Role Of Glutathione Reductase Research Articles

The Role of Glutathione Reductase in Influenza Infection.

Influenza infection induces lung epithelial cell injury via programmed cell death. Glutathione, a potent antioxidant, has been reported to be associated with influenza infection. We hypothesized that lung epithelial cell death during influenza infection is regulated by glutathione metabolism. Eight-week-old male and female BALB/c mice were infected with influenza (PR8: A/PR/8/34 [H1N1]) via intranasal instillation. Metabolomic analyses were performed on whole lung lysate after influenza infection. For invitro analysis, Beas-2B cells were infected with influenza. RNA was extracted, and QuantiTect Primer Assay was used to assess gene expression. Glutathione concentrations were assessed by colorimetric assay. Influenza infection resulted in increased inflammation and epithelial cell injury in our murine model, leading to increased morbidity and mortality. In both our invivo and invitro models, influenza infection was found to induce apoptosis and necroptosis. Influenza infection led to decreased glutathione metabolism and reduced glutathione reductase activity in lung epithelial cells. Genetic inhibition of glutathione reductase suppressed apoptosis and necroptosis of lung epithelial cells. Pharmacologic inhibition of glutathione reductase reduced airway inflammation, lung injury, and cell death in our murine influenza model. Our results demonstrate that glutathione reductase activity is suppressed during influenza. Glutathione reductase inhibition prevents epithelial cell death and morbidity in our murine influenza model. Our results suggest that glutathione reductase-dependent glutathione metabolism may play an important role in the host response to viral infection by regulating lung epithelial cell death.

BcGR1.1, a Cytoplasmic Localized Glutathione Reductase, Enhanced Tolerance to Copper Stress in Arabidopsis thaliana.

Copper is a mineral element, which is necessary for the normal growth and development of plants, but high levels of copper will seriously damage plants. Studies have shown that AtGR1 improves the tolerance of Arabidopsis to aluminum and cadmium stress. However, the role of GR in the copper stress response of plants is still unclear. Here, we identified four genes (named BcGR1.1, BcGR1.2, BcGR2.1 and BcGR2.2, respectively) encoding glutathione reductase (GR) in non-heading Chinese cabbage (Brassica campestris (syn. Brassica rapa) ssp. chinensis), which could be divided into two types based on the subcellular localization. Among them, BcGR1.1, which belonged to the cytoplasmic localization type, was significantly upregulated under copper stress. Compared to WT (the wild type), Arabidopsis thaliana heterologously overexpressed BcGR1.1 had longer roots, higher fresh weight, higher GSH levels and GSH/GSSG (oxidized form of GSH) ratio, and accumulated more superoxide dismutase and peroxidase under copper stress. However, in the AsA-GSH cycle under copper stress, the contents of AsA and AsA/DHA were significantly downregulated, and the contents of DHA and T-AsA (total AsA) were upregulated, in the BcGR1.1-overexpressing Arabidopsis. Therefore, BcGR1.1 could improve the scavenging ability of reactive oxygen species (ROS) by increasing the activity of GR, antioxidant enzymes and the utilization of AsA, and then enhance the copper stress tolerance of plants.

Exploration of glutathione reductase for abiotic stress response in bread wheat (Triticum aestivum L.)

