Interaction of nitric oxide with glutathione or cysteine generates reactive oxygen species causing DNA single strand breaks

It was found that reactive oxygen species (ROS) were generated in the interactions of nitric oxide (NO) with glutathione (GSH) or cysteine (CySH) under aerobic conditions. When supercoiled DNA was incubated with a mixture of NO/GSH, NO/CySH, NOC-7 (a NO donor)/GSH or NOC-7/CySH under aerobic conditions, DNA single-strand breaks were observed on agarose gel electrophoresis. The strand breaks were inhibited by common ROS scavengers: superoxide dismutase+catalase, the spin trapping agent 5,5-dimethyl-1-pyrroline-N oxide (DMPO), ethanol, and EDTA. The strand breaks were also caused by incubation with a mixture of S-nitrosoglutathione (GSNO) with GSH or CySH, which was inhibited by ROS scavengers. In the reaction of NO/GSH, GSNO rapidly formed and then gradually decreased, and in the reaction of GSNO/GSH, GSNO was gradually decreased. The decrease inf GSNO was accelerated in the presence of superoxide+catalase. Hydroxyl radical was detected in the mixtures of NO with GSH or CySH under aerobic conditions, and thiyl radicals were detected in the mixtures of GSNO with GSH or CySH under anaerobic conditions as examined in electron spin resonance studies using DMPO as a spin trap. The results indicate that the interaction of NO with thiols in the presence of O2 generates ROS that caused DNA single-strand breaks.     

Nitrofurazone: kinetics and oxidative stress in the singlepass isolated perfused rat liver

The disposition of the antibiotic nitrofurazone was studied in the singlepass isolated perfused rat liver. Both the effects of the steady-state level of drug and the composition of the perfusate were evaluated. The higher level (120 micrograms/ml) of nitrofurazone in a perfusion medium lacking the glutathione (GSH) precursors, glycine, glutamic acid and cysteine, caused a marked increase in bile flow (from 1.01 +/- 0.07 to 2.33 +/- 1.07 microliters/min/g), massive biliary efflux of glutathione disulfide (GSSG) (from 0.55 +/- 0.07 to 60.6 +/- 25.4 nmol/min/g) and a sharp decline in the caval efflux of GSH (to undetectable levels) and the tissue level of GSH (from 5.74 +/- 0.20 to 2.68 +/- 0.13 mumol/g). Even after the drug was discontinued, these parameters were not restored to control levels. The lower level (30 micrograms/ml) of nitrofurazone with or without amino acid supplementation and the higher level with supplementation induced less dramatic effects. Using [35S]methionine, a new conjugated metabolite of nitrofurazone and glutathione was detected. The data suggest that the toxicity of the reactive oxygen species generated by the redox cycling of the nitro group and the reactive metabolites generated by further reduction of nitrofurazone can be mitigated by adequate glutathione levels, but that livers lacking sufficient glutathione to scavenge these reactive species may be damaged.     

Apoptosis/necrosis switch in two different cancer cell lines: Influence of benzoquinone- and hydrogen peroxide-induced oxidative stress intensity, and glutathione.

Depending on the strength of oxidative stress, cells exhibit proliferative, apoptotic or necrotic responses. We have investigated whether the severity of glutathione (GSH) depletion could determine the type of cell death using 1,4-benzoquinone (BQ) and H(2)O(2) in two different tumor cell lines (human mammary gland carcinoma MCF-7 and rat hepatoma H5-6). BQ-treated surviving cells showed an increase in GSH, but no detectable oxidized glutathione (GSSG) nor reactive oxygen species (ROS) augmentation. Alternatively, H(2)O(2) depressed GSH. BQ induced mostly apoptosis, up to 90% cell elimination, while necrosis was prominent in H(2)O(2)-treated cultures. The resistance of BQ-treated cells to necrosis could be due to increased cellular GSH and formation of BQ-GSH conjugates which are less toxic than free BQ, minimal toxicity being provided by GS4-BHQ. This ability of certain cancer cells to tightly keep the apoptotic pathway may have therapeutic applications for oxidation-based drugs.     

Redox regulation of PTEN by S-nitrosothiols

PTEN (phosphatase with sequence homology to tensin) is a phosphatidylinositol 3,4,5-trisphosphate phosphatase that regulates many cellular processes. Activity of the enzyme is dependent on the redox state of the active site cysteine such that oxidation by H2O2 leads to inhibition. Because S-nitrosothiols are known to modify enzymes containing reactive cysteines, we hypothesized that S-nitrosothiols would oxidize PTEN and inhibit its phosphatase activity. In the present study, we show that S-nitrosocysteine (CSNO), S-nitrosoglutathione (GSNO), and S-nitroso-N-acetylpenicillamine (SNAP) reversibly oxidized recombinant PTEN. In addition, CSNO led to concentration- and time-dependent oxidation of endogenous cellular PTEN. However, in contrast, GSNO and SNAP were effective only when coincubated with cysteine, suggesting that these nitrosothiols must react with cysteine to form CSNO, which can be transferred across cell membranes. Oxidation of cellular PTEN resulted from thiol modification and led to reversible inhibition of phosphatase activity. Although oxidation of PTEN by H2O2 led to formation of an intramolecular disulfide, oxidation of PTEN by CSNO seemed to lead to formation of a mixed disulfide. Glutathionylation of cellular proteins by incubating cells with diamide or incubating cellular extracts with GSSG oxidized PTEN in a manner similar to that of CSNO. Overall, these data demonstrate for the first time that S-nitrosothiols oxidatively modify PTEN, leading to reversible inhibition of its phosphatase activity, and suggest that the oxidized species is a mixed disulfide.     

The role of GSH efflux in staurosporine-induced apoptosis in colonic epithelial cells

Staurosporine (STP) was shown to induce cell apoptosis through formation of reactive oxygen species, but a role for cellular redox has not been defined. In this study, we report that STP (2 microM) caused apoptosis (24+/-3% at 24 h) of human colon adenocarcinoma epithelial cell line HT29 that was preceded by significant glutathione (GSH) and glutathione disulfide (GSSG) efflux (6 h), but independent of changes in cellular glutathione/glutathione disulfide (GSH/GSSG) redox status. The blockade of GSH efflux by gamma-glutamyl glutamate (gamma-GG) or ophthalmic acid was associated with apoptosis attenuation; however, gamma-GG administration after peak GSH efflux (8 h) did not confer cytoprotection. Moreover, lowering cellular GSH through inhibition of its synthesis prevented extracellular GSH accumulation and cell apoptosis, thus validating a link between cellular GSH export and the trigger of cell apoptosis. Inhibition of gamma-glutamyl transferase (GGT1, EC 2.3.2.2)-catalyzed extracellular GSH degradation with acivicin significantly blocked GSH efflux, suggesting that GSH breakdown is a driving force for GSH export. Interestingly, acivicin treatment enhanced extracellular GSSG accumulation, consistent with GSH oxidation. STP-induced HT29 cell apoptosis was associated with caspase-3 activation independent of caspase-8 or caspase-9 activity; accordingly, inhibitors of the latter caspases were without effect on STP-induced apoptosis. STP similarly induced GSH efflux and apoptosis in a non-malignant human NCM460 colonic cell line in association with caspase-3 activation. Collectively, our results demonstrate that STP induction of apoptosis in malignant and non-malignant colonic cells is temporally linked to the export of cellular GSH and the activation of caspase-3 without caspase-8 or -9 involvement.     

人参皂苷保护小鼠精原细胞氧化损伤的研究

目的观察人参皂苷对活性氧引起的小鼠睾丸生殖细胞氧化损伤的保护作用。方法利用体外培养的小鼠精原细胞建立氧化应激模型,通过检测生殖细胞活性、脂质过氧化产物丙二醛(MDA)生成、超氧化物歧化酶(SOD)活性和谷胱甘肽(GSH)水平评价人参皂苷对精原细胞氧化损伤的缓解作用。结果次黄嘌呤/黄嘌呤氧化酶(HX/XO)体系产生的活性氧可引起生殖细胞活性降低、MDA的生成量增加、SOD活性和GSH水平降低,而添加人参皂苷(10mg·L-1)能恢复HX/XO引起的生殖细胞活性、SOD活性和GSH水平的下降以及MDA生成的增加。结论人参皂苷可通过抗氧化作用保护活性氧引起的小鼠精原细胞氧化损伤。     

Oxidative stress mediated cytotoxicity of cyanide in LLC-MK2 cells and its attenuation by alpha-ketoglutarate and N-acetyl cysteine

Cyanide is a rapidly acting mitochondrial poison that inhibits cellular respiration and energy metabolism leading to histotoxic hypoxia followed by cell death. Cyanide is predominantly a neurotoxin but its toxic manifestations in non-neuronal cells are also documented. This study addresses the oxidative stress mediated cytotoxicity of cyanide in Rhesus monkey kidney epithelial cells (LLC-MK2). Cells were treated with various concentrations of potassium cyanide (KCN) for different time intervals and cytotoxicity was evidenced by increased leakage of intracellular lactate dehydrogenase, mitochondrial dysfunction (MTT assay) and depleted energy status of cells (ATP assay). Cytotoxicity was accompanied by lipid peroxidation indicated by elevated levels of malondialdehyde (MDA), reactive oxygen species (ROS) and reactive nitrogen species (RNS) (DCF-DA staining), diminished cellular antioxidant status (reduced glutathione (GSH), glutathione peroxidase, superoxide dismutase and catalase). These cascading events triggered an apoptotic kind of cell death characterized by oligonucleosomal DNA fragmentation and nuclear fragmentation (Hoechst 33342 staining). Apoptosis was further confirmed by increased caspase-3 activity. Cyanide-induced cytotoxicity, oxidative stress, and DNA fragmentation were prevented by alpha-ketoglutarate (A-KG) and N-acetyl cysteine (NAC). A-KG is a potential cyanide antidote that confers protection by interacting with cyanide to form cyanohydrin complex while NAC is a free radical scavenger and enhances the cellular GSH levels. The study reveals cytotoxicity of cyanide in cells of renal origin and the protective efficacy of A-KG and NAC.     

Roles of oxidative stress and glutathione depletion in JP-8 jet fuel-induced apoptosis in rat lung epithelial cells

The toxic jet fuel JP-8 induces morphological and biochemical changes characteristic of apoptosis in rat lung epithelial (RLE-6TN) cells. The mechanism of JP-8 toxicity in these cells was further investigated in an attempt to identify potential therapeutic interventions. Given that oxidative stress and changes in the concentrations of endogenous antioxidants, such as glutathione (GSH), have been associated with the cellular damage elicited by numerous toxicants, the possibility that JP-8 induces cellular oxidative stress was investigated. Experimentally induced depletion of intracellular GSH or exposure of cells to a low concentration of H(2)O(2) markedly enhanced JP-8-induced cell death. A significant reduction in intracellular concentrations of GSH was noted in RLE-6TN cells shortly after exposure to JP-8. Furthermore, JP-8 induced the generation of reactive oxygen species (ROS) in RLE-6TN cells. Consistent with the notion that JP-8 toxicity is mediated by generation of ROS and depletion of intracellular GSH, JP-8-induced cell death was inhibited by exogenous GSH or the thiol-containing antioxidant N-acetyl-cysteine. This protective effect was associated with marked inhibition of both the activation of caspase-3 and the loss of the mitochondrial membrane potential induced by JP-8. Inhibition of the JP-8-induced activation of poly(ADP-ribose) polymerase by 3-aminobenzamide did not protect cells against JP-8 toxicity. Together, these results indicate that thiol antioxidants are highly effective in rescuing cells from JP-8-induced cell death and that they may provide a basis for new therapeutic approaches to counteract JP-8 toxicity.     

Mechanisms of N-acetyl-p-benzoquinone imine cytotoxicity

N-Acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, rapidly reacts at physiological pH with glutathione (GSH) forming an acetaminophen-glutathione conjugate and stoichiometric amounts of acetaminophen and glutathione disulfide (GSSG). The same reaction products are formed in isolated hepatocytes incubated with NAPQI. In hepatocytes which have been treated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) in order to inhibit glutathione reductase, the initial rise in GSSG concentration in the presence of NAPQI is maintained, whereas GSSG is rapidly reduced back to GSH in untreated hepatocytes. Oxidation by NAPQI of GSH to GSSG and the reduction of GSSG back to GSH by the NADPH-dependent glutathione reductase appear to be responsible for the rapid oxidation of NADPH that occurs in hepatocytes incubated with NAPQI in that the effect is blocked by pretreatment of cells with BCNU. When added to hepatocytes, NAPQI not only reacts with GSH but also causes a loss in protein thiol groups. The loss in protein thiols occurs more rapidly in cells pretreated with BCNU or diethylmaleate. Whereas both of these treatments enhance cytotoxicity caused by NAPQI, BCNU pretreatment has