorbate peroxidase and glutathione S-transferase activities; and decreased the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and catalase. The activities of glyoxalase I (Gly I) and glyoxalase II (Gly II) increased due to drought stress. In contrast to drought stress alone, exogenous GSH enhanced most of the components of the antioxidant and glyoxalase systems in drought-affected mung bean seedlings at 24 h, but GSH did not significantly affect AsA, Pro, RWC, leaf succulence and the activities of Gly I and DHAR after 48 h of stress. Thus, exogenous GSH supplementation with drought significantly enhanced the antioxidant components and successively reduced oxidative damage, and GSH up-regulated the glyoxalase system and reduced MG toxicity, which played a significant role in improving the physiological features and drought

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.

Spirulina improves antioxidant status by reducing oxidative stress in rabbits fed a high-cholesterol diet.

PubMed

Kim, Mi Yeon; Cheong, Sun Hee; Lee, Jeung Hee; Kim, Min Ji; Sok, Dai-Eun; Kim, Mee Ree

2010-04-01

The beneficial effect of Spirulina (Spirulina platensis) on tissue lipid peroxidation and oxidative DNA damage was tested in the hypercholesterolemic New Zealand White rabbit model. After hypercholesterolemia was induced by feeding a high cholesterol (0.5%) diet (HCD) for 4 weeks, then HCD supplemented with 1% or 5% Spirulina (SP1 or SP5, respectively) was provided for an additional 8 weeks. Spirulina supplementation significantly reduced the increased lipid peroxidation level in HCD-fed rabbits, and levels recovered to control values. Oxidative stress biomarkers such as glutathione, glutathione peroxidase, glutathione reductase, and glutathione S-transferase were significantly improved in the liver and red blood cells of rabbits fed SP1. Furthermore, SP5 induced antioxidant enzyme activity by 3.1-fold for glutathione, 2.5-fold for glutathione peroxidase, 2.7-fold for glutathione reductase, and 2.3-fold for glutathione S-transferase in liver, compared to the HCD group. DNA damage in lymphocytes was significantly reduced in both the SP1 and SP5 groups, based on the comet assay. Findings from the present study suggest that dietary supplementation with Spirulina may be useful to protect the cells from lipid peroxidation and oxidative DNA damage.

Effect of chemical form of selenium on tissue glutathione peroxidase activity in developing rats

NASA Technical Reports Server (NTRS)

Lane, Helen W.; Strength, Ralph; Johnson, Janet; White, Marguerite T.

1991-01-01

The hypothesis that the stage of development of rats may affect the availability of various forms of selenium for the activity of glutathione peroxidase (GSHPx) in the rat was experimentally investigated. One experiment evaluated the availability of selenium as selenite or selenomethionine for GSPHx activity during three developmental states in rats: fetus and 7-day old and 14-day old nursing pups. In all tissues studied, GSHPx activity was highest in the 14-day-old pups whose dams were in the selenomethionine group. Rat pups given intraperitoneal selenite had higher liver and kidney GSHPx activity than pups given the same amount of selenium as intraperitoneal selenomethionine. In a second experiment, all dams were fed the same basal diet and pups were weaned to diets containing one of two levels of selenium and one of three forms of selenium (selenite, selenomethionine, or selenocystine). The results also supported the hypothesis these dietary forms of selenium are differentially available for GSHPx activity.

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

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

PubMed Central

Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

2014-01-01

Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

L-cysteine efflux in erythrocytes as a function of human age: correlation with reduced glutathione and total anti-oxidant potential.

PubMed

Kumar, Prabhanshu; Maurya, Pawan Kumar

2013-06-01

Thiol compounds such as cysteine (Cys) and reduced glutathione (GSH) play an important role in human aging and age-related diseases. In erythrocytes, GSH is synthesized by glutamic acid, cysteine, and glycine, but the rate of GSH synthesis is determined only by the availability of L-cysteine. Cysteine supplementation has been shown to ameliorate several parameters that are known to degenerate during human aging. We have studied L-cysteine efflux in vitro in human erythrocytes as a function of age by suspending cells in solution containing 10 mM L-cysteine for uptake; later cells were re-suspended in phosphate-buffered saline (PBS)-glucose to allow efflux. Change in the free sulfhydryl (-SH) concentration was then measured to calculate the rate of efflux. The GSH/oxidized glutathione (GSSG) ratio was taken as a control to study the oxidation/reduction state of the erythrocyte. The total anti-oxidant potential of plasma was measured in terms of ferric reducing ability of plasma (FRAP) values. We have shown a significant (p<0.0001) decline in the efflux of L-cysteine in erythrocytes during human aging, and the GSH/GSSG ratio decreases as a function of human age. The decline in L-cysteine efflux during aging correlates with the decrease in GSH and the FRAP value. This finding may help to explain the shift in the redox status and low GSH concentration that might determine the rate of L-cysteine efflux observed in erythrocytes and an important factor in the development of oxidative stress in erythrocytes during aging.

The relation of blood pressure and carotid intima-media thickness with the glutathione cycle in a young bi-ethnic population: the African-PREDICT study.

PubMed

Myburgh, Caitlynd; Huisman, Hugo W; Mels, Catharina M C

2018-04-01

Oxidative stress has been implicated in the development of hypertension, arterial stiffness and atherosclerosis. Optimal functioning of the enzymatic antioxidant system is central to prevent increased oxidative stress and its consequences. We aimed to investigate the relationships of ambulatory blood pressure and carotid intima-media thickness with enzyme activities of the glutathione cycle in 396 young, black and white South Africans of the African-PREDICT study. Ambulatory blood pressure and carotid intima-media thickness were measured and glutathione peroxidase and glutathione reductase activities were analyzed. Black participants had higher reactive oxygen species (men: p = 0.019; women: borderline p = 0.064) and total glutathione (both p < 0.001), but lower glutathione peroxidase activity and total antioxidant status (all p < 0.001). In black men, ambulatory pulse pressure was negatively associated with glutathione peroxidase activity (R 2  = 0.19; β = -0.25; p = 0.06). Black and white women displayed positive associations of ambulatory systolic blood pressure (black: R 2  = 0.25; β = 0.21; p = 0.048; white: R 2  = 0.44; β = 0.18; p = 0.016) with glutathione reductase activity, whereas white men displayed a positive association of ambulatory pulse pressure with glutathione reductase activity (R 2  = 0.25; β = 0.29; p = 0.01). The lower glutathione peroxidase activity and total antioxidant status, the higher reactive oxygen species, as well as the negative association between ambulatory pulse pressure and glutathione peroxidase activity in the black men suggest that oxidative stress may be associated with early vascular changes in this group. In the other three groups, the positive associations of blood pressure with glutathione reductase activity suggest a possible role for adequate glutathione reductase activity in preventing or delaying the development of hypertension.

Effect on post-cryopreserved semen characteristics of Holstein bulls of adding combinations of vitamin C and either catalase or reduced glutathione to Tris extender.

PubMed

Eidan, Sajeda M

2016-04-01

This study was undertaken to investigate the influence of adding combinations of vitamin C to Tris extender with either catalase or reduced glutathione on post-cryopreserved semen characteristics of Holstein bulls for different preservation periods (cooling at 5°C, 48 h, 1, 2 and 3 months post cryopreservation, PC). Seven Holstein bulls of 2.5-3 years of age were used in this experiment. Semen was collected via artificial vagina in one ejaculate per bull per week for the 7 week experimental period. Pooled semen was equally divided into three treatments using Tris extender. Combinations of vitamin C (2.5mM) were added with either catalase (100 IU/ml, T2) or reduced glutathione (2mM, T3) to Tris extender and comparisons in response were made with the control group (Tris extender, T1). Individual sperm motility (IM), viability (V), plasma membrane integrity (PMI), and acrosome integrity (AI) were assessed during all periods of the study along with Malondialdehyde (MDA) concentrations and freezing ability. The IM was greater (P ≤ 0.01) in the T2 as compared with the T1 group at all periods of the study. Furthermore, the IM were greater (P ≤ 0.01) in the T3 as compared with the T1 group at the 48 h time period and at 3 months PC. The V, PMI and AI were greater (P ≤ 0.01) in T2 and T3 as compared with the T1 group at all the experimental periods. The MDA was greater (P ≤ 0.01) in the T2 as compared with the T1 group at 3 months PC. In conclusion, there was improved semen quality if semen of Holstein bulls was collected and stored in combinations of vitamin C with either catalase (T2) or reduced glutathione (T3) being added to Tris extender. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of activated NR2B gene- and caspase-3 protein-expression by glutathione following traumatic brain injury in a rat model

PubMed Central

Arifin, Muhammad Zafrullah; Faried, Ahmad; Shahib, Muhammad Nurhalim; Wiriadisastra, Kahdar; Bisri, Tatang

2011-01-01

Background. Traumatic brain injury (TBI) remains a major cause of death and disability. Oxidative stress is an important element of the injury cascade following TBI. Progressive compromise of antioxidant defenses and free radical-mediated lipid peroxidation are one of the major mechanisms of secondary TBI. NR2B is a glutamate receptor and its activation is caused by TBI increasing a brain cell death, along with caspase-3 as a hall mark of apoptosis. Glutathione is a potent free radical scavenger that might prevent secondary TBI damage and inhibited apoptosis. Materials and Methods. In the present study, it aims to demonstrate the effect of glutathione on inhibition of brain oxidative damage in a TBI rat model. Results. In this study, the expressions of mRNA NR2B in placebo group and groups with glutathione administration at 0, 3, and 6 hours after TBI were 328.14, 229.90, 178.50, and 136.14, respectively (P<0.001). The highest caspase-3 expression was shown in placebo group with 66.7% showing strong positive results (>80%); as expected, glutathione administered in 0, 3, and 6 hours groups had lower strong positive results of 50%, 16.7%, and 16.7%, respectively, (P=0.025). Conclusion. In conclusion, this study showed that glutathione administration in a TBI rat model decreased NR2B gene- and caspase-3 protein-expression that lead to the inhibition of brain cell death. Our results suggest that glutathione, as a potent free radical scavenger, has a brain cell protective effect against oxidative damage and cell death induced by TBI in rat model. PMID:22347327

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

PubMed Central

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

2016-01-01

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

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

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

[Effects of organic selenium supplement on glutathione peroxidase activities: a meta-analysis of randomized controlled trials].

PubMed

Jiang, Xia; Dong, Jiayi; Wang, Bo; Yin, Xuebin; Qin, Liqiang

2012-01-01

To study the effects of organic selenium supplementation on glutathione peroxidase (GPx) activities. Randomized controlled trials (RCT) published from January 1988 to December 2010 on the relationship between organic selenium supplementation and GPx activities were collected. Meta-analysis was applied to estimate the combined standardized mean difference (SMD) and 95% confidence interval (95% CI). A total of 10 RCTs were included. The number of studies observing GPx activities in plasma, erythrocyte and platelet was 8, 5 and 5, respectively. Compared with the controls, the combined SMD (95% CI) of GPx activities in plasma, erythrocyte and platelet of subjects supplemented with organic selenium was 0.46 (0.09 - 0.83), 0.36 (0.02 - 0.69) and 0.56 (-0.02 - 1.15). Supplementation with organic selenium increases GPx activities in healthy adults.

Glutathione regulation-based dual-functional upconversion sensing-platform for acetylcholinesterase activity and cadmium ions.

PubMed

Fang, Aijin; Chen, Hongyu; Li, Haitao; Liu, Meiling; Zhang, Youyu; Yao, Shouzhuo

2017-01-15

A dual-functional platform for the sensing of acetylcholinesterase (AChE) activity and cadmium ions (Cd 2+ ) was developed based on the fluorescence resonance energy transfer (FRET) between NaYF 4 :Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs) via glutathione regulation. The detection mechanism is based on the fact that AuNPs can quench the fluorescence of UCNPs. AChE catalyzes the hydrolysis of acetylthiocholine (ATC) into thiocholine which reacts with AuNPs by S-Au conjunction and results the aggregation of AuNPs and change in fluorescence of UCNPs. Therefore, the AChE activity can be detected through the changes of the color of solution and fluorescence recovery of UCNPs. However, the presence of glutathione (GSH) can protect AuNPs from aggregation and enlarge the inter-particle distance between AuNPs and UCNPs. When Cd 2+ is added into the stable mixture of AuNPs, GSH and AChE/ATC, Cd 2+ could interact with GSH to form a spherical shaped (GSH) 4 Cd complex, which decreases the free GSH on the surface of AuNPs to weaken the stability of AuNPs and lead to the easily aggregation of them in the system. The aggregated-AuNPs are released from the surface of UCNPs, which results in the fluorescence of UCNPs gradually recovered. Under the optimized conditions, the detection limits of AChE activity and Cd 2+ are estimated to be 0.015mU/mL and 0.2µM, respectively. The small molecules regulated dual-functional platform based on UCNPs/AuNPs is a simple, label-free method and can be applied for the turn-on fluorescence detection of AChE activity in human serum and Cd 2+ in real water samples. The present work demonstrates a general strategy for the design of small molecules regulated multifunctional platform and will be expanded for different areas in the future. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

The relationship between leaf rolling and ascorbate-glutathione cycle enzymes in apoplastic and symplastic areas of Ctenanthe setosa subjected to drought stress.

PubMed

Saruhan, Neslihan; Terzi, Rabiye; Saglam, Aykut; Kadioglu, Asim

2009-01-01

The ascorbate-glutathione (ASC-GSH) cycle has an important role in defensive processes against oxidative damage generated by drought stress. In this study, the changes that take place in apoplastic and symplastic ASC-GSH cycle enzymes of the leaf and petiole were investigated under drought stress causing leaf rolling in Ctenanthe setosa (Rose.) Eichler (Marantaceae). Apoplastic and symplastic extractions of leaf and petiole were performed at different visual leaf rolling scores from 1 to 4 (1 is unrolled, 4 is tightly rolled and the others are intermediate forms). Glutathione reductase (GR), a key enzyme in the GSH regeneration cycle, and ascorbate (ASC) were present in apoplastic spaces of the leaf and petiole, whereas dehydroascorbate reductase (DHAR), which uses glutathione as reductant, monodehydroascorbate reductase (MDHAR), which uses NAD(P)H as reductant, and glutathione were absent. GR, DHAR and MDHAR activities increased in the symplastic and apoplastic areas of the leaf. Apoplastic and symplastic ASC and dehydroascorbate (DHA), the oxidized form of ascorbate, rose at all scores except score 4 of symplastic ASC in the leaf. On the other hand, while reduced glutathione (GSH) content was enhanced, oxidized glutathione (GSSG) content decreased in the leaf during rolling. As for the petiole, GR activity increased in the apoplastic area but decreased in the symplastic area. DHAR and MDHAR activities increased throughout all scores, but decreased to the score 1 level at score 4. The ASC content of the apoplast increased during leaf rolling. Conversely, symplastic ASC content increased at score 2, however decreased at the later scores. While the apoplastic DHA content declined, symplastic DHA rose at score 2, but later was down to the level of score 1. While GSH content enhanced during leaf rolling, GSSG content did not change except at score 2. As well, there were good correlations between leaf rolling and ASC-GSH cycle enzyme activities in the leaf (GR and DHAR

Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding

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

Gibson, Lydia M.; Celeste, Lesa R.; Lovelace, Leslie L.

Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181-197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed thatmore » hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.« less

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.

Redox regulation of antioxidant enzymes: post-translational modulation of catalase and glutathione peroxidase activity by resveratrol in diabetic rat liver.

PubMed

Sadi, Gökhan; Bozan, Davut; Yildiz, Huseyin Bekir

2014-08-01

Resveratrol is a strong antioxidant that exhibits blood glucose-lowering effects, which might contribute to its usefulness in preventing complications associated with diabetes. The present study aimed to investigate resveratrol effects on catalase (CAT) and glutathione peroxidase (GPx) gene and protein expression, their phosphorylation states and activities in rat liver of STZ-induced diabetes. Diabetes increased the levels of total protein phosphorylation and p-CAT, while mRNA expression, protein levels, and activity were reduced. Although diabetes induced transcriptional repression over GPx, it did not affect the protein levels and activity. When resveratrol was administered to diabetic rats, an increase in activity was associated with an increase in p-GPx levels. Decrease in Sirtuin1 (SIRT1) and nuclear factor erythroid 2-related factor (Nrf2) and increase in nuclear factor kappa B (NFκB) gene expression in diabetes were associated with a decrease in CAT and GPx mRNA expression. A possible compensatory mechanism for reduced gene expression of antioxidant enzymes is proved to be nuclear translocation of redox-sensitive Nrf2 and NFκB in diabetes which is confirmed by the increase in nuclear and decrease in cytoplasmic protein levels of Nrf2 and NFκB. Taken together, these findings revealed that an increase in the oxidized state in diabetes intricately modified the cellular phosphorylation status and regulation of antioxidant enzymes. Gene regulation of antioxidant enzymes was accompanied by nuclear translocation of Nrf2 and NFκB. Resveratrol administration also activated a coordinated cytoprotective response against diabetes-induced changes in liver tissues.

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...

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...

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...

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.

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

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.

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

PubMed Central

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

2011-01-01

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

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.

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

S-Nitrosation destabilizes glutathione transferase P1-1.

PubMed

Balchin, David; Stoychev, Stoyan H; Dirr, Heini W

2013-12-23

Protein S-nitrosation is a post-translational modification that regulates the function of more than 500 human proteins. Despite its apparent physiological significance, S-nitrosation is poorly understood at a molecular level. Here, we investigated the effect of S-nitrosation on the activity, structure, stability, and dynamics of human glutathione transferase P1-1 (GSTP1-1), an important detoxification enzyme ubiquitous in aerobes. S-Nitrosation at Cys47 and Cys101 reduces the activity of the enzyme by 94%. Circular dichroism spectroscopy, acrylamide quenching, and amide hydrogen-deuterium exchange mass spectrometry experiments indicate that the loss of activity is caused by the introduction of local disorder at the active site of GSTP1-1. Furthermore, the modification destabilizes domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. In contrast, S-nitrosation at Cys101 alone introduces a refolding defect in domain 1 but compensates by stabilizing the domain kinetically. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation and provide general insight into the consequences of S-nitrosation on protein stability and dynamics.

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.

An energy-saving glutathione production method from low-temperature cooked rice using amylase-expressing Saccharomyces cerevisiae.

PubMed

Hara, Kiyotaka Y; Kim, Songhee; Kiriyama, Kentaro; Yoshida, Hideyo; Arai, Shogo; Ishii, Jun; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

2012-05-01

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is industrially produced by fermentation using Saccharomyces cerevisiae. Before the glutathione fermentation process with S. cerevisiae, a glucose extraction process from starchy materials is required. This glucose extraction is usually carried out by converting starchy materials to starch using high-temperature cooking and subsequent hydrolysis by amylases to convert starch to glucose. In this study, to develop an energy-saving glutathione production process by reducing energy consumption during the cooking step, we efficiently produced glutathione from low-temperature cooked rice using amylase-expressing S. cerevisiae. The combination of the amylase-expressing yeast with low-temperature cooking is potentially applicable to a variety of energy-saving bio-production methods of chemicals from starchy bio-resources. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

SKN-1-independent transcriptional activation of glutathione S-transferase 4 (GST-4) by EGF signaling

PubMed Central

Van de Walle, Pieter; Schoofs, Liliane

2016-01-01

ABSTRACT In C. elegans research, transcriptional activation of glutathione S-transferase 4 (gst-4) is often used as a read-out for SKN-1 activity. While many heed an assumed non-exclusivity of the GFP reporter signal driven by the gst-4 promoter to SKN-1, this is also often ignored. We here show that gst-4 can also be transcriptionally activated by EOR-1, a transcription factor mediating effects of the epidermal growth factor (EGF) pathway. Along with enhancing exogenous oxidative stress tolerance, EOR-1 inde-pendently of SKN-1 increases gst-4 transcription in response to augmented EGF signaling. Our findings caution researchers within the C. elegans community to always rely on sufficient experimental controls when assaying SKN-1 transcriptional activity with a gst-4p::gfp reporter, such as SKN-1 loss-of-function mutants and/or additional target genes next to gst-4. PMID:28090393

Unbalanced activation of glutathione metabolic pathways suggests potential involvement in plant defense against the gall midge mayetiola destructor in wheat

USDA-ARS?s Scientific Manuscript database

Glutathione, a thiol tripeptide of '-glutamylcysteinylglycine, exists abundantly in nearly all organisms. Glutathione participates in various physiological processes involved in redox reactions by serving as an electron donor/acceptor. In this study, we found that the abundance of total glutathion...

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

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

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.

Hepatic glutathione metabolism and lipid peroxidation in response to excess dietary selenomethionine and selenite in mallard ducklings

USGS Publications Warehouse

Hoffman, D.J.; Heinz, G.H.; Krynitsky, A.J.

1989-01-01

Selenium from selenomethionine accumulated in a dose-dependent manner in the liver, resulting in a decrease in hepatic-reduced glutathione with a corresponding decrease in total hepatic thiols. There was a dose-dependent increase in the oxidized to reduced glutathione ratio, and an increase in lipid peroxidation. These findings indicate that Se in the diet at 10 ppm and higher causes significant sublethal alterations in mallard ducklings, and 20-40 ppm causes significant hepatotoxicity.

Decreased glutathione S-transferase expression and activity and altered sex steroids in Lake Apopka brown bullheads (Ameriurus nebulosus)

USGS Publications Warehouse

Gallagher, E.P.; Gross, T.S.; Sheehy, K.M.

2001-01-01

A number of freshwater lakes and reclaimed agricultural sites in Central Florida have been the receiving waters for agrochemical and municipal runoff. One of these sites, Lake Apopka, is also a eutrophic system that has been the focus of several case studies reporting altered reproductive activity linked to bioaccumulation of persistent organochlorine chemicals in aquatic species. The present study was initiated to determine if brown bullheads (Ameriurus nebulosus) from the north marsh of Lake Apopka (Lake Apopka Marsh) exhibit an altered capacity to detoxify environmental chemicals through hepatic glutathione S-transferase (GST)-mediated conjugation as compared with bullheads from a nearby reference site (Lake Woodruff). We also compared plasma sex hormone concentrations (testosterone, 17-?? estradiol, and 11 keto-testosterone) in bullheads from the two sites. Female bullheads from Lake Apopka had 40% lower initial rate GST conjugative activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 50% lower activity towards p-nitrobutyl chloride (NBC), 33% lower activity toward ethacrynic acid (ECA), and 43% lower activity toward ??5-androstene-3,17-dione (??5-ADI), as compared with female bullheads from Lake Woodruff. Enzyme kinetic analyses demonstrated that female bullheads from Lake Apopka had lower GST-catalyzed CDNB clearance than did female Lake Woodruff bullheads. Western blotting studies of bullhead liver cytosolic proteins demonstrated that the reduced GST catalytic activities in female Lake Apopka bullheads were accompanied by lower expression of hepatic GST protein. No site differences were observed with respect to GST activities or GST protein expression in male bullheads. Female Lake Apopka bullheads also had elevated concentrations of plasma androgens (testosterone and 11-ketotestosterone) as compared with females from Lake Woodruff. In contrast, male Lake Apopka bullheads had elevated levels of plasma estrogen but similar levels of androgens as compared with

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

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

Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS: An insight into the redox state of hematopoietic stem cells.

PubMed

Carroll, Dustin; Howard, Diana; Zhu, Haining; Paumi, Christian M; Vore, Mary; Bondada, Subbarao; Liang, Ying; Wang, Chi; St Clair, Daret K

2016-08-01

Cellular redox balance plays a significant role in the regulation of hematopoietic stem-progenitor cell (HSC/MPP) self-renewal and differentiation. Unregulated changes in cellular redox homeostasis are associated with the onset of most hematological disorders. However, accurate measurement of the redox state in stem cells is difficult because of the scarcity of HSC/MPPs. Glutathione (GSH) constitutes the most abundant pool of cellular antioxidants. Thus, GSH metabolism may play a critical role in hematological disease onset and progression. A major limitation to studying GSH metabolism in HSC/MPPs has been the inability to measure quantitatively GSH concentrations in small numbers of HSC/MPPs. Current methods used to measure GSH levels not only rely on large numbers of cells, but also rely on the chemical/structural modification or enzymatic recycling of GSH and therefore are likely to measure only total glutathione content accurately. Here, we describe the validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) in HSC/MPPs isolated from bone marrow. The lower limit of quantitation (LLOQ) was determined to be 5.0ng/mL for GSH and 1.0ng/mL for GSSG with lower limits of detection at 0.5ng/mL for both glutathione species. Standard addition analysis utilizing mouse bone marrow shows that this method is both sensitive and accurate with reproducible analyte recovery. This method combines a simple extraction with a platform for the high-throughput analysis, allows for efficient determination of GSH/GSSG concentrations within the HSC/MPP populations in mouse, chemotherapeutic treatment conditions within cell culture, and human normal/leukemia patient samples. The data implicate the importance of the modulation of GSH/GSSG redox couple in stem cells related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

PubMed

Cihil, Kristine M; Swiatecka-Urban, Agnieszka

2013-12-13

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

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.

Glutathione S-transferase π complexes with and stimulates Na⁺,K⁺-ATPase.

PubMed

Ochiai, Hideo; Eguchi, Hiroshi; Noguchi, Shunsuke; Hayashi, Yutaro; Nishino, Hideaki; Kawamura, Masaru; Wu, Chau H

2013-01-01

Glutathione S-transferase (GST) was found to complex with the Na⁺,K⁺-ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α-subunit of the Na⁺,K⁺-ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺-ATPase. Co-immunoprecipitation experiments, using the anti-α-subunit antiserum to precipitate the GST-Na⁺,K⁺-ATPase complex and then using antibodies specific to an isoform of GST to identify the co-precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺-ATPase. GST stimulated the Na⁺,K⁺-ATPase activity up to 1.4-fold. The level of stimulation exhibited a saturable dose-response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺-ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺-ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺-ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase π

PubMed Central

Ralat, Luis A.; Colman, Roberta F.

2003-01-01

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase π (GST π): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST π with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST π at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzyme’s use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST π with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites. PMID:14573868

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase pi.

PubMed

Ralat, Luis A; Colman, Roberta F

2003-11-01

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase pi (GST pi): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST pi with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST pi at pH 7.0 and 25 degrees C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST pi with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.

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.

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.

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.

Effect of oral contraceptive use on the erythrocytic glutathione reductase and aspartate aminotransferase activities in women with or without clinical signs of vitamin deficiency.

PubMed

Tovar, A; Bourges, H; Canto, T; Torres, N; Lopez-castro, B R

1985-07-01

The effect of the chronic use of combined oral contraceptives (OCs) on the "activity coefficients" (alpha = coenzyme-stimulated activity/basal activity) of erythrocytic glutathione reductase and aspartate aminotransferase was studied in 2 groups of 90 female volunteers each; 1 of the groups, from the state of Yucatan in southeast Mexico, presented clinical lesions of vitamin deficiency, while the other group, from Mexico City, did not have any clinical evidence of vitamin deficiency. One half of the women (45) in each group were chronic OC users and the other half were not. The results were analyzed comparing OC users with non-users in each location. For both glutathione reductase and aspartate aminotransferase, the Mexico City OC users had significantly higher (p 0.001) alpha values than nonusers, while in the Yucatan women, the alpha values were similarly high independent of OC use.

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.

GSTP1 Polymorphisms and their Association with Glutathione Transferase and Peroxidase Activities in Patients with Motor Neuron Disease.

PubMed

Gajewska, Beata; Kaźmierczak, Beata; Kuźma-Kozakiewicz, Magdalena; Jamrozik, Zygmunt; Barańczyk-Kuźma, Anna

2015-01-01

Glutathione S-transferase pi (GSTP1) is a crucial enzyme in detoxification of electrophilic compounds and organic peroxides. Together with Se-dependent glutathione peroxidase (Se-GSHPx) it protects cells against oxidative stress which may be a primary factor implicated in motor neuron disease (MND) pathogenesis. We investigated GSTP1 polymorphisms and their relationship with GST and Se-GSTPx activities in a cohort of Polish patients with MND. Results were correlated with clinical phenotypes. The frequency of genetic variants for GSTP1 exon 5 (I105V) and exon 6 (A114V) was studied in 104 patients and 100 healthy controls using real-time polymerase chain reaction. GST transferase activity was determined in serum with 1-chloro-2,4-dinitrobenzene, its peroxidase activity with cumene hydroperoxide, and Se-GSHPx activity with hydrogen peroxide. There were no differences in the prevalence of GSTP1 polymorphism I105V and A114V between MND and controls, however the occurrence of CT variant in codon 114 was associated with a higher risk for MND. GSTP1 polymorphisms were less frequent in classic ALS than in progressive bulbar palsy. In classic ALS C* (heterozygous I /V and A /V) all studied activities were significantly lower than in classic ALS A* (homozygous I /I and A/A). GST peroxidase activity and Se-GSHPx activity were lower in classic ALS C* than in control C*, but in classic ALS A* Se-GSHPx activity was significantly higher than in control A*. It can be concluded that the presence of GSTP1 A114V but not I105V variant increases the risk of MND, and combined GSTP1 polymorphisms in codon 105 and 114 may result in lower protection of MND patients against the toxicity of electrophilic compounds, organic and inorganic hydroperoxides.

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.

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.

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

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

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

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.

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

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.

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.

Enhanced memory in Wistar rats by virgin coconut oil is associated with increased antioxidative, cholinergic activities and reduced oxidative stress.

PubMed

Rahim, Nur Syafiqah; Lim, Siong Meng; Mani, Vasudevan; Abdul Majeed, Abu Bakar; Ramasamy, Kalavathy

2017-12-01

Virgin coconut oil (VCO) has been reported to possess antioxidative, anti-inflammatory and anti-stress properties. Capitalizing on these therapeutic effects, this study investigated for the first time the potential of VCO on memory improvement in vivo. Thirty male Wistar rats (7-8 weeks old) were randomly assigned to five groups (n = six per group). Treatment groups were administered with 1, 5 and 10 g/kg VCO for 31 days by oral gavages. The cognitive function of treated-rats were assessed using the Morris Water Maze Test. Brains were removed, homogenized and subjected to biochemical analyses of acetylcholine (ACh) and acetylcholinesterase (AChE), antioxidants [superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx) and glutathione reductase (GRx)], lipid peroxidase [malondialdehyde (MDA)] as well as nitric oxide (NO). α-Tocopherol (αT; 150 mg/kg) was also included for comparison purposes. VCO-fed Wistar rats exhibited significant (p < 0.05) improvement of cognitive functions [reduced escape latency (≥ 1.8 s), reduced escape distance (≥ 0.3 m) and increased total time spent on platform (≥ 1 s)]. The findings were accompanied by elevation of ACh (15%), SOD (8%), CAT (≥ 54%), GSH (≥ 20%) and GPx (≥ 12%) and reduction of AChE (≥17%), MDA (> 33%) and NO (≥ 34%). Overall, memory improvement by VCO was comparable to αT. VCO has the potential to be used as a memory enhancer, the effect of which was mediated, at least in part, through enhanced cholinergic activity, increased antioxidants level and reduced oxidative stress.

A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

PubMed Central

Rajatileka, Shavanti; Steven, Andrew; Hemingway, Janet; Ranson, Hilary; Paine, Mark; Vontas, John

2010-01-01

Background Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. Methodology/Principal Findings We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. Conclusions/Significance The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control. PMID:20824165

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

Nrf1 CNC-bZIP protein promotes cell survival and nucleotide excision repair through maintaining glutathione homeostasis.

PubMed

Han, Weinong; Ming, Mei; Zhao, Rui; Pi, Jingbo; Wu, Chunli; He, Yu-Ying

2012-05-25

Skin cancer is the most common cancer in the United States. Its major environmental risk factor is UVB radiation in sunlight. In response to UVB damage, epidermal keratinocytes activate a specific repair pathway, i.e. nucleotide excision repair, to remove UVB-induced DNA lesions. However, the regulation of UVB response is not fully understood. Here we show that the long isoform of the nuclear factor erythroid 2-related factor 1 (Nrf1, also called NFE2L1), a cytoprotective transcription factor critical for the expression of multiple antioxidant response element-dependent genes, plays an important role in the response of keratinocytes to UVB. Nrf1 loss sensitized keratinocytes to UVB-induced apoptosis by up-regulating the expression of the proapoptotic Bcl-2 family member Bik through reducing glutathione levels. Knocking down Bik reduced UVB-induced apoptosis in Nrf1-inhibited cells. In UVB-irradiated surviving cells, however, disruption of Nrf1 impaired nucleotide excision repair through suppressing the transcription of xeroderma pigmentosum C (XPC), a factor essential for initiating the global genome nucleotide excision repair by recognizing the DNA lesion and recruiting downstream factors. Nrf1 enhanced XPC expression by increasing glutathione availability but was independent of the transcription repressor of XPC. Adding XPC or glutathione restored the DNA repair capacity in Nrf1-inhibited cells. Finally, we demonstrate that Nrf1 levels are significantly reduced by UVB radiation in mouse skin and are lower in human skin tumors than in normal skin. These results indicate a novel role of Nrf1 in UVB-induced DNA damage repair and suggest Nrf1 as a tumor suppressor in the skin.

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 ...

Roles of sedentary aging and lifelong physical activity in exchange of glutathione across exercising human skeletal muscle.

PubMed

Nyberg, Michael; Mortensen, Stefan P; Cabo, Helena; Gomez-Cabrera, Mari-Carmen; Viña, Jose; Hellsten, Ylva

2014-08-01

Reactive oxygen species (ROS) are important signaling molecules with regulatory functions, and in young and adult organisms, the formation of ROS is increased during skeletal muscle contractions. However, ROS can be deleterious to cells when not sufficiently counterbalanced by the antioxidant system. Aging is associated with accumulation of oxidative damage to lipids, DNA, and proteins. Given the pro-oxidant effect of skeletal muscle contractions, this effect of age could be a result of excessive ROS formation. We evaluated the effect of acute exercise on changes in blood redox state across the leg of young (23 ± 1 years) and older (66 ± 2 years) sedentary humans by measuring the whole blood concentration of the reduced (GSH) and oxidized (GSSG) forms of the antioxidant glutathione. To assess the role of physical activity, lifelong physically active older subjects (62 ± 2 years) were included. Exercise increased the venous concentration of GSSG in an intensity-dependent manner in young sedentary subjects, suggesting an exercise-induced increase in ROS formation. In contrast, venous GSSG levels remained unaltered during exercise in the older sedentary and active groups despite a higher skeletal muscle expression of the superoxide-generating enzyme NADPH oxidase. Arterial concentration of GSH and expression of antioxidant enzymes in skeletal muscle of older active subjects were increased. The potential impairment in exercise-induced ROS formation may be an important mechanism underlying skeletal muscle and vascular dysfunction with sedentary aging. Lifelong physical activity upregulates antioxidant systems, which may be one of the mechanisms underlying the lack of exercise-induced increase in GSSG. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

Exogenous glutathione improves high root-zone temperature tolerance by modulating photosynthesis, antioxidant and osmolytes systems in cucumber seedlings

PubMed Central

Ding, Xiaotao; Jiang, Yuping; He, Lizhong; Zhou, Qiang; Yu, Jizhu; Hui, Dafeng; Huang, Danfeng

2016-01-01

To investigate the physiological responses of plants to high root-zone temperature (HT, 35 °C) stress mitigated by exogenous glutathione (GSH), cucumber (Cucumis sativus L.) seedlings were exposed to HT with or without GSH treatment for 4 days and following with 4 days of recovery. Plant physiological variables, growth, and gene expression related to antioxidant enzymes and Calvin cycle were quantified. The results showed that HT significantly decreased GSH content, the ratio of reduced to oxidized glutathione (GSH/GSSG), chlorophyll content, photosynthesis and related gene expression, shoot height, stem diameter, as well as dry weight. The exogenous GSH treatment clearly lessened the HT stress by increasing the above variables. Meanwhile, HT significantly increased soluble protein content, proline and malondialdehyde (MDA) content as well as O2•− production rate, the gene expression and activities of antioxidant enzymes. The GSH treatment remarkably improved soluble protein content, proline content, antioxidant enzymes activities, and antioxidant enzymes related gene expression, and reduced the MDA content and O2•− production rate compared to no GSH treatment in the HT condition. Our results suggest that exogenous GSH enhances cucumber seedling tolerance of HT stress by modulating the photosynthesis, antioxidant and osmolytes systems to improve physiological adaptation. PMID:27752105

Glutathione S-transferases as antioxidant defence agents confer pyrethroid resistance in Nilaparvata lugens.

PubMed Central

Vontas, J G; Small, G J; Hemingway, J

2001-01-01

Selection of a laboratory colony of the brown planthopper Nilaparvata lugens with the pyrethroids permethrin and lambda-cyhalothrin increased its resistance to both insecticides. Biochemical analysis and synergistic studies with metabolic inhibitors indicated that elevated glutathione S-transferases (GSTs) with a predominant peroxidase activity conferred resistance to both pyrethroids, whereas esterases conferred part of the resistance to permethrin. Purified esterases hydrolysed permethrin at a slow rate, but incubation of either pyrethroid or their primary metabolites with partially purified GSTs had no effect on the metabolic profile. Although GSTs were sensitive to inhibition by both pyrethroids, they did not serve as binding proteins, as previously hypothesized [Grant and Matsumura (1988) Insect Biochem. 18, 615-622]. We demonstrate that pyrethroids, in addition to their neurotoxic effect, induce oxidative stress and lipid peroxidation in insects. Pyrethroid exposure induced lipid peroxides, protein oxidation and depleted reduced glutathione. Elevated GSTs in the resistant strains attenuated the pyrethroid-induced lipid peroxidation and reduced mortality, whereas their in vivo inhibition eliminated their protective role. We therefore hypothesize that the main role of elevated GSTs in conferring resistance in N. lugens is through protecting tissues from oxidative damage. Our study extends the GSTs' range of efficacy to pyrethroid insecticides and possibly explains the role of elevated GSTs in other pyrethroid-resistant insects. PMID:11415437

Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells.

PubMed

Jones, Jane T; Qian, Xi; van der Velden, Jos L J; Chia, Shi Biao; McMillan, David H; Flemer, Stevenson; Hoffman, Sidra M; Lahue, Karolyn G; Schneider, Robert W; Nolin, James D; Anathy, Vikas; van der Vliet, Albert; Townsend, Danyelle M; Tew, Kenneth D; Janssen-Heininger, Yvonne M W

2016-08-01

Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKβ), among other NF-κB proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKβ and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKβ. SiRNA-mediated knockdown of GSTP decreased IKKβ-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKβ-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor. Copyright �

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

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

Pleiotropic Functions of Glutathione S-Transferase P

PubMed Central

Zhang, Jie; Grek, Christina; Ye, Zhi-Wei; Manevich, Yefim; Tew, Kenneth D.; Townsend, Danyelle M.

2016-01-01

Glutathione S-transferase P (GSTP) is one member of the GST superfamily that is prevalently expressed in mammals. Known to possess catalytic activity through deprotonating glutathione allowing formation of thioether bonds with electrophilic substrates, more recent discoveries have broadened our understanding of the biological roles of this protein. In addition to catalytic detoxification, other properties so far ascribed to GSTP include chaperone functions, regulation of nitric oxide pathways, regulation of a variety of kinase signaling pathways, and participation in the forward reaction of protein S-glutathionylation. The expression of GSTP has been linked with cancer and other human pathologies and more recently even with drug addiction. With respect to human health, polymorphic variants of GSTP may determine individual susceptibility to oxidative stress and/or be critical in the design and development of drugs that have used redox pathways as a discovery platform. PMID:24974181

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.

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.

Glutathione determination by the Tietze enzymatic recycling assay and its relationship to cellular radiation response.

PubMed Central

Eady, J. J.; Orta, T.; Dennis, M. F.; Stratford, M. R.; Peacock, J. H.

1995-01-01

Large fluctuations in glutathione content were observed on a daily basis using the Tietze enzyme recycling assay in a panel of six human cell lines of varying radiosensitivity. Glutathione content tended to increase to a maximum during exponential cell proliferation, and then decreased at different rates as the cells approached plateau phase. By reference to high-performance liquid chromatography and flow cytometry of the fluorescent bimane derivative we were able to verify that these changes were real. However, the Tietze assay was occasionally unable to detect glutathione in two of our cell lines (MGH-U1 and AT5BIVA), although the other methods indicated its presence. The existence of an inhibitory activity responsible for these anomalies was confirmed through spiking our samples with known amounts of glutathione. We were unable to detect a direct relationship between cellular glutathione concentration and aerobic radiosensitivity in our panel of cell lines. PMID:7577452

In vivo toxicity, biodistribution, and clearance of glutathione-coated gold nanoparticles.

PubMed

Simpson, Carrie A; Salleng, Kenneth J; Cliffel, David E; Feldheim, Daniel L

2013-02-01

Gold nanoparticles are emerging as promising materials from which to construct nanoscale therapeutics and therapeutic delivery systems. However, animal studies have shown that gold nanoparticles modified with certain thiol monolayers such as tiopronin can cause renal complications and morbidity. Although these effects may be eliminated by coadsorbing small amounts of polyethylene glycol (PEG) onto the nanoparticle surface, PEG can also lower cellular internalization efficiency and binding interactions with protein disease targets, significantly reducing the potential for using gold nanoparticles as therapeutics. Using ICP-MS analysis of blood, urine, and several organs, we show in this article that glutathione-coated gold nanoparticles (1.2 nm ± 0.9 nm) cause no morbidity at any concentration up to and including 60 μM and target primary organs although providing gradual dissipation and clearance over time. This study suggests that glutathione may be an attractive alternative to PEG in the design of gold nanoparticle therapeutics. This study describes the utility and toxicity of glutathione coated gold nanoparticles in comparison to PEGylated counterparts that are commonly used to increase "Stealth" properties and lower cytotoxicity. Too much PEG on the NPs can lead to lower cellular internalization efficiency and less efficient binding interactions with protein disease targets, significantly reducing the potential for using gold nanoparticles as therapeutics. Copyright © 2013 Elsevier Inc. All rights reserved.

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

Revisiting the thiosemicarbazonecopper(II) reaction with glutathione. Activity against colorectal carcinoma cell lines.

PubMed

García-Tojal, Javier; Gil-García, Rubén; Fouz, Víctor Ivo; Madariaga, Gotzon; Lezama, Luis; Galletero, María S; Borrás, Joaquín; Nollmann, Friederike I; García-Girón, Carlos; Alcaraz, Raquel; Cavia-Saiz, Mónica; Muñiz, Pilar; Palacios, Òscar; Samper, Katia G; Rojo, Teófilo

2018-03-01

Thiosemicarbazones (TSCs), and their copper derivatives, have been extensively studied mainly due to the potential applications as antitumor compounds. A part of the biological activity of the TSC-Cu II complexes rests on their reactivity against cell reductants, as glutathione (GSH). The present paper describes the structure of the [Cu(PTSC)(ONO 2 )] n compound (1) (HPTSC=pyridine-2-carbaldehyde thiosemicarbazone) and its spectroscopic and magnetic properties. ESI studies performed on the reaction of GSH with 1 and the analogous [{Cu(PTSC*)(ONO 2 )} 2 ] derivative (2, HPTSC*=pyridine-2-carbaldehyde 4N-methylthiosemicarbazone) show the absence of peaks related with TSC-Cu-GSH species. However GSH-Cu ones are detected, in good agreement with the release of Cu I ions after reduction in the experimental conditions. The reactivity of 1 and 2 with cytochrome c and myoglobin and their activities against HT-29 and SW-480 colon carcinoma cell lines are compared with those shown by the free HPTSC and HPTSC* ligands. Copyright © 2017 Elsevier Inc. All rights reserved.

Variants of glutathione s-transferase pi 1 exhibit differential enzymatic activity and inhibition by heavy metals.

PubMed

Goodrich, Jaclyn M; Basu, Niladri

2012-06-01

Nonsynonymous single nucleotide polymorphisms in glutathione s-transferase pi 1 (GSTP1; Ile/Val 105, Ala/Val 114) have been associated with altered toxicant metabolism in epidemiological cohorts. We explored the impact of GSTP1 genotype on enzyme kinetics and heavy metal inhibition in vitro. Four GSTP1 allozymes (105/114: Ile/Ala, Val/Ala, Ile/Val, Val/Val) were expressed in and purified from Escherichia coli. Enzyme activity assays quantifying the rate of glutathione conjugation with 1-chloro-2,4-dinitrobenzene (CDNB) revealed significant differences in kinetic parameters depending on genotype (p<0.01). Allozymes with Ile105 had better catalytic efficiency and greater affinity for CDNB (mean ± SEM: Ile105 Ala114 K(m)=0.33 ± 0.07 mM vs. Val105 Ala114 K(m)=1.15 ± 0.07 mM). Inhibition of GSTP1 activity by heavy metals was assessed following treatment with mercury (inorganic-HgCl(2), methylmercury-MeHg), selenium, cadmium, lead, arsenic, and manganese. All allozymes were inhibited by HgCl(2) (IC(50) range: 24.1-172 μM), MeHg (93.9-480 μM), and selenium (43.7-62.8 μM). Genotype significantly influenced the potency of mercury with GSTP1 Ile105 Val114 the least sensitive and Val105 Ala114 the most sensitive to inhibition by HgCl(2) and MeHg. Overall, genotype of two nonsynonymous polymorphisms in GSTP1 influenced enzyme kinetics pertaining to an electrophilic substrate and inhibition by two mercury species. Published by Elsevier Ltd.

Variants of glutathione s-transferase pi 1 exhibit differential enzymatic activity and inhibition by heavy metals

PubMed Central

Goodrich, Jaclyn M.; Basu, Niladri

2012-01-01

Nonsynonymous single nucleotide polymorphisms in glutathione s-transferase pi 1 (GSTP1; Ile/Val 105, Ala/Val 114) have been associated with altered toxicant metabolism in epidemiological cohorts. We explored the impact of GSTP1 genotype on enzyme kinetics and heavy metal inhibition in vitro. Four GSTP1 allozymes (105/114: Ile/Ala, Val/Ala, Ile/Val, Val/Val) were expressed in and purified from E. coli. Enzyme activity assays quantifying the rate of glutathione conjugation with 1-chloro-2,4-dinitrobenzene (CDNB) revealed significant differences in kinetic parameters depending on genotype (p<0.01). Allozymes with Ile105 had better catalytic efficiency and greater affinity for CDNB (mean ±SEM: Ile105 Ala114 Km= 0.33±0.07 mM vs. Val105 Ala114 Km=1.15±0.07 mM). Inhibition of GSTP1 activity by heavy metals was assessed following treatment with mercury (inorganic- HgCl2, methylmercury- MeHg), selenium, cadmium, lead, arsenic, and manganese. All allozymes were inhibited by HgCl2 (IC50 range: 24.1–172 μM), MeHg (93.9–480 μM), and selenium (43.7–62.8 μM). Genotype significantly influenced the potency of mercury with GSTP1 Ile105 Val114 the least sensitive and Val105 Ala114 the most sensitive to inhibition by HgCl2 and MeHg. Overall, genotype of two nonsynonymous polymorphisms in GSTP1 influenced enzyme kinetics pertaining to an electrophilic substrate and inhibition by two mercury species. PMID:22401947

Novel amphiphilic PEG-hydroxycamptothecin conjugates as glutathione-responsive prodrug nanocapsules for cancer chemotherapy

NASA Astrophysics Data System (ADS)

Guo, Na; Hao, Tiantian; Shang, Xiuzhuan; Zhang, Tianle; Liu, Huan; Zhang, Qian; Wang, Jing; Jiang, Du; Rong, Yao; Teng, Yuou; Yu, Peng

2017-06-01

A series of novel hydroxycamptothecin (HCPT) conjugates ( 13a-14d), which contained a polyethylene glycol moiety and disulfide bond, were designed and synthesized in five to six steps, with overall yields of 20-39%. The anticancer activities and toxicities of these new conjugates were evaluated using an in vitro MTT assay in K562, HepG2, and HT-29 cell lines and HUVECs. The conjugates displayed enhanced antitumor activity and reduced toxicity in comparison with their parent molecule, HCPT. Among these conjugates, compound 13a exhibited 100-fold better selectivity to the tumor cells than to HUVECs. TEM and DLS experiments demonstrated that 13a formed nanosized micelles with a diameter of approximately 200 nm in aqueous solution and that the conjugate could undergo glutathione-responsive degradation to release HCPT at the tumor site. The improved potency and reduced toxicity of these conjugates may be caused by the enhanced permeation and retention (EPR) effect of nanoparticles.

Nrf2-mediated antioxidant response by ethanolic extract of Sida cordifolia provides protection against alcohol-induced oxidative stress in liver by upregulation of glutathione metabolism.

PubMed

Rejitha, S; Prathibha, P; Indira, M

2015-03-01

Objective The study aimed to evaluate the antioxidant property of ethanolic extract of Sida cordifolia (SAE) on alcohol-induced oxidative stress and to elucidate its mechanism of action. Methods Male albino rats of the Sprague-Dawley strain were grouped into four: (1) control, (2) alcohol (4 g/kg body weight), (3) SAE (50 mg/100 g body weight), and (4) alcohol (4 g/kg body weight) + SAE (50 mg/100 g body weight). Alcohol and SAE were given orally each day by gastric intubation. The duration of treatment was 90 days. Results The activities of toxicity markers in liver and serum increased significantly in alcohol-treated rats and to a lesser extent in the group administered SAE + alcohol. The activity of alcohol dehydrogenase and the reactive oxygen species level were increased significantly in alcohol-treated rats but attenuated in the SAE co-administered group. Oxidative stress was increased in alcohol-treated rats as evidenced by the lowered activities of antioxidant enzymes, decreased level of reduced glutathione (GSH), increased lipid peroxidation products, and decreased expression of γ-glutamyl cysteine synthase in liver. The co-administration of SAE with alcohol almost reversed these changes. The activity of glutathione-S-transferase and translocation of Nrf2 from cytosol to nucleus in the liver was increased in both the alcohol and alcohol + SAE groups, but the maximum changes were observed in the latter group. Discussion The SAE most likely elicits its antioxidant potential by reducing oxidative stress, enhancing the translocation of Nrf2 to nucleus and thereby regulating glutathione metabolism, leading to enhanced GSH content.

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.

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

Reactive oxygen species scavenger N-acetyl cysteine reduces methamphetamine-induced hyperthermia without affecting motor activity in mice

PubMed Central

Sanchez-Alavez, Manuel; Bortell, Nikki; Galmozzi, Andrea; Conti, Bruno; Marcondes, Maria Cecilia G.

2014-01-01

Hyperthermia is a potentially lethal side effect of Methamphetamine (Meth) abuse, which involves the participation of peripheral thermogenic sites such as the Brown Adipose Tissue (BAT). In a previous study we found that the anti-oxidant N-acetyl cysteine (NAC) can prevent the high increase in temperature in a mouse model of Meth-hyperthermia. Here, we have further explored the ability of NAC to modulate Meth-induced hyperthermia in correlation with changes in BAT. We found that NAC treatment in controls causes hypothermia, and, when administered prior or upon the onset of Meth-induced hyperthermia, can ameliorate the temperature increase and preserve mitochondrial numbers and integrity, without affecting locomotor activity. This was different from Dantrolene, which decreased motor activity without affecting temperature. The effects of NAC were seen in spite of its inability to recover the decrease of mitochondrial superoxide induced in BAT by Meth. In addition, NAC did not prevent the Meth-induced decrease of BAT glutathione. Treatment with S-adenosyl-L-methionine, which improves glutathione activity, had an effect in ameliorating Meth-induced hyperthermia, but also modulated motor activity. This suggests a role for the remaining glutathione for controlling temperature. However, the mechanism by which NAC operates is independent of glutathione levels in BAT and specific to temperature. Our results show that, in spite of the absence of a clear mechanism of action, NAC is a pharmacological tool to examine the dissociation between Meth-induced hyperthermia and motor activity, and a drug of potential utility in treating the hyperthermia associated with Meth-abuse. PMID:26346736

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.

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

Signaling molecule methylglyoxal ameliorates cadmium injury in wheat (Triticum aestivum L) by a coordinated induction of glutathione pool and glyoxalase system.

PubMed

Li, Zhong-Guang; Nie, Qian; Yang, Cong-Li; Wang, Yue; Zhou, Zhi-Hao

2018-03-01

Methylglyoxal (MG) now is found to be an emerging signaling molecule. It can relieve the toxicity of cadmium (Cd), however its alleviating mechanism still remains unknown. In this study, compared with the Cd-stressed seedlings without MG treatment, MG treatment could stimulate the activities of glutathione reductase (GR) and gamma-glutamylcysteine synthetase (γ-ECS) in Cd-stressed wheat seedlings, which in turn induced an increase of reduced glutathione (GSH). Adversely, the activated enzymes related to GSH biosynthesis and increased GSH were weakened by N-acetyl-L-cysteine (NAC, MG scavenger), 2,4-dihydroxy-benzylamine (DHBA) and 1,3-bischloroethyl-nitrosourea (BCNU, both are specific inhibitors of GR), buthionine sulfoximine (BSO, a specific inhibitors of GSH biosynthesis), and N-ethylmaleimide (NEM, GSH scavenger), respectively. In addition, MG increased the activities of glyoxalase I (Gly I) and glyoxalase II (Gly II) in Cd-treated seedlings, followed by declining an increase in endogenous MG as comparision to Cd-stressed seedlings alone. On the contrary, the increased glyoxalase activity and decreased endogenous MG level were reversed by NAC and specific inhibitors of Gly I (isoascorbate, IAS; squaric acid, SA). Furthermore, MG alleviated an increase in hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) in Cd-treated wheat seedlings. These results indicated that MG could alleviate Cd toxicity and improve the growth of Cd-stressed wheat seedlings by a coordinated induction of glutathione pool and glyoxalase system. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Purification and Thermal Dependence of Glutathione Reductase from Two Forage Legume Species 1

PubMed Central

Kidambi, Saranga P.; Mahan, James R.; Matches, Arthur G.

1990-01-01

Alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.) are forage legumes that differ in their responses to high and low temperature stresses. Thermal limitations on the function of glutathione reductase (EC 1.6.4.2) could adversely affect the ability of the plant to cope with adverse temperatures. Our objectives were to (a) purify glutathione reductase from `Cimarron' alfalfa and `PI 212241' sainfoin and (b) investigate the intraspecies variation in the thermal dependency of glutathione reductase from each of three cultivars of alfalfa and two cultivars and an introduction of sainfoin. Glutathione reductase was purified 1222-and 1948-fold to a specific activity of 281 and 273 units per milligram of protein, from one species each of alfalfa and sainfoin, respectively. The relative molecular mass of the protein was approximately 140 kilodaltons with subunits of 57 and 37 kilodaltons under denaturing conditions. The activation energies were approximately 50 kilojoules per mole for both species. Over a 5 to 45°C temperature gradient, large variation among species and genotypes within species was found for: (a) the minimum apparent Michaelis constant (0.6-2.1 micromoles of NADPH), (b) the temperature at which the minimum apparent Michaelis constant was observed (10-25°C), and (c) the thermal kinetic windows (6-19°C width). Future studies will focus on relating the thermal dependence of the Michaelis constant of the glutathione reductases and plant growth rates and forage quality of these species throughout the growing season. PMID:16667283

Proteomics analysis of human placenta reveals glutathione metabolism dysfunction as the underlying pathogenesis for preeclampsia.

PubMed

Jin, Xiaohan; Xu, Zhongwei; Cao, Jin; Shao, Ping; Zhou, Maobin; Qin, Zhe; Liu, Yan; Yu, Fang; Zhou, Xin; Ji, Wenjie; Cai, Wei; Ma, Yongqiang; Wang, Chengyan; Shan, Nana; Yang, Ning; Chen, Xu; Li, Yuming

2017-09-01

Hypertensive disorder in pregnancy (HDP) refers to a series of diseases that cause the hypertension during pregnancy, including HDP, preeclampsia (PE) and eclampsia. This study screens differentially expressed proteins of placenta tissues in PE cases using 2D LC-MS/MS quantitative proteomics strategy. A total of 2281 proteins are quantified, of these, 145 altering expression proteins are successfully screened between PE and control cases (p<0.05). Bioinformatics analysis suggests that these proteins are mainly involved in many biological processes, such as oxidation reduction, mitochondrion organization, and acute inflammatory response. Especially, the glutamine metabolic process related molecules, GPX1, GPX3, SMS, GGCT, GSTK1, NFκB, GSTT2, SOD1 and GCLM, are involved in the switching process from oxidized glutathione (GSSG) conversion to the reduced glutathione (GSH) by glutathione, mercapturic acid and arginine metabolism process. Results of this study revealed that glutathione metabolism disorder of placenta tissues may contribute to the occurrence of PE disease. Copyright © 2017. Published by Elsevier B.V.

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...

Auranofin-induced oxidative stress causes redistribution of the glutathione pool in Taenia crassiceps cysticerci.

PubMed

Martínez-González, J J; Guevara-Flores, A; Rendón, J L; Arenal, I P Del

2015-05-01

Previously, we have studied the effect of the gold-compound auranofin (AF) on both thioredoxin-glutathione reductasa (TGR) activity and viability of Taenia crassiceps cysticerci. It was demonstrated that micromolar concentrations of AF were high enough to fully inhibit TGR and kill the parasites. In this work, the dynamics of changes in the glutathione pool of T. crassiceps cysticerci following the addition of AF, was analyzed. A dose-dependent decrease in the internal glutathione concentration, concomitant with an increase in ROS production was observed. These changes were simultaneous with the formation of glutathione-protein complexes and the export of glutathione disulfide (GSSG) to the culture medium. Incubation of cysticerci in the presence of both AF and N-acetyl cysteine (NAC) prevents all the above changes, maintaining cysticerci viability. By contrast, the presence of both AF and buthionine sulfoximine (BSO) resulted in a potentiation of the effects of the gold compound, jeopardizing cysticerci viability. These results suggest the lethal effect of AF on T. crassiceps cysticerci, observed at micromolar concentrations, can be explained as a consequence of major changes in the glutathione status, which results in a significant increase in the oxidative stress of the parasites. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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

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

Glutathione transferases P1/P2 regulate the timing of signaling pathway activations and cell cycle progression during mouse liver regeneration

PubMed Central

Pajaud, J; Ribault, C; Ben Mosbah, I; Rauch, C; Henderson, C; Bellaud, P; Aninat, C; Loyer, P; Morel, F; Corlu, A

2015-01-01

Glutathione transferases (GST) are phase II enzymes catalyzing the detoxification of endogenous noxious compounds and xenobiotics. They also regulate phosphorylation activities of MAPKinases in a catalytic-independent manner. Previous studies have demonstrated the regulation of JNK-dependent pathway by GSTP1/2. Considering the crucial role of JNK in the early steps of the hepatocyte cell cycle, we sought to determine whether GSTP1/2 were essential for hepatocyte proliferation following partial hepatectomy (PH). Using a conventional double knockout mouse model for the Gstp1 and Gstp2 genes, we found that the lack of GSTP1/P2 reduced the rate of DNA replication and mitotic index during the first wave of hepatocyte proliferation. The lowered proliferation was associated with the decrease in TNFalpha and IL-6 plasma concentrations, reduced hepatic HGF expression and delayed and/or altered activation of STAT3, JNK and ERK1/2 signaling pathways. In addition, the expression and/or activation of cell cycle regulators such as Cyclin D1, CDK4, E2F1 and MCM7 was postponed demonstrating that the absence of GSTP1/2 delayed the entry into and progression through the G1 phase of the cell cycle and impaired the synchrony of proliferation in hepatocytes following PH. Furthermore, while JNK and its downstream targets c-Jun and ATF2 were activated during the early steps of the liver regeneration in wild-type animals, the constitutively active JNK found in the quiescent liver of Gstp1/2 knockout mice underwent a decrease in its activity after PH. Transient induction of antioxidant enzymes and nitric oxide synthase were also delayed or repressed during the regenerative response. Altogether our results demonstrate that GSTP1/2 are a critical regulators of hepatocyte proliferation in the initial phases of liver regeneration. PMID:25590808

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

PubMed

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

2009-01-01

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

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

PubMed Central

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

2017-01-01

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

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

[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.

Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation

PubMed Central

Begara-Morales, Juan C.; Sánchez-Calvo, Beatriz; Chaki, Mounira; Mata-Pérez, Capilla; Valderrama, Raquel; Padilla, María N.; Luque, Francisco; Corpas, Francisco J.; Barroso, Juan B.

2015-01-01

The ascorbate–glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO– and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO–. The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO–. These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO– or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate–glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement

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

Chlortetracycline detoxification in maize via induction of glutathione S-transferases after antibiotic exposure.

PubMed

Farkas, Michael H; Berry, James O; Aga, Diana S

2007-02-15

Soil contamination with nonmetabolized antibiotics is an emerging environmental concern, especially on agricultural croplands that receive animal manure as fertilizer. In this study, phytotoxicity of chlortetracycline (CTC) antibiotics on pinto beans (Phaseolus vulgaris) and maize (Zea mays) was investigated under controlled conditions. When grown in CTC-treated soil, a significant increase in the activities of the plant stress proteins glutathione S-transferases (GST) and peroxidases (POX) were observed in maize plants, but not in pinto beans. In vitro conjugation reactions demonstrated that the induced GST in maize catalyzed the conjugation of glutathione (GSH) with CTC, producing stable conjugates that were structurally characterized using liquid chromatography/mass spectrometry. The antibiotic-induced GST produced CTC-glutathione conjugate at relative concentrations 2-fold higher than that produced by constitutively expressed GST extracted from untreated maize. On the other hand, GST extracted from pinto beans (both treated and untreated) did not efficiently catalyze glutathione conjugation with CTC. These results suggest that maize is able to detoxify chlortetracycline via the glutathione pathway, whereas pinto beans cannot. This may explain the observed stunted growth of pinto beans after antibiotic treatment. This study demonstrates the importance of plant uptake in determining the fate of antibiotics in soil and their potential phytotoxicity to susceptible plants.

[The selenium haemostasis during experimental anaphylaxis reaction in rats treated with reduced glutathione and selenium enriched spirulina].

PubMed

Golubkina, N A; Mazo, V K; Gmoshinskiĭ, I V; Zorin, S N; Tambiev, A Kh; Kirikova, N N

2000-01-01

The main events caused by anaphilaxis in selenium haemostasis in rats include significant increase of selenium excretion with urine (6.36 +/- 1.18 nM Se/18 h., n = 10, compared with 1.72 +/- 0.38 nM Se/18 h., n = 10) and decrease of selenium plasma/selenium erythrocytes ratio from 0.939 to 0.791. Reduced glutathione (G-SH) administration led to 1.5-fold decrease of plasma selenium level and 1.3-fold increase of selenium concentration in intestinal walls of sensitized rats (r = -0.720, P < 0.001). Chromatographic separation of plasma proteins showed that intragastric intubation of G-SH to sensibilized rats significantly decreased the protein P content and did not influence the concentration of Se-GSHPx, thus indicating the local selenium acceptor role of G-SH. G-SH administration did not influence the intestinal permeability in sensitised rats while use of complex additive: G-SH and selenium enriched spirulina--normalized the latter parameter and the ratio of protein P/Se-GSHPx in plasma.

The effect of intermittent hypobaric-hypoxia treatments on renal glutathione peroxidase activity of rats

NASA Astrophysics Data System (ADS)

Paramita, I. A.; Jusman, S. W. A.

2017-08-01

Many people living at high altitudes experiencing a condition called intermittent hypobaric hypoxia (IHH). Some people even create IHH condition as an exercise for pilots, athletes, and mountaineers. In this experiment, we aimed to determine whether the protective effect of IHH is mediated through glutathione peroxidase (GPX) enzyme. The experiment’s sample is two-month-old healthy Sprague-Dawley rat kidneys weighing 200-250 g. Intermittent hypobaric hypoxia treatment is done using a Hypobaric Chamber type I that can mimic air pressure at certain altitudes: 35,000 (one minute), 30,000 (three minutes), 25,000 (five minutes), and 18,000 (30 minutes) feet. The rats were divided into five treatment groups, including a control group, hypobaric-hypoxia group, and intermittent hypobaric-hypoxia 1x, 2x, and 3x groups with each group consisting of three rats. The specific activity of GPX was measured using RANDOX and RANSEL methods. The statistical analysis of one way-ANOVA did not show significant differences between the groups (p > 0.05), although specific activities of the renal GPX of rats exposed to hypobaric-hypoxia were higher than the control group. This may be caused by the other antioxidants’ activities. In conclusion, the IHH treatment did not affect GPX activity in the rat kidneys.

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

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.

Nuclear glutathione S-transferase pi prevents apoptosis by reducing the oxidative stress-induced formation of exocyclic DNA products.

PubMed

Kamada, Kensaku; Goto, Shinji; Okunaga, Tomohiro; Ihara, Yoshito; Tsuji, Kentaro; Kawai, Yoshichika; Uchida, Koji; Osawa, Toshihiko; Matsuo, Takayuki; Nagata, Izumi; Kondo, Takahito

2004-12-01

We previously found that nuclear glutathione S-transferase pi (GSTpi) accumulates in cancer cells resistant to anticancer drugs, suggesting that it has a role in the acquisition of resistance to anticancer drugs. In the present study, the effect of oxidative stress on the nuclear translocation of GSTpi and its role in the protection of DNA from damage were investigated. In human colonic cancer HCT8 cells, the hydrogen peroxide (H(2)O(2))-induced increase in nuclear condensation, the population of sub-G(1) peak, and the number of TUNEL-positive cells were observed in cells pretreated with edible mushroom lectin, an inhibitor of the nuclear transport of GSTpi. The DNA damage and the formation of lipid peroxide were dependent on the dose of H(2)O(2) and the incubation time. Immunological analysis showed that H(2)O(2) induced the nuclear accumulation of GSTpi but not of glutathione peroxidase. Formation of the 7-(2-oxo-hepyl)-substituted 1,N(2)-etheno-2'-deoxyguanosine adduct by the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with 2'-deoxyguanosine was inhibited by GSTpi in the presence of glutathione. The conjugation product of 4-oxo-2-nonenal, a lipid aldehyde of 13-HPODE, with GSH in the presence of GSTpi, was identified by LS/MS. These results suggested that nuclear GSTpi prevents H(2)O(2)-induced DNA damage by scavenging the formation of lipid-peroxide-modified DNA.

Glutathione-supported arsenate reduction coupled to arsenolysis catalyzed by ornithine carbamoyl transferase

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

Nemeti, Balazs; Gregus, Zoltan

2009-09-01

Three cytosolic phosphorolytic/arsenolytic enzymes, (purine nucleoside phosphorylase [PNP], glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase) have been shown to mediate reduction of arsenate (AsV) to the more toxic arsenite (AsIII) in a thiol-dependent manner. With unknown mechanism, hepatic mitochondria also reduce AsV. Mitochondria possess ornithine carbamoyl transferase (OCT), which catalyzes phosphorolytic or arsenolytic citrulline cleavage; therefore, we examined if mitochondrial OCT facilitated AsV reduction in presence of glutathione. Isolated rat liver mitochondria were incubated with AsV, and AsIII formed was quantified. Glutathione-supplemented permeabilized or solubilized mitochondria reduced AsV. Citrulline (substrate for OCT-catalyzed arsenolysis) increased AsV reduction. The citrulline-stimulated AsV reduction was abolished bymore » ornithine (OCT substrate inhibiting citrulline cleavage), phosphate (OCT substrate competing with AsV), and the OCT inhibitor norvaline or PALO, indicating that AsV reduction is coupled to OCT-catalyzed arsenolysis of citrulline. Corroborating this conclusion, purified bacterial OCT mediated AsV reduction in presence of citrulline and glutathione with similar responsiveness to these agents. In contrast, AsIII formation by intact mitochondria was unaffected by PALO and slightly stimulated by citrulline, ornithine, and norvaline, suggesting minimal role for OCT in AsV reduction in intact mitochondria. In addition to OCT, mitochondrial PNP can also mediate AsIII formation; however, its role in AsV reduction appears severely limited by purine nucleoside supply. Collectively, mitochondrial and bacterial OCT promote glutathione-dependent AsV reduction with coupled arsenolysis of citrulline, supporting the hypothesis that AsV reduction is mediated by phosphorolytic/arsenolytic enzymes. Nevertheless, because citrulline cleavage is disfavored physiologically, OCT may have little role in AsV reduction in vivo.« less

Evaluation of the antioxidant properties of N-acetylcysteine in human platelets: prerequisite for bioconversion to glutathione for antioxidant and antiplatelet activity.

PubMed

Gibson, Kyle R; Neilson, Ilene L; Barrett, Fiona; Winterburn, Tim J; Sharma, Sushma; MacRury, Sandra M; Megson, Ian L

2009-10-01

N-Acetylcysteine (NAC) is a frequently used "antioxidant" in vitro, but the concentrations applied rarely correlate with those encountered with oral dosing in vivo. Here, we investigated the in vitro antioxidant and antiplatelet properties of NAC at concentrations (10-100 microM) that are achievable in plasma with tolerable oral dosing. The impact of NAC pretreatment (2 hours) on aggregation of platelets from healthy volunteers in response to thrombin and adenosine diphosphate and on platelet-derived nitric oxide (NO) was examined. NAC was found to be a weak reducing agent and a poor antioxidant compared with glutathione (reduced form) (GSH). However, platelets treated with NAC showed enhanced antioxidant activity and depression of reactive oxygen species generation associated with increases in intraplatelet GSH levels. An approximately 2-fold increase in NO synthase-derived nitrite was observed with 10 microM NAC treatment, but the effect was not concentration dependent. Finally, NAC significantly reduced both thrombin-induced and adenosine diphosphate-induced platelet aggregation. NAC should be considered a weak antioxidant that requires prior conversion to GSH to convey antioxidant and antithrombotic benefit at therapeutically relevant concentrations. Our results suggest that NAC might be an effective antiplatelet agent in conditions where increased oxidative stress contributes to heightened risk of thrombosis but only if the intraplatelet machinery to convert it to GSH is functional.

Development and characterization of glutathione-conjugated albumin nanoparticles for improved brain delivery of hydrophilic fluorescent marker.

PubMed

Patel, Prerak J; Acharya, Niyati S; Acharya, Sanjeev R

2013-01-01

The glutathione-conjugated bovine serum albumin (BSA) nanoparticles were constructed in the present exploration as a novel biodegradable carrier for brain-specific drug delivery with evaluation of its in vitro and in vivo delivery properties. BSA nanocarriers were activated and conjugated to the distal amine functions of the glutathione via carbodiimide chemistry using EDAC as a mediator. These nanoparticles were characterized for particle shape, average size, SPAN value, drug entrapment and in vitro drug release. Further, presence of glutathione on the surface of BSA nanoparticles was confirmed by Ellman's assay, which has suggested that approximately 750 units of glutathione were conjugated per BSA nanoparticle. To evaluate the brain delivery properties of the glutathione-conjugated BSA nanoparticles fluorescein sodium was used as a model hydrophilic compound. Permeability and neuronal uptake properties of developed formulations were evaluated against the MDCK-MDR1 endothelial and neuro-glial cells, respectively. The permeability of glutathione-conjugated BSA nanoparticles across the monolayer of MDCK-MDR1 endothelial tight junction was shown significantly higher than that of unconjugated nanoparticles and fluorescein sodium solution. Similarly, glutathione-conjugated nanoparticles exhibited considerably higher uptake by neuro-glial cells which was inferred by high fluorescence intensity under microscope in comparison to unconjugated nanoparticles and fluorescein sodium solution. Following an intravenous administration, nearly three folds higher fluorescein sodium was carried to the rat brain by glutathione-conjugated nanoparticles as compared to unconjugated nanoparticles. The significant in vitro and in vivo results suggest that glutathione-conjugated BSA nanoparticles is a promising brain drug delivery system with low toxicity.

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.

Glutathione homeostasis as an important and novel factor controlling blossom-end rot development in calcium-deficient tomato fruits.

PubMed

Mestre, Teresa C; Garcia-Sanchez, Francisco; Rubio, Francisco; Martinez, Vicente; Rivero, Rosa M

2012-11-15

Based on previous results in which oxidative metabolism was suggested as a possible inducer of blossom-end rot (BER), the main questions addressed here were whether calcium deficiency is the main factor that induces BER or whether this physiological disorder a general stress-related phenomenon? Tomato plants were grown under optimal or deficient calcium concentrations. Only the application of 0.1mM calcium resulted in BER induction, although only half of the fruits grown under this treatment had this disorder. Having fruits showing or not showing BER in the same plant and treatment provided us with a powerful tool that we used to investigate whether calcium deficiency operates alongside another mechanism in the induction of BER. Whether or not this other mechanism was the one controlling BER incidence was also investigated. We performed a complete study of the oxidative metabolism in the pericarp of healthy fruits and in the healthy portion of BER-affected fruits. Calcium deficiency led to an induction of NADPH oxidase, superoxide dismutase, dehydro- and monodehydroascorbate reductase, and to an inhibition of catalase, ascorbate peroxidase and glutathione reductase, with a concomitant accumulation of hydrogen peroxide and an increase in lipid peroxidation. While the ascorbate redox state was not affected by calcium deficiency, the glutathione redox state was markedly reduced. We conclude that calcium deficiency fundamentally affected the activity of the ascorbate-glutathione enzymes, with special importance to the inhibition of GR, which lead to a reduction of the glutathione redox state. This could cause the breakdown of cellular homeostasis, the inhibition of other enzymes responsible for H(2)O(2) detoxification, and ultimately an increase of lipid peroxidation. Therefore, BER is defined here as the visual symptom of a massive lipid peroxidation event caused by the breakdown of cellular glutathione homeostasis. Copyright © 2012 Elsevier GmbH. All rights reserved.

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

l-Arginine induces antioxidant response to prevent oxidative stress via stimulation of glutathione synthesis and activation of Nrf2 pathway.

PubMed

Liang, Mingcai; Wang, Zhengxuan; Li, Hui; Cai, Liang; Pan, Jianghao; He, Hongjuan; Wu, Qiong; Tang, Yinzhao; Ma, Jiapei; Yang, Lin

2018-05-01

Arginine is a conditionally essential amino acid. To elucidate the influence of l-arginine on the activation of endogenous antioxidant defence, male Wistar rats were orally administered daily with l-arginine at different levels of 25, 50, 100 mg/100 g body weight. After 7 and 14 days feeding, the antioxidative capacities and glutathione (GSH) contents in the plasma and in the liver were uniformly enhanced with the increasing consumption of l-arginine, whereas the oxidative stress was effectively suppressed by l-arginine treatment. After 14 days feeding, the mRNA levels and protein expressions of Keap1 and Cul3 were gradually reduced by increasing l-arginine intake, resulting that the nuclear factor Nrf2 was activated. Upon activation of Nrf2, the expressions of antioxidant responsive element (ARE)-dependent genes and proteins (GCLC, GCLM, GS, GR, GST, GPx, CAT, SOD, NQO1, HO-1) were up-regulated by l-arginine feeding, indicating an upward trend in antioxidant capacity uniformly with the increasing consumption of l-arginine. The present study demonstrates that the supplementation of l-arginine stimulates GSH synthesis and activates Nrf2 pathway, leading to the up-regulation of ARE-driven antioxidant expressions via Nrf2-Keap1 pathway. Results suggest the availability of l-arginine is a critical factor to suppress oxidative stress and induce an endogenous antioxidant response. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparative assay of glutathione S-transferase (GSTs) activity of excretory-secretory materials and somatic extract of Fasciola spp parasites.

PubMed

Alirahmi, Heshmatollah; Farahnak, Ali; Golmohamadi, Taghi; Esharghian, Mohammad Reza

2010-01-01

Fascioliasis is a worldwide parasitic disease in human and domestic animals. The causative agents of fascioliasis are Fasciola hepatica and Fasciola gigantica. In the recent years, fasciola resistance to drugs has been reported in the many of publications. Fasciola spp has detoxification system including GST enzyme which may be responsible for its resistance. Therefore , the aim of the study was to assay of GST enzyme activity in fasciola parasites. Fasciola gigantica and Fasciola hepatica helminths were collected from abattoir as a live and cultured in buffer media for 4 h at 37 °C. Excretory-Secretory products were collected and stored in -80◦C. F. gigantica and Fasciola hepatica were homogenized with homogenizing buffer in a glass homogenizer to prepare of somatic extract. Suspension was then centrifuged and supernatant was stored at -80°C. In order to assay the enzyme activity, excretory-secretory and somatic extracts in the form of cocktails (potassium phosphate buffer, reduced glutathione and 1-chloro-2,4-dinitrobenzene substrates) were prepared and their absorbance recorded for 5 minutes at 340 nm. The total and specific GST activity of F. gigantica somatic and ES products were obtained as 2916.00, 272.01 micromole/minute and 1.33, 1.70 micromole/minute/mg protein, respectively. Fasciola hepatica also showed 2705.00, 276.86 micromole/minute and 1.33, 1.52 micromole/minute/mg protein, respectively. These results are important for analysis of parasite survival / resistance to drugs which use for treatment of fascioliasis.

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

Selenium-Enriched Foods Are More Effective at Increasing Glutathione Peroxidase (GPx) Activity Compared with Selenomethionine: A Meta-Analysis

PubMed Central

Bermingham, Emma N.; Hesketh, John E.; Sinclair, Bruce R.; Koolaard, John P.; Roy, Nicole C.

2014-01-01

Selenium may play a beneficial role in multi-factorial illnesses with genetic and environmental linkages via epigenetic regulation in part via glutathione peroxidase (GPx) activity. A meta-analysis was undertaken to quantify the effects of dietary selenium supplementation on the activity of overall GPx activity in different tissues and animal species and to compare the effectiveness of different forms of dietary selenium. GPx activity response was affected by both the dose and form of selenium (p < 0.001). There were differences between tissues on the effects of selenium supplementation on GPx activity (p < 0.001); however, there was no evidence in the data of differences between animal species (p = 0.95). The interactions between dose and tissue, animal species and form were significant (p < 0.001). Tissues particularly sensitive to changes in selenium supply include red blood cells, kidney and muscle. The meta-analysis identified that for animal species selenium-enriched foods were more effective than selenomethionine at increasing GPx activity. PMID:25268836

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.

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...

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...

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...

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...

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...

Characterization of bifunctional L-glutathione synthetases from Actinobacillus pleuropneumoniae and Actinobacillus succinogenes for efficient glutathione biosynthesis.

PubMed

Yang, Jianhua; Li, Wei; Wang, Dezheng; Wu, Hui; Li, Zhimin; Ye, Qin

2016-07-01

Glutathione (GSH), an important bioactive substance, is widely applied in pharmaceutical and food industries. In this work, two bifunctional L-glutathione synthetases (GshF) from Actinobacillus pleuropneumoniae (GshFAp) and Actinobacillus succinogenes (GshFAs) were successfully expressed in Escherichia coli BL-21(DE3). Similar to the GshF from Streptococcus thermophilus (GshFSt), GshFAp and GshFAs can be applied for high titer GSH production because they are less sensitive to end-product inhibition (Ki values 33 and 43 mM, respectively). The active catalytic forms of GshFAs and GshFAp are dimers, consistent with those of GshFPm (GshF from Pasteurella multocida) and GshFSa (GshF from Streptococcus agalactiae), but are different from GshFSt (GshF from S. thermophilus) which is an active monomer. The analysis of the protein sequences and three dimensional structures of GshFs suggested that the binding sites of GshFs for substrates, L-cysteine, L-glutamate, γ-glutamylcysteine, adenosine-triphosphate, and glycine are highly conserved with only very few differences. With sufficient supply of the precursors, the recombinant strains BL-21(DE3)/pET28a-gshFas and BL-21(DE3)/pET28a-gshFap were able to produce 36.6 and 34.1 mM GSH, with the molar yield of 0.92 and 0.85 mol/mol, respectively, based on the added L-cysteine. The results showed that GshFAp and GshFAs are potentially good candidates for industrial GSH production.

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

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

The role of glutathione S-transferases in the detoxification of some organophosphorus insecticides in larvae and pupae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae).

PubMed

Kostaropoulos, I; Papadopoulos, A I; Metaxakis, A; Boukouvala, E; Papadopoulou-Mourkidou, E

2001-06-01

The correlation between the natural levels of glutathione S-transferase (GST) and the tolerance to the organophosphorus insecticides parathion-methyl and paraoxon-methyl, as well as the interaction of affinity-purified enzyme and the insecticides were investigated in order to collect further information on the role of the glutathione S-transferase system as a mechanism of defence against insecticides in insects. The studies were carried out on the larvae and pupae of the coleopteran Tenebrio molitor L, which exhibit varying natural levels of GST activity. Stage-dependent susceptibility of the insect against insecticides was observed during the first 24 h. However, 48 h after treatment, the KD50 value increased significantly due to the recovery of some individuals. Simultaneous injection of insecticide with compounds which inhibit GST activity in vitro caused an alteration in susceptibility of insects 24 or 48 h post-treatment, depending on stage and insecticide used. Inhibition studies combined with competitive fluorescence spectroscopy revealed that the insecticides probably bind to the active site of the enzyme, thus inhibiting its activity towards 1-chloro-2,4-dinitrobenzene in a competitive manner. High-performance liquid chromatography and gas chromatography revealed that T molitor GST catalyses the conjugation of the insecticides studied to a reduced form of glutathione (GSH). From the above experimental results, it is considered that GST offers a protection against the organophosphorus insecticides studied by active site binding and subsequent conjugation with GSH.

Glutathione redox potential in the mitochondrial intermembrane space is linked to the cytosol and impacts the Mia40 redox state

PubMed Central

Kojer, Kerstin; Bien, Melanie; Gangel, Heike; Morgan, Bruce; Dick, Tobias P; Riemer, Jan

2012-01-01

Glutathione is an important mediator and regulator of cellular redox processes. Detailed knowledge of local glutathione redox potential (EGSH) dynamics is critical to understand the network of redox processes and their influence on cellular function. Using dynamic oxidant recovery assays together with EGSH-specific fluorescent reporters, we investigate the glutathione pools of the cytosol, mitochondrial matrix and intermembrane space (IMS). We demonstrate that the glutathione pools of IMS and cytosol are dynamically interconnected via porins. In contrast, no appreciable communication was observed between the glutathione pools of the IMS and matrix. By modulating redox pathways in the cytosol and IMS, we find that the cytosolic glutathione reductase system is the major determinant of EGSH in the IMS, thus explaining a steady-state EGSH in the IMS which is similar to the cytosol. Moreover, we show that the local EGSH contributes to the partially reduced redox state of the IMS oxidoreductase Mia40 in vivo. Taken together, we provide a comprehensive mechanistic picture of the IMS redox milieu and define the redox influences on Mia40 in living cells. PMID:22705944

Hydrogen sulfide alleviates toxic effects of arsenate in pea seedlings through up-regulation of the ascorbate-glutathione cycle: Possible involvement of nitric oxide.

PubMed

Singh, Vijay Pratap; Singh, Samiksha; Kumar, Jitendra; Prasad, Sheo Mohan

2015-06-01

In plants, hydrogen sulfide (H2S) is an emerging novel signaling molecule that is involved in growth regulation and abiotic stress responses. However, little is known about its role in the regulation of arsenate (As(V)) toxicity. Therefore, hydroponic experiments were conducted to investigate whether sodium hydrosulfide (NaHS; a source of H2S) is involved in the regulation of As(V) toxicity in pea seedlings. Results showed that As(V) caused decreases in growth, photosynthesis (measured as chlorophyll fluorescence) and nitrogen content, which was accompanied by the accumulation of As. As(V) treatment also reduced the activities of cysteine desulfhydrase and nitrate reductase, and contents of H2S and nitric oxide (NO). However, addition of NaHS ameliorated As(V) toxicity in pea seedlings, which coincided with the increased contents of H2S and NO. The cysteine level was higher under As(V) treatment in comparison to all other treatments (As-free; NaHS; As(V)+NaHS). The content of reactive oxygen species (ROS) and damage to lipids, proteins and membranes increased by As(V) while NaHS alleviated these effects. Enzymes of the ascorbate-glutathione cycle (AsA-GSH cycle) showed inhibition of their activities following As(V) treatment while their activities were increased by application of NaHS. The redox status of ascorbate and glutathione was disturbed by As(V) as indicated by a steep decline in their reduced/oxidized ratios. However, simultaneous NaHS application restored the redox status of the ascorbate and glutathione pools. The results of this study demonstrated that H2S and NO might both be involved in reducing the accumulation of As and triggering up-regulation of the AsA-GSH cycle to counterbalance ROS-mediated damage to macromolecules. Furthermore, the results suggest a crucial role of H2S in plant priming, and in particular for pea seedlings in mitigating As(V) stress. Copyright © 2015 Elsevier GmbH. All rights reserved.

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

Effect of natural ageing and antioxidant inhibition on liver antioxidant enzymes, glutathione system, peroxidation, and oxygen consumption in Rana perezi.

PubMed

López-Torres, M; Pérez-Campo, R; Barja de Quiroga, G

1991-01-01

A study of the physiological role of oxygen free radicals in relation to the ageing process was performed using the liver of Rana perezi, an animal with a moderate rate of oxygen consumption and a life span substantially longer than that of laboratory rodents. Among the five different antioxidant enzymes only superoxide dismutase (SOD) showed an age-dependent decrease. Cytochrome oxidase (COX), glutathione status, in vivo and in vitro liver peroxidation, and metabolic rate did not vary as a function of age. Long-term (2.5 months) treatment with aminotriazole and diethyldithiocarbamate depleted catalase (CAT) activity and did not change both glutathione peroxidases (GPx), COX, reduced (GSH) and oxidized (GSSG) glutathione, or metabolic rate. This treatment resulted in great compensatory increases in SOD (to 250-460% of controls) and glutathione reductase (GR) (to 200%) which are possibly responsible for the lack of increase of in vivo and in vitro liver peroxidation and for the absence of changes in survival rate. The comparison of these results with previous data from other species suggests the possibility that decreases in antioxidant capacity in old age are restricted to animal species with high metabolic rates. Nevertheless, ageing can still be due to the continuous presence of small concentrations of O2 radicals in the tissues throughout life in animals with either high or low metabolic rates, because radical scavenging can not be 100% effective. Compensatory homeostasis among antioxidants seems to be a general phenomenon in different species.

Crystal structure and functional characterization of selenocysteine-containing glutathione peroxidase 4 suggests an alternative mechanism of peroxide reduction.

PubMed

Borchert, Astrid; Kalms, Jacqueline; Roth, Sophia R; Rademacher, Marlena; Schmidt, Andrea; Holzhutter, Hermann-Georg; Kuhn, Hartmut; Scheerer, Patrick

2018-06-05

Glutathione peroxidases (GPX) are anti-oxidative enzymes that reduce organic and inorganic hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. The human genome involves eight GPX genes and five of them encode for selenocysteine-containing enzymes. Among the human GPX-isoforms, GPX4 is unique since it is capable of reducing complex hydroperoxy ester lipids such as hydroperoxy phospholipids and hydroperoxy cholesterolesters. Using a number of genetically modified mouse strains the biological role of GPX4 has comprehensively characterized but the molecular enzymology is less well explored. This lack of knowledge is partly related to the fact that mammalian selenoproteins are not high-level expressed in conventional overexpression systems. To explore the structural and functional properties of human GPX4 we expressed this selenoprotein in a cysteine-auxotrophic E. coli strain using a semi-chemical expression strategy. The recombinant enzyme was purified in mg amounts from the bacterial lysate to electrophoretic homogeneity and characterized with respect to its protein-chemical and enzymatic properties. Its crystal structure was solved at 1.3 Å resolution and the X-ray data indicated a monomeric protein, which contains the catalytic selenium at the redox level of the seleninic acid. These data suggest an alternative reaction mechanism involving three different redox states (selenol, selenenic acid, seleninic acid) of the catalytically active selenocysteine. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Differential effect of UV-B radiation on growth, oxidative stress and ascorbate-glutathione cycle in two cyanobacteria under copper toxicity.

PubMed

Singh, Vijay Pratap; Srivastava, Prabhat Kumar; Prasad, Sheo Mohan

2012-12-01

Effects of low (UV-B(L); 0.1 μmol m(-2) s(-1)) and high (UV-B(H); 1.0 μmol m(-2) s(-1)) fluence rates of UV-B radiation on growth, oxidative stress and ascorbate-glutathione cycle (AsA-GSH cycle) were investigated in two cyanobacteria viz. Phormidium foveolarum and Nostoc muscorum under copper (2 and 5 μM) toxicity after 24 and 72 h of experiments. Cu at 2 and 5 μM and UV-B(H) irradiation decreased growth in both the organisms and the effect was more pronounced in N. muscorum. Superoxide radical (SOR) and hydrogen peroxide (H(2)O(2)) productions were significantly enhanced by Cu and UV-B(H) which was accompanied by accelerated lipid peroxidation (malondialdehyde; MDA) and protein oxidation (reactive carbonyl groups; RCG). The components of AsA-GSH cycle, i.e. ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascobate reductase (MDHAR) and dehydroascorbate reductase (DHAR) activities as well as total ascorbate and glutathione contents and their reduced/oxidized ratios were decreased considerably by Cu and UV-B(H). Further, combined treatments of Cu and UV-B(H) exacerbated damaging effects in both the cyanobacteria. Unlike UV-B(H), UV-B(L) irradiation rather than damaging cyanobacteria caused alleviation in Cu-induced toxicity by down-regulating the levels of SOR, H(2)O(2), MDA and RCG due to enhanced activity of APX, GR, MDHAR and DHAR, and contents of ascorbate and glutathione. Results revealed that UV-B radiation at low fluence rate (UV-B(L)) stimulated protective responses in both the organisms under Cu toxicity while UV-B(H) irradiation caused damage alone as well as together with Cu, and the components of AsA-GSH cycle play significant role in these responses. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Characterisation of Dermanyssus gallinae glutathione S-transferases and their potential as acaricide detoxification proteins.

PubMed

Bartley, Kathryn; Wright, Harry W; Bull, Robert S; Huntley, John F; Nisbet, Alasdair J

2015-06-26

Glutathione S-transferases (GSTs) facilitate detoxification of drugs by catalysing the conjugation of the reduced glutathione (GSH) to electrophilic xenobiotic substrates and therefore have a function in multi-drug resistance. As a result, knowledge of GSTs can inform both drug resistance in, and novel interventions for, the control of endo- and ectoparasite species. Acaricide resistance and the need for novel control methods are both pressing needs for Dermanyssus gallinae, a highly economically important haematophagous ectoparasite of poultry. A transcriptomic database representing D. gallinae was examined and 11 contig sequences were identified with GST BlastX identities. The transcripts represented by 3 contigs, designated Deg-GST-1, -2 and -3, were fully sequenced and further characterized by phylogenetic analysis. Recombinant versions of Deg-GST-1, -2 and -3 (rDeg-GST) were enzymically active and acaricide-binding properties of the rDeg-GSTs were established by evaluating the ability of selected acaricides to inhibit the enzymatic activity of rDeg-GSTs. 6 of the identified GSTs belonged to the mu class, followed by 3 kappa, 1 omega and 1 delta class molecules. Deg-GST-1 and -3 clearly partitioned with orthologous mu class GSTs and Deg-GST-2 partitioned with delta class GSTs. Phoxim, permethrin and abamectin significantly inhibited rDeg-GST-1 activity by 56, 35 and 17% respectively. Phoxim also inhibited rDeg-2-GST (14.8%) and rDeg-GST-3 (20.6%) activities. Deg-GSTs may have important roles in the detoxification of pesticides and, with the increased occurrence of acaricide resistance in this species worldwide, Deg-GSTs are attractive targets for novel interventions.

Radioprotective activity of glutathione on cognitive ability in X-ray radiated tumor-bearing mice.

PubMed

Lu, Lina; Li, Zongli; Zuo, Yanhua; Zhao, Libo; Liu, Bin

2018-05-30

The use of X-ray for therapeutics always raises the problem of radiation hazards to living beings. In this research, we explored the radioprotective activity of glutathione (GSH) on cognitive ability of X-ray radiated tumor-bearing mice. Forty C57BL/6 mice were chosen to establish the GL261 glioma model and randomly divided into four groups: Model group, X-ray group, Pre-GSH group and Pos-GSH group. Morris water maze test was used to test cognitive ability. Moreover, histopathological observation of hippocampus was observed by hematoxylin and eosin (HE) staining. The protein expression of choline acetyl transferase (ChAT) was measured by western blot, simultaneously the contents of acetylcholinesterase (Ach), superoxide dismutase (SOD), methane dicarboxylic aldehyde (MDA),TNF-α and IL-6 were detected by the respective kit. There was a significant difference in X-ray group of the escape latency from the Model group (P<0.05). Besides, HE staining revealed that nucleus in hippocampus cells were pyknotic, glial cells were hyperplastic and the nerve cells were swelling in X-ray group. In X-ray group the expression of ChAT and Ache were decreased versus Model group. Finally, the cognitive ability in Pre-GSH and Pos-GSH group was enhanced than X-ray group, in which the cognitive ability of Pos-GSH group was higher than the Pre-GSH group. X-ray impaired the brain tissues and cognitive ability of tumor-bearing mice. The damages of brain tissues were alleviated by Pre-GSH and Pos-GSH protection and the efficacy of Pos-GSH protection was superior to Pre-GSH protection. Abbreviation Ach: Acetylcholinesterase; GSH: Glutathione; HE: Hematoxylin and eosin; MDA: methane dicarboxylic aldehyde; SOD: Superoxide dismutase; TV: Tumor volume; TW: Tumor weight.

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.

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.

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.

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.

Therapeutic Hypothermia Reduces Oxidative Damage and Alters Antioxidant Defenses after Cardiac Arrest

PubMed Central

Hackenhaar, Fernanda S.; Medeiros, Tássia M.; Heemann, Fernanda M.; Behling, Camile S.; Putti, Jordana S.; Mahl, Camila D.; Verona, Cleber; da Silva, Ana Carolina A.; Guerra, Maria C.; Gonçalves, Carlos A. S.; Oliveira, Vanessa M.; Riveiro, Diego F. M.; Vieira, Silvia R. R.

2017-01-01

After cardiac arrest, organ damage consequent to ischemia-reperfusion has been attributed to oxidative stress. Mild therapeutic hypothermia has been applied to reduce this damage, and it may reduce oxidative damage as well. This study aimed to compare oxidative damage and antioxidant defenses in patients treated with controlled normothermia versus mild therapeutic hypothermia during postcardiac arrest syndrome. The sample consisted of 31 patients under controlled normothermia (36°C) and 11 patients treated with 24 h mild therapeutic hypothermia (33°C), victims of in- or out-of-hospital cardiac arrest. Parameters were assessed at 6, 12, 36, and 72 h after cardiac arrest in the central venous blood samples. Hypothermic and normothermic patients had similar S100B levels, a biomarker of brain injury. Xanthine oxidase activity is similar between hypothermic and normothermic patients; however, it decreases posthypothermia treatment. Xanthine oxidase activity is positively correlated with lactate and S100B and inversely correlated with pH, calcium, and sodium levels. Hypothermia reduces malondialdehyde and protein carbonyl levels, markers of oxidative damage. Concomitantly, hypothermia increases the activity of erythrocyte antioxidant enzymes superoxide dismutase, glutathione peroxidase, and glutathione S-transferase while decreasing the activity of serum paraoxonase-1. These findings suggest that mild therapeutic hypothermia reduces oxidative damage and alters antioxidant defenses in postcardiac arrest patients. PMID:28553435

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

Alleviation of lead-induced physiological, metabolic, and ultramorphological changes in leaves of upland cotton through glutathione.

PubMed

Khan, Mumtaz; Daud, M K; Basharat, Ali; Khan, Muhammad Jamil; Azizullah, Azizullah; Muhammad, Niaz; Muhammad, Noor; Ur Rehman, Zia; Zhu, Shui Jin

2016-05-01

Plants face changes in leaves under lead (Pb) toxicity. Reduced glutathione (GSH) has several functions in plant metabolism, but its role in alleviating Pb toxicity in cotton leaves is still unknown. In the present study, cotton seedlings (28 days old) were exposed to 500 μM Pb and 50 μM GSH, both alone and in combination, for a period of 10 days, in the Hoagland solution under controlled growth conditions. Results revealed Pb-induced changes in cotton's leaf morphology, photosynthesis, and oxidative metabolism. However, exogenous application of GSH restored leaf growth. GSH triggered build up of chlorophyll a, chlorophyll b, and carotenoid contents and boosted fluorescence ratios (F v/F m and F v/F 0). Moreover, GSH reduced the malondialdehyde (MDA), hydrogen peroxide (H2O2), and Pb contents in cotton leaves. Results further revealed that total soluble protein contents were decreased under Pb toxicity; however, exogenously applied GSH improved these contents in cotton leaves. Activities of antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), glutathione reductase (GR), and ascorbate peroxidase (APX)) were also increased by GSH application under Pb toxicity. Microscopic analysis showed that excess Pb shattered thylakoid membranes in chloroplasts. However, GSH stabilized ultrastructure of Pb-stressed cotton leaves. These findings suggested that exogenously applied GSH lessened the adverse effects of Pb and improved cotton's tolerance to oxidative stress.

Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

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

Low, Wai Yee; Feil, Susanne C.; Ng, Hooi Ling

2010-06-14

GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 {angstrom} resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model ofmore » the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional {sup 1}H,{sup 15}N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.« less

Cardiovascular and renal manifestations of glutathione depletion induced by buthionine sulfoximine.

PubMed

Vargas, Félix; Rodríguez-Gómez, Isabel; Pérez-Abud, Rocío; Vargas Tendero, Pablo; Baca, Yolanda; Wangensteen, Rosemary

2012-06-01

Oxidative stress contributes to the development of several cardiovascular diseases, including diabetes, renal insufficiency, and arterial hypertension. Animal studies have evidenced the association between higher blood pressure (BP) and increased oxidative stress, and treatment with antioxidants has been shown to reduce BP, while BP reduction due to antihypertensive drugs is associated with reduced oxidative stress. In 2000, it was first reported that oxidative stress and arterial hypertension were produced in normal Sprague-Dawley rats by oral administration of buthionine sulfoximine (BSO), which induces glutathione (GSH) depletion, indicating that oxidative stress may induce hypertension. The contribution of several potential pathogenic factors has been evaluated in the BSO rat model, the prototype of oxidative stress-induced hypertension, including vascular reactivity, endothelium-derived factors, renin-angiotensin system activity, TXA(2)-PGH(2) production, sodium sensitivity, renal dopamine-induced natriuresis, and sympathetic tone. This review summarizes the main factors implicated in the pathogenesis of BSO-induced hypertension and the alterations associated with GSH depletion that are related to renal function or BP control.

Glutathione reductase-mediated synthesis of tellurium-containing nanostructures exhibiting antibacterial properties.

PubMed

Pugin, Benoit; Cornejo, Fabián A; Muñoz-Díaz, Pablo; Muñoz-Villagrán, Claudia M; Vargas-Pérez, Joaquín I; Arenas, Felipe A; Vásquez, Claudio C

2014-11-01

Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Glutathione Reductase-Mediated Synthesis of Tellurium-Containing Nanostructures Exhibiting Antibacterial Properties

PubMed Central

Pugin, Benoit; Cornejo, Fabián A.; Muñoz-Díaz, Pablo; Muñoz-Villagrán, Claudia M.; Vargas-Pérez, Joaquín I.; Arenas, Felipe A.

2014-01-01

Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells. PMID:25193000

Oligo-carrageenan kappa-induced reducing redox status and activation of TRR/TRX system increase the level of indole-3-acetic acid, gibberellin A3 and trans-zeatin in Eucalyptus globulus trees.

PubMed

González, Alberto; Contreras, Rodrigo A; Zúiga, Gustavo; Moenne, Alejandra

2014-08-20

Eucalyptus globulus trees treated with oligo-carrageenan (OC) kappa showed an increase in NADPH, ascorbate and glutathione levels and activation of the thioredoxin reductase (TRR)/thioredoxin (TRX) system which enhance photosynthesis, basal metabolism and growth. In order to analyze whether the reducing redox status and the activation of thioredoxin reductase (TRR)/thioredoxin (TRX) increased the level of growth-promoting hormones, trees were treated with water (control), with OC kappa, or with inhibitors of ascorbate synthesis, lycorine, glutathione synthesis, buthionine sulfoximine (BSO), NADPH synthesis, CHS-828, and thioredoxin reductase activity, auranofine, and with OC kappa, and cultivated for four additional months. Eucalyptus trees treated with OC kappa showed an increase in the levels of the auxin indole 3-acetic acid (IAA), gibberellin A3 (GA3) and the cytokinin trans-zeatin (t-Z) as well as a decrease in the level of the brassinosteroid epi-brassinolide (EB). In addition, treatment with lycorine, BSO, CHS-828 and auranofine inhibited the increase in IAA, GA3 and t-Z as well as the decrease in EB levels. Thus, the reducing redox status and the activation of TRR/TRX system induced by OC kappa increased the levels of IAA, GA3 and t-Z levels determining, at least in part, the stimulation of growth in Eucalyptus trees.

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.

Insights into ligand binding to a Glutathione S-transferase from mango: structure, thermodynamics and kinetics

PubMed Central

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.; Serrano-Posada, Hugo; Lopez-Zavala, Alonso A.; García-Orozco, Karina D.; Hernandez-Paredes, Javier; Rudiño-Piñera, Enrique; Stojanoff, Vivian; Sotelo-Mundo, Rogerio R.; Islas-Osuna, Maria A.

2017-01-01

We studied a mango glutathione S-transferase (GST) (Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a Km, Vmax and kcat for CDNB of 0.792 mM, 80.58 mM·min−1 and 68.49 s−1 respectively and 0.693 mM, 105.32 mM·min−1 and 89.57 s−1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 μM) or GSX (7.8 μM). The crystal structure of the MiGSTU in apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes. PMID:28104507

Characterization of glutathione transferases involved in the pathogenicity of Alternaria brassicicola.

PubMed

Calmes, Benoit; Morel-Rouhier, Mélanie; Bataillé-Simoneau, Nelly; Gelhaye, Eric; Guillemette, Thomas; Simoneau, Philippe

2015-06-18

Glutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection. Mining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were 'orphans'. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues. Among the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs.

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.

Caribbean yellow band disease compromises the activity of catalase and glutathione S-transferase in the reef-building coral Orbicella faveolata exposed to anthracene.

PubMed

Montilla, Luis Miguel; Ramos, Ruth; García, Elia; Cróquer, Aldo

2016-05-03

Healthy and diseased corals are threatened by different anthropogenic sources, such as pollution, a problem expected to become more severe in the near future. Despite the fact that coastal pollution and coral diseases might represent a serious threat to coral reef health, there is a paucity of controlled experiments showing whether the response of diseased and healthy corals to xenobiotics differs. In this study, we exposed healthy and Caribbean yellow band disease (CYBD)-affected Orbicella faveolata colonies to 3 sublethal concentrations of anthracene to test if enzymatic responses to this hydrocarbon were compromised in CYBD-affected tissues. For this, a 2-factorial fully orthogonal design was used in a controlled laboratory bioassay, using tissue condition (2 levels: apparently healthy and diseased) and pollutant concentration (4 levels: experimental control, 10, 30 and 100 ppb concentration) as fixed factors. A permutation-based ANOVA (PERMANOVA) was used to test the effects of condition and concentration on the specific activity of 3 enzymatic biomarkers: catalase, glutathione S-transferase, and glutathione peroxidase. We found a significant interaction between the concentration of anthracene and the colony condition for catalase (Pseudo-F = 3.84, df = 3, p < 0.05) and glutathione S-transferase (Pseudo-F = 3.29, df = 3, p < 0.05). Moreover, our results indicated that the enzymatic response to anthracene in CYBD-affected tissues was compromised, as the activity of these enzymes decreased 3- to 4-fold compared to healthy tissues. These results suggest that under a potential scenario of increasing hydrocarbon coastal pollution, colonies of O. faveolata affected with CYBD might become more vulnerable to the deleterious effects of chemical pollution.

Energetics of Glutathione Binding to Human Eukaryotic Elongation Factor 1 Gamma: Isothermal Titration Calorimetry and Molecular Dynamics Studies.

PubMed

Tshabalala, Thabiso N; Tomescu, Mihai-Silviu; Prior, Allan; Balakrishnan, Vijayakumar; Sayed, Yasien; Dirr, Heini W; Achilonu, Ikechukwu

2016-12-01

The energetics of ligand binding to human eukaryotic elongation factor 1 gamma (heEF1γ) was investigated using reduced glutathione (GSH), oxidised glutathione (GSSG), glutathione sulfonate and S-hexylglutathione as ligands. The experiments were conducted using isothermal titration calorimetry, and the findings were supported using computational studies. The data show that the binding of these ligands to heEF1γ is enthalpically favourable and entropically driven (except for the binding of GSSG). The full length heEF1γ binds GSSG with lower affinity (K d  = 115 μM), with more hydrogen-bond contacts (ΔH = -73.8 kJ/mol) and unfavourable entropy (-TΔS = 51.7 kJ/mol) compared to the glutathione transferase-like N-terminus domain of heEF1γ, which did not show preference to any specific ligand. Computational free binding energy calculations from the 10 ligand poses show that GSSG and GSH consistently bind heEF1γ, and that both ligands bind at the same site with a folded bioactive conformation. This study reveals the possibility that heEF1γ is a glutathione-binding protein.

Interactive effects of herbicide and enhanced UV-B on growth, oxidative damage and the ascorbate-glutathione cycle in two Azolla species.

PubMed

Prasad, Sheo Mohan; Kumar, Sushil; Parihar, Parul; Singh, Rachana

2016-11-01

A field experiment was conducted to investigate the impact of alone and combined exposures of herbicide pretilachlor (5, 10 and 20μgml(-1)) and enhanced UV-B radiation (UV-B1; ambient +2.2kJm(-2) day(-1) and UV-B2; ambient +4.4kJm(-2) day(-1)) on growth, oxidative stress and the ascorbate-glutathione (AsA-GSH) cycle in two agronomically important Azolla spp. viz., Azolla microphylla and Azolla pinnata. Decreased relative growth rate (RGR) in both the species under tested stress could be linked to enhanced oxidative stress, thus higher H2O2 accumulation was observed, that in turn might have caused severe damage to lipids and proteins, thereby decreasing membrane stability. The effects were exacerbated when spp. were exposed to combined treatments of enhanced UV-B and pretilachlor. Detoxification of H2O2 is regulated by enzymes/metabolites of AsA-GSH cycle such as ascorbate peroxidase (APX) and glutathione reductase (GR) activity that were found to be stimulated. While, dehydroascorabte reductase (DHAR) activity, and the amount of metabolites: ascorbate (AsA), glutathione (GSH) and ratios of reduced/oxidized AsA (AsA/DHA) and GSH (GSH/GSSG), showed significant reduction with increasing doses of both the stressors, either applied alone or in combination. Glutathione-S-transferase (GST), an enzyme involved in scavenging of xenobiotics, was found to be stimulated under the tested stress. This study suggests that decline in DHAR activity and in AsA/DHA ratio might have led to enhanced H2O2 accumulation, thus decreased RGR was noticed under tested stress in both the species and the effect was more pronounced in A. pinnata. Owing to better performance of AsA-GSH cycle in A. microphylla, this study substantiates the view that A. microphylla is more tolerant than A. pinnata. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

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

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.

Alleviation of isoproturon toxicity to wheat by exogenous application of glutathione.

PubMed

Nemat Alla, Mamdouh M; Hassan, Nemat M

2014-06-01

Treatment with the recommended field dose of isoproturon to 7-d-old wheat seedlings significantly decreased shoot height, fresh and dry weights during the subsequent 15days. Meanwhile contents of carotenoids, chlorophylls and anthocyanin as well as activities of δ-aminolevulinate dehydratase (ALA-D), phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) were significantly inhibited. On the other hand, the herbicide significantly increased malondialdehyde (MDA), a naturally occurring product of lipid peroxidation and H2O2, while it significantly decreased the contents of glutathione (GSH) and ascorbic acid (AsA) and reduced the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). These findings indicate an induction of a stress status in wheat seedlings following isoproturon treatment. However, exogenous GSH appeared to limit the toxic effects of isoproturon and seemed to overcome this stress status. Most likely, contents of pigment and activities of enzymes were raised to approximate control levels. Moreover, antioxidants were elevated and the oxidative stress indices seemed to be alleviated by GSH application. These results indicate that exogenous GSH enhances enzymatic and nonenzymatic antioxidants to alleviate the effects of isoproturon. Copyright © 2014 Elsevier Inc. All rights reserved.

Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees.

PubMed

González, Alberto; Moenne, Fabiola; Gómez, Melissa; Sáez, Claudio A; Contreras, Rodrigo A; Moenne, Alejandra

2014-01-01

In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control), with OC kappa at 1 mg mL(-1), or treated with inhibitors of NAD(P)H, ascorbate (ASC), and glutathione (GSH) syntheses and thioredoxin reductase (TRR) activity, CHS-828, lycorine, buthionine sulfoximine (BSO), and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX) activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS), adenosine 5'-phosphosulfate reductase (APR), involved in C, N, and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH, and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH, and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle, and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC, and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses, and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism, and growth in Eucalyptus trees.

Glutathione-mediated detoxification of halobenzoquinone drinking water disinfection byproducts in T24 cells.

PubMed

Li, Jinhua; Wang, Wei; Zhang, Hongquan; Le, X Chris; Li, Xing-Fang

2014-10-01

Halobenzoquinones (HBQs) are a new class of drinking water disinfection byproducts (DBPs) and are capable of producing reactive oxygen species and causing oxidative damage to proteins and DNA in T24 human bladder carcinoma cells. However, the exact mechanism of the cytotoxicity of HBQs is unknown. Here, we investigated the role of glutathione (GSH) and GSH-related enzymes including glutathione S-transferase (GST) and glutathione peroxidase (GPx) in defense against HBQ-induced cytotoxicity in T24 cells. The HBQs are 2,6-dichloro-1,4-benzoquinone (DCBQ), 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ), 2,3,6-trichloro-1,4-benzoquinone (TriCBQ), and 2,6-dibromobenzoquinone (DBBQ). We found that depletion of cellular GSH could sensitize cells to HBQs and extracellular GSH supplementation could attenuate HBQ-induced cytotoxicity. HBQs caused significant cellular GSH depletion and increased cellular GST activities in a concentration-dependent manner. Our mass spectrometry study confirms that HBQs can conjugate with GSH, explaining in part the mechanism of GSH depletion by HBQs. The effects of HBQs on GPx activity are compound dependent; DCMBQ and DBBQ decrease cellular GPx activities, whereas DCBQ and TriCBQ have no significant effects. Pearson correlation analysis shows that the cellular GSH level is inversely correlated with ROS production and cellular GST activity in HBQ-treated cells. These results support a GSH and GSH-related enzyme-mediated detoxification mechanism of HBQs in T24 cells. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics

DOE PAGES

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.; ...

2017-01-17

We studied a mango glutathione S-transferase (GST) ( Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a K m, V max and k cat for CDNB of 0.792 mM, 80.58 mM min -1 and 68.49 s -1 respectively and 0.693 mM, 105.32 mM min -1 and 89.57 s -1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 mM) or GSX (7.8 mM). As a result, the crystal structure of the MiGSTU inmore » apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.« less

Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics

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

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.

We studied a mango glutathione S-transferase (GST) ( Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a K m, V max and k cat for CDNB of 0.792 mM, 80.58 mM min -1 and 68.49 s -1 respectively and 0.693 mM, 105.32 mM min -1 and 89.57 s -1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 mM) or GSX (7.8 mM). As a result, the crystal structure of the MiGSTU inmore » apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.« less

Effects of methodological variation on assessment of riboflavin status using the erythrocyte glutathione reductase activation coefficient assay.

PubMed

Hill, Marilyn H E; Bradley, Angela; Mushtaq, Sohail; Williams, Elizabeth A; Powers, Hilary J

2009-07-01

Riboflavin status is usually measured as the in vitro stimulation with flavin adenine dinucleotide of the erythrocyte enzyme glutathione reductase, and expressed as an erythrocyte glutathione reductase activation coefficient (EGRAC). This method is used for the National Diet and Nutrition Surveys (NDNS) of the UK. In the period between the 1990 and 2003 surveys of UK adults, the estimated prevalence of riboflavin deficiency, expressed as an EGRAC value > or = 1.30, increased from 2 to 46 % in males and from 1 to 34 % in females. We hypothesised that subtle but important differences in the detail of the methodology between the two NDNS accounted for this difference. We carried out an evaluation of the performance of the methods used in the two NDNS and compared against an 'in-house' method, using blood samples collected from a riboflavin intervention study. Results indicated that the method used for the 1990 NDNS gave a significantly lower mean EGRAC value than both the 2003 NDNS method and the 'in-house' method (P < 0.0001). The key differences between the methods relate to the concentration of FAD used in the assay and the duration of the period of incubation of FAD with enzyme. The details of the EGRAC method should be standardised for use in different laboratories and over time. Additionally, it is proposed that consideration be given to re-evaluating the basis of the EGRAC threshold for riboflavin deficiency.

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.

[Effect of nano-selenium on the activities of glutathione peroxidase and type-I deiodinase in the liver of weanling pigs].

PubMed

Zhang, Hongmei; Xia, Meisheng; Hu, Caihong

2007-02-01

To study the effects of nano elemental selenium (Nano-Se) or sodium selenite (Na2SeO3) on the activities of glutathione peroxidase (GSH-Px) and Type-I deiodinase in the liver. A total of 234 weanling pigs (Duroc x Landrace x Yorkshire) at an average initial body weight of 8.3 kg were allocated to 13 treatments. The thirteen dietary treatments were basal diet only (containing 0.04 mg/kg Se), basal diet + 0.1, 0.2, 0.3, 0.4, 0.5, 1.0 mg/kg Se as Na2SeO3 or Nano-Se, respectively. The results were as follows: Supplementation with 1.0 mg/ kg Se as Na2SeO3 reduced (P < 0.05) growth performance and GSH-Px activities as compared with the addition of a concentration range of 0.20-0.40 mg/kg Se. When Nano-Se was added to the diet, the growth and GSH-Px activities remained steady at the peak value as at a concentration of 1.0 mg/kg Se; There were no difference in the activities of GSH-Px between the treatments of Nano-Se and Na2SeO3 when added concentration of Se was 0.10-0.40 mg/kg. The pigs had higher (P < 0.05) activities of GSH-Px at a concentration range of 0.50 and 1.0 mg/kg as Nano-Se than Na2SeO3; Supplentation with Se increased the activity of Type- I deiodinase in liver, however, the increased extent was affected by neither Se sources nor added concentration of Se. The results implicated that for the best concentration range of Weinberg curve, Nano-Se is wider than Na2SeO3.

Glutathione Efflux and Cell Death

PubMed Central

2012-01-01

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

Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines.

PubMed

Sawers, L; Ferguson, M J; Ihrig, B R; Young, H C; Chakravarty, P; Wolf, C R; Smith, G

2014-09-09

Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity. Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT-PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays. Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC50, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers. Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients.

The role of strong electrostatic interactions at the dimer interface of human glutathione synthetase.

PubMed

De Jesus, Margarita C; Ingle, Brandall L; Barakat, Khaldoon A; Shrestha, Bisesh; Slavens, Kerri D; Cundari, Thomas R; Anderson, Mary E

2014-10-01

The obligate homodimer human glutathione synthetase (hGS) provides an ideal system for exploring the role of protein-protein interactions in the structural stability, activity and allostery of enzymes. The two active sites of hGS, which are 40 Å apart, display allosteric modulation by the substrate γ-glutamylcysteine (γ-GC) during the synthesis of glutathione, a key cellular antioxidant. The two subunits interact at a relatively small dimer interface dominated by electrostatic interactions between S42, R221, and D24. Alanine scans of these sites result in enzymes with decreased activity, altered γ-GC affinity, and decreased thermal stability. Molecular dynamics simulations indicate these mutations disrupt interchain bonding and impact the tertiary structure of hGS. While the ionic hydrogen bonds and salt bridges between S42, R221, and D24 do not mediate allosteric communication in hGS, these interactions have a dramatic impact on the activity and structural stability of the enzyme.

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

Impact of uranium (U) on the cellular glutathione pool and resultant consequences for the redox status of U.

PubMed

Viehweger, Katrin; Geipel, Gerhard; Bernhard, Gert

2011-12-01

Uranium (U) as a redox-active heavy metal can cause various redox imbalances in plant cells. Measurements of the cellular glutathione/glutathione disulfide (GSH/GSSG) by HPLC after cellular U contact revealed an interference with this essential redox couple. The GSH content remained unaffected by 10 μM U whereas the GSSG level immediately increased. In contrast, higher U concentrations (50 μM) drastically raised both forms. Using the Nernst equation, it was possible to calculate the half-cell reduction potential of 2GSH/GSSG. In case of lower U contents the cellular redox environment shifted towards more oxidizing conditions whereas the opposite effect was obtained by higher U contents. This indicates that U contact causes a consumption of reduced redox equivalents. Artificial depletion of GSH by chlorodinitrobenzene and measuring the cellular reducing capacity by tetrazolium salt reduction underlined the strong requirement of reduced redox equivalents. An additional element of cellular U detoxification mechanisms is the complex formation between the heavy metal and carboxylic functionalities of GSH. Because two GSH molecules catalyze electron transfers each with one electron forming a dimer (GSSG) two UO(2) (2+) are reduced to each UO(2) (+) by unbound redox sensitive sulfhydryl moieties. UO(2) (+) subsequently disproportionates to UO(2) (2+) and U(4+). This explains that in vitro experiments revealed a reduction to U(IV) of only around 33% of initial U(VI). Cellular U(IV) was transiently detected with the highest level after 2 h of U contact. Hence, it can be proposed that these reducing processes are an important element of defense reactions induced by this heavy metal.