A total of seven glutathione reductase (GR) genes were identified in Triticum aestivum, which were used for comparative structural characterization, phylogenetic analysis and expression profiling with the GR genes of other cereal plants. The modulated gene expression and enzyme activity revealed the role of GRs in abiotic stress response in T. aestivum. Glutathione reductase (GR) is an enzymatic antioxidant that converts oxidized glutathione (GSSG) into reduced glutathione (GSH) through the ascorbate-glutathione cycle. In this study, a total of seven GR genes forming two homeologous groups were identified in the allohexaploid genome of bread wheat (Triticum aestivum). Besides, we identified three GR genes in each Aegilops tauschii, Brachypodium distachyon, Triticum urartu and Sorghum bicolor, which were used for comparative characterization. Phylogenetic analysis revealed the clustering of GR proteins into two groups; class I and class II, which were predicted to be localized in cytoplasm and chloroplast, respectively. The exon-intron and conserved motif patterns were almost conserved in each group, in which a maximum of 10 and 17 exons were present in chloroplastic and cytoplasmic GRs, respectively. The protein structure analysis confirmed the occurrence of conserved pyridine nucleotide disulfide oxidoreductase (Pyr_redox) and pyridine nucleotide disulfide oxidoreductase dimerization (Pyr_redox_dim) domains in each GR. The active site of GR proteins consisted of two conserved cysteine residues separated by four amino acid residues. Promoter analysis revealed the occurrence of growth and stress-related cis-active elements. Tissue-specific expression profiling suggested the involvement of GRs in both vegetative and reproductive tissue development in various plants. The differential expression of TaGR genes and enhanced GR enzyme activity suggested their roles under drought, heat, salt and arsenic stress. Interaction of GRs with other proteins and chemical compounds of the ascorbate-glutathione cycle revealed their coordinated functioning. The current study will provide a foundation for the validation of the precise role of each GR gene in future studies.

New insight into the role of glutathione reductase in glutathione peroxidase-like activity determination by coupled reductase assay: Molecular Docking Study

Previously we have shown that among 15 substituted salicyloyl (2-hydroxybenzoyl) 5-seleninic acids (SSAs) 4 compounds with longer side chains or a cyclohexyl group exhibit no glutathione peroxidase (GPx)-like activity in the coupled reductase assay. Experimental inhibition of glutathione reductase (GR) by the selenenylsulfide (a main intermediate in the catalytic cycle for GPx-like activity determination) of one of the inactive compounds led us to assess the interactions between 15 selenenylsulfide compounds and the active site of GR by molecular docking. Docking results showed that S and Se atoms in selenenylsulfides of the compounds with no GPx-like activity were beyond 5 Å from S atom of Cys-58 or N atom of imidazole ring of His-467 (Root Mean Square Distances for general assessment of 3 major distances were over 4.8 Å) in the active site, so that they could not be catalyzed to be reduced by GR. Furthermore, their docking scores over 89 Kcal/mol meant that the selenenylsulfides were bound too strongly to the active site to leave it, leading eventually to inhibition of GR. We also applied the molecular docking to other GPx mimics such as ebselen, cyclic seleninate esters and di(propylaminomethylphenyl) diselenides to explain the differences in their GPx-like activity depending to the assays used. Our results suggest that the reduction of a selenenylsulfide by GR plays a positive role in GPx-like activity of GPx mimics in the coupled assay and recommended the prediction of possibility and strength of GPx-like activity by molecular docking before entering experimental research.

Role of glutathione reductase and catalase enzyme in antioxidant defense mechanism in controlling fluoride-induced oxidative stress

Antioxidant enzymes play a protective role in plant with oxidative stress. . Fluoride negatively affects plants by increasing the level of ROS species and by negatively regulating the growth of plants. Fluoride stress this is one of the most important abiotic stresses. In this studio; Vigna radiata L was exposed to fluoride stress in a half-diluted Hoagland solution. In this study, a concentration-dependent analysis (0, 2.5 and 5, 7.5, 10, 12.5 and 15 mM NaF) of antioxidant enzyme activity performed against the most significant inhibition of growth levels and content of MDA, the decrease in chlorophyll content, was reported in seedlings treated with NaF compared to controls. Antioxidant expression (CAT, GR) showed the highest activity at 7.5 mM NaF and showed a significant defense against fluoride stress during the harvest. This study would help to understand the role of antioxidants in the survival of plants against fluoride stress

Correlation of glutathione reductase activity distribution in the blood serum and tissues of white unborn rats

It is known that the majority of pathological processes take place against the background of formation of active oxygen species and intensification of free radical oxidation of bio-substrates. In response to this, the antioxidant system of the cell is activated, and the glutathione system is an important link in this system. The latter can take part in the maintenance of the optimal state of biomembranes, in the processes of detoxification, antioxidant protection, etc. The biological role of glutathione reductase is to maintain high intracellular concentration of reduced glutathione. The aim of our study was to study the relationship between the distribution of glutathione reductase activity in blood serum and rat tissues. In order to achieve the goal of the study the following tasks were solved: the activity of glutathione reductase in blood serum and tissues of liver, brain, heart, as well as in skeletal muscle tissues of rats was determined; the interrelation of the activity distribution of glutathione reductase in blood serum and tissues of rats was revealed. The article presents the results of nonparametric correlation analysis to assess the relationship between the distribution of glutathione reductase activity in blood serum and tissues of small experimental animals.

EVALUATION OF GLUTATHIONE REDUCTASE (GR) ACTIVITY IN LIVER OF FISH, CHANNA PUNCTATUS (BLOCH, 1793) EXPOSED TO HEXAVALENT CHROMIUM

Antioxidant defence system is known to be a sensitive biomarker of metal exposure widely employed in predicting toxicity after sub-lethal exposure of Cr6+in fish. The present study conducted to assess the impact of Cr6+on Glutathione reductase (GR) activity in liver of fish, Channa punctatus. Fish were exposed to two different sub-lethal concentrations of Cr6+viz.,3.93 mg/L (5% of 96h LC50) (T1) and 7.85 mg/L (10% of 96h LC50) (T2) along with one control group without any toxicant exposure, for 15, 30 and 45 d. Results showa significant (p <0.05) increase in GR activity with an increase in dose and exposure period. This study emphasized the role of GR in monitoring of metal toxicity in aquatic regimes as an early warning tool.

Cloning and characterization of a thermostable glutathione reductase from a psychrophilic Arctic bacterium Sphingomonas sp.

Glutathione reductase is an important oxidoreductase that helps maintain redox homeostasis by catalyzing the conversion of glutathione disulfide to glutathione using NADPH as a cofactor. In this study, we cloned and characterized a glutathione reductase (hereafter referred to as SpGR) from Sphingomonas sp. PAMC 26621, an Arctic bacterium. SpGR comprises 449 amino acids, and functions as a dimer. Surprisingly, SpGR exhibits characteristics of thermophilic enzymes, showing optimum activity at 60°C and thermal stability up to 70°C with ∼50% residual activity at 70°C for 2 h. The amino acid composition analysis of SpGR showed a 1.9-fold higher Arg content (6%) and a 2.7-fold lower Lys/Arg ratio (0.75) compared to the Arg content (3.15%) and the Lys/Arg ratio (2.01) of known psychrophilic glutathione reductases. SpGR also exhibits its activity at 4°C, and circular dichroism and fluorescence spectroscopy results indicate that SpGR maintains its secondary and tertiary structures within the temperature range of 4-70°C. Taken together, the results of this study indicate that despite its origin from a psychrophilic bacterium, SpGR has high thermal stability. Our study provides an insight into the role of glutathione reductase in maintaining the reducing power of an Arctic bacterium in a broad range of temperatures.

The role of ginsenoside Rb1, a potential natural glutathione reductase agonist, in preventing oxidative stress-induced apoptosis of H9C2 cells

BackgroundOxidative stress-induced cardiomyocytes apoptosis is a key pathological process in ischemic heart disease. Glutathione reductase (GR) reduces glutathione disulfide to glutathione (GSH) to alleviate oxidative stress. Ginsenoside Rb1 (GRb1) prevents the apoptosis of cardiomyocytes; however, the role of GR in this process is unclear. Therefore, the effects of GRb1 on GR were investigated in this study. MethodsThe antiapoptotic effects of GRb1 were evaluated in H9C2 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, annexin V/propidium iodide staining, and Western blotting. The antioxidative effects were measured by a reactive oxygen species assay, and GSH levels and GR activity were examined in the presence and absence of the GR inhibitor 1,3-bis-(2-chloroethyl)-1-nitrosourea. Molecular docking and molecular dynamics simulations were used to investigate the binding of GRb1 to GR. The direct influence of GRb1 on GR was confirmed by recombinant human GR protein. ResultsGRb1 pretreatment caused dose-dependent inhibition of tert-butyl hydroperoxide-induced cell apoptosis, at a level comparable to that of the positive control N-acetyl-L-cysteine. The binding energy between GRb1 and GR was positive (−6.426 kcal/mol), and the binding was stable. GRb1 significantly reduced reactive oxygen species production and increased GSH level and GR activity without altering GR protein expression in H9C2 cells. Moreover, GRb1 enhanced the recombinant human GR protein activity in vitro, with a half-maximal effective concentration of ≈2.317 μM. Conversely, 1,3-bis-(2-chloroethyl)-1-nitrosourea co-treatment significantly abolished the GRb1's apoptotic and antioxidative effects of GRb1 in H9C2 cells. ConclusionGRb1 is a potential natural GR agonist that protects against oxidative stress–induced apoptosis of H9C2 cells.

Exogenously applied salicylic acid maintains redox homeostasis in salt-stressed Arabidopsis gr1 mutants expressing cytosolic roGFP1

Exogenous salicylic acid (SA) can be used for chemical hardening to alleviate oxidative stress in plants exposed to salinity. The treatment of 5-week-old Arabidopsis thaliana plants with increasing doses of SA alters the ascorbate (ASC) and glutathione (GSH) pools, and modulates their redox status and the activity of several antioxidant enzymes, such as ascorbate peroxidase (APX) and glutathione reductase (GR). To investigate the role of GR in the maintenance of cytoplasmic redox homeostasis after hardening by SA, wild type (WT) and gr1 mutant plants, expressing the cytoplasmic redox-sensitive green fluorescent protein (c-roGFP1), were pre-treated with 10−7 and 10−5 M SA for 2 weeks and subsequently exposed to 100 mM NaCl. The redox status of the salt-stressed WT plants became more oxidized, which was prevented by pretreatment with 10−5 M SA. The gr1 mutants showed more positive redox potential than WT plants, which could be reversed by treatment with 10−5 M SA. In mutants, the increased GSH levels may have compensated for the deleterious effect of GR deficiency and stabilized the redox potential in plants exposed to salinity. The ASC regeneration in WT plants shifted from the GSH-dependent dehydroascorbate reductase (DHAR) reaction to the NAD(P)H-dependent monodehydroascorbate reductase (MDHAR) activity during chemical hardening, which contributed to the preservation of the GSH pool in plants under salt stress. Our results suggest that the maintenance of GSH levels and redox homeostasis by SA-mediated hardening play a major role in priming and defending against salt stress.

A role for glutathione reductase and glutathione in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress.

The role of glutathione reductase (GR; EC 1.6.4.2) in the tolerance of Chlamydomonas reinhardtii P.A. Dangeard to high-intensity light stress (HL, 1400 μmol m-2 s-1 ) was examined. Cells survived under high light (HL) stress, although their growth was inhibited after long-term treatment (9-24 h). GR activity increased 1 h after HL treatment. The contents of total glutathione, reduced glutathione (GSH) and glutathione disulfide (GSSG) increased 1-3 h after HL treatment and then decreased after 24 h, while the GSH:GSSG ratio (glutathione redox potential) decreased after 3-9 h and recovered after 24 h. The transcript abundance of GR, CrGR1 (Cre06.g262100) and CrGR2 (Cre09.g396252) as well as glutathione synthesis-related genes, CrGSH1 (Cre02g077100.t1.1) and CrGSH2 (Cre17.g70800.t1.1), increased with a peak near 1 h after HL treatment. Except for enhanced glutathione synthesis, the GR-mediated glutathione redox machinery is also critical for the tolerance of C. reinhardtii cells to HL stress. Therefore, GR was downregulated or upregulated to investigate the importance of GR in HL tolerance. The CrGR1 knockdown amiRNA line exhibited low GR transcript abundance, GR activity and GSH:GSSG ratio and could not survive under HL conditions. Over-expression of CrGR1 or CrGR2 driven by a HSP70A:RBCS2 fusion promoter resulted in a higher GR transcript abundance, GR activity and GSH:GSSG ratio and led to cell survival when exposed to high-intensity illumination, i.e. 1800 μmol m-2 s-1 . In conclusion, GR-mediated modulation of the glutathione redox potential plays a role in the tolerance of Chlamydomonas cells to photo-oxidative stress.

Methylglyoxal-Induced Protection Response and Toxicity: Role of Glutathione Reductase and Thioredoxin Systems.

Thioredoxin (Trx) and glyoxalase (Glo) systems have been suggested to be molecular targets of methylglyoxal (MGO). This highly reactive endogenous compound has been associated with the development of neurodegenerative pathologies and cell death. In the present study, the glutathione (GSH), Trx, and Glo systems were investigated to understand early events (0.5-3h) that may determine cell fate. It is shown for the first time that MGO treatment induces an increase in glutathione reductase (GR) protein in hippocampal slices (1h) and HT22 nerve cells (0.5 and 2.5h). Thioredoxin interacting protein (Txnip), thioredoxin reductase (TrxR), Glo1, and Glo2 were markedly increased (2- to 4-fold) in hippocampal slices and 1.2- to 1.3-fold in HT22 cells. This increase in protein levels in hippocampal slices was followed by a corresponding increase in GR, TrxR, and Glo1 activities, but not in HT22 cells. In these cells, GR and TrxR activities were decreased by MGO. This result is in agreement with the idea that MGO can affect the Trx/TrxR reducing system, and now we show that GR and Txnip can also be affected by MGO. Impairment in the GR or TrxR reducing capacity can impair peroxide removal by glutathione peroxidase and peroxiredoxin, as both peroxidases depend on reduced GSH and Trx, respectively. In this regard, inhibition of GR and TrxR by 2-AAPA or auranofin, respectively, potentiated MGO toxicity in differentiated SH-SY5Y cells. Overall, MGO not only triggers a clear defense response in hippocampal slices and HT22 cells but also impairs the Trx/TrxR and GSH/GR reducing couples in HT22 cells. The increased MGO toxicity caused by inhibition of GR and TrxR with specific inhibitors, or their inhibition by MGO treatment, supports the notion that both reducing systems are relevant molecular targets of MGO.

Gene cloning and expression analysis of mitochondrial glutathione reductase from Arabian camel (Camelus dromedarius) liver in Escherichia coli

Glutathione reductase (GR) is highly conserved among diverse taxa and has important biochemical functions. These functions may facilitate survival in harsh conditions, but the role of GR from the liver of the Arabian camel (Camelus dromedarius) is unknown. In this study, the mitochondrial glutathione reductase gene (Gsr) from C. dromedarius liver was cloned and highly expressed in Escherichia coli (Jm109) to gain insight into GR functions in the liver. After amplification of the cDNA encoding the functional unit of Gsr (1.2 kb), the products were cloned into the PGEM-T Easy and PET28a vectors. Gsr expression was confirmed using an immunoblotting technique (45 kDa). Recombinant GR was purified to homogeneity using Ni-NTA resins, with an overall yield of 7.23% and a specific activity of 0.3063 U/mg. The optimum pH of recombinant GR was 7 and the optimum temperature was 35 °C in 50 mM K3PO4 buffer. The Michaelis constant, Km, for the substrates glutathione disulfide (GSSG) and NADPH was 45.6 μM and 63.5 μM, respectively; moreover, maximal velocity (Vmax) values were 3.969 x 10-2 U/mg and 1.497 x 10-1 U/mg. This partial characterization of camel liver GR extends our insight into the ability of camels to cope with harsh environmental conditions.

The Role of Glutathione Reductase in Neutrophil Respiratory Burst and ERK Signaling

Abstract Respiratory burst plays a crucial role in the killing of bacteria by phagocytes. During the respiratory burst, NADPH oxidase complexes are assembled on the membrane to produce superoxide, which is then converted to H2O2 for pathogen killing. Glutathione (GSH) is crucial for the detoxification of H2O2 that has diffused into the cytosol. GSH is generated via both de novo synthesis and regeneration from glutathione disulfide. The rate-limiting step in GSH synthesis is catalyzed by γ-glutamate cysteine ligase, composed of a catalytic subunit (Gclc) and a regulatory subunit (Gclm), whereas GSH regeneration is catalyzed by glutathione reductase (Gsr). Mice with a mutation in the Gsr gene exhibit a profound defect in immune defense against bacteria, which is associated with compromised respiratory burst activity. To address the mechanisms underlying the compromised respiratory burst, we purified neutrophils from wildtype (WT) and Gsr−/− and Gclm−/− mice, and assessed their respiratory burst in response to phorbol ester (PMA). Compared to WT neutrophils, Gsr−/− neutrophils exhibited a dramatic decrease in respiratory burst activity. A similar trend was observed in neutrophils derived from Gclm−/− mice, which have 10–40% of normal tissue GSH levels. However, no significant defect in immune defense against E. coli was found in Gclm−/− mice. Cellular fractionation indicated that the NADPH oxidase subunits underwent membrane translocation in response to PMA in both WT and Gsr−/− neutrophils. Interestingly, Gsr−/− neutrophils also displayed a dramatic enhancement in activation of the ERK MAP kinase pathway. Our results indicate that Gsr plays an important role in the regulation of both the respiratory burst and cell signaling in neutrophils.

Effect of glutathione reductase knockdown in response to UVB-induced oxidative stress in human lung adenocarcinoma

BackgroundGlutathione reductase (GR) plays a critical role in the maintenance of physiological redox status in cells. However, the comprehensive investigations of GR-modulated oxidative stress have not been reported.MethodsIn the present study, we cultured a human lung adenocarcinoma line CL1-0 and its GR-knockdown derivative CL1-0ΔGR to evaluate their differential responses to UVB-irradiation.ResultsWe identified 18 proteins that showed significant changes under UVB-irradiation in CL1-0ΔGR cells rather than in CL1-0 cells. Several proteins involving protein folding, metabolism, protein biosynthesis and redox regulation showed significant changes in expression.ConclusionsIn summary, the current study used a comprehensive lung adenocarcinoma-based proteomic approach for the identification of GR-modulated protein expression in response to UVB-irradiation. To our knowledge, this is the first global proteomic analysis to investigate the role of GR under UVB-irradiation in mammalian cell model.

Proteomic and redox‐proteomic study on the role of glutathione reductase in human lung cancer cells

Glutathione reductase (GR), a cytosolic protein, plays a vital role in maintaining a correct redox status in cells. However, comprehensive investigations of GR-modulated cellular responses, including protein level alteration and redox regulation, have yet to be performed. In this study, we cultured a human lung adenocarcinoma line transfected with empty pLKO.1 vector as a control, CL1-0shControl, and its GR-knockdown derivative, CL1-0shΔGR, to evaluate differential protein level alteration and redox regulation of these two cell lines. We identified 34 spots that exhibited marked changes in intensities, and 13 proteins showing significant changes in thiol reactivity, in response to GR depletion. Several proteins involved in redox regulation, calcium signaling, cytoskeleton regulation, and protein folding showed significant changes in expression, whereas proteins involved in redox regulation, protein folding, and glycolysis displayed changes in thiol reactivity. Interestingly, GR knockdown induces peroxiredoxin-1 overexpression in the air-exposed tissue and high oxygen consuming tissue such as cornea and liver, but not in the low oxygen consuming tissues such as breast and uterine. In summary, we used a comprehensive lung adenocarcinoma based proteomic approach for identifying GR-modulated protein expression alteration and redox modification. Based on our research, this is the first comprehensive proteomic and redox-proteomic analysis used to investigate the role of GR in a mammalian cell model.

Influence of abiotic stresses on the antioxidant enzyme activity of cereals

The unfavourable effects of climate change were studied in terms of changes in the stress tolerance of cereals. Changes in the antioxidant enzyme activities were analysed as a function of the weather in a 2-year field experiment in order to determine the effect of extreme temperatures and rainfall conditions on the enzyme activity. The enzyme responses of two winter wheat genotypes to drought stress, simulated by withholding water completely for 7 days, were analysed under phytotronic conditions in three phenophases. The plants were raised either at ambient CO2 concentration or at a doubled level. The quantities of glutathione reductase (GR), glutathione-S-transferase (GST), catalase (CAT), guaiacol peroxidase (POD) and ascorbate peroxidase (APX) were determined from leaf samples. The peroxidases had the most intense activity during the winter and early spring periods, with guaiacol peroxidase being dominant until the end of the winter. CAT generally became more active in late spring and summer, the activity being correlated with the development of water deficiency. The activity of GR, GST, POD and CAT was found to increase during the dry period, while the role of GR and POD was extremely important for resistance to low temperature.

The role of glutathione reductase in the interplay between oxidative stress response and turnover of cytosolic NADPH in Kluyveromyces lactis

The phosphoglucose isomerase mutant of the respiratory yeast Kluyveromyces lactis (rag2) is forced to metabolize glucose through the oxidative pentose phosphate pathway and shows an increased respiratory chain activity and reactive oxygen species production. We have proved that the K. lactis rag2 mutant is more resistant to oxidative stress (OS) than the wild type, and higher activities of glutathione reductase (GLR) and catalase contribute to this phenotype. Resistance to OS of the rag2 mutant is reduced when the gene encoding GLR is deleted. The reduction is higher when, in addition, catalase activity is inhibited. In K. lactis, catalase activity is induced by peroxide-mediated OS but GLR is not. We have found that the increase of GLR activity is correlated with that of glucose-6-phosphate dehydrogenase (G6PDH) activity that produces NADPH. G6PDH is positively regulated by an active respiratory chain and GLR plays a role in the reoxidation of the NADPH from the pentose phosphate pathway in these conditions. Cytosolic NADPH is also used by mitochondrial external alternative dehydrogenases. Neither GLR overexpression nor induction of the OS response restores growth on glucose of the rag2 mutant when the mitochondrial reoxidation of cytosolic NADPH is blocked.

Molecular mechanism of glutathione-mediated protection from oxidized low-density lipoprotein-induced cell injury in human macrophages: Role of glutathione reductase and glutaredoxin

Macrophage death is a hallmark of advanced atherosclerotic plaque, and oxidized low-density lipoprotein (OxLDL) found in these lesions is believed to contribute to macrophage injury. However, the underlying mechanisms of this phenomenon are only poorly understood. Here we show that in human monocyte-derived macrophages, OxLDL depleted intracellular glutathione (GSH) and inhibited glutathione reductase, resulting in a marked diminution of the glutathione/glutathione disulfide ratio. In the absence of OxLDL, an 80% depletion of intracellular GSH levels did not affect cell viability, but glutathione depletion dramatically increased OxLDL-induced cell death. Conversely, supplementation of intracellular GSH stores with glutathione diethyl ester substantially diminished OxLDL toxicity. OxLDL also promoted protein- S-glutathionylation, which was increased in macrophages pretreated with the glutathione reductase inhibitor BCNU. Knockdown experiments with siRNA directed against glutathione reductase and glutaredoxin showed that both enzymes are essential for the protection of macrophages against OxLDL. Finally, the peroxyl-radical scavenger Trolox did not prevent GSH depletion but completely blocked OxLDL-induced protein- S-glutathionylation and cell death. These data suggest that OxLDL promotes ROS formation and protein- S-glutathionylation by a mechanism independent from its effect on GSH depletion. Neither mechanism was sufficient to induce macrophage injury, but when stimulated concurrently, these pathways promoted the accumulation of protein-glutathione mixed disulfides and cell death.

The role of glutathione reductase in the cytotoxicity of chromium (VI) in isolated rat hepatocytes

Chromium (VI) is an environmental and occupational carcinogen, and it is accepted that intracellular reduction is necessary for DNA damage and cytotoxicity. We have investigated the interaction of Cr(VI) with hepatocytes in vitro to determine the contribution of various hepatic enzymes to the reduction of Cr(VI). Cr(VI) caused a dose-dependent decrease in cell viability and intracellular reduced glutathione (GSH) levels between 100 and 500 μM within 3 h exposure of hepatocytes. Both DT-diaphorase and cytochrome P450 play only a minor role in detoxifying Cr(VI) and/or its metabolites. (GSH) appears to act as a non-enzymatic reductant, reducing Cr(VI) to a toxic form. The evidence for this is two-fold. Firstly, GSH was depleted during the metabolism of Cr(VI) and, secondly, pretreatment of the cells with diethylmaleate to deplete GSH levels, partially protected the cells from Cr(VI) toxicity. Glutathione reductase appears to play an important role in the enzymatic reduction of Cr(VI) as inhibition of this enzyme by carmustine (BCNU) markedly protected the cells from cytotoxicity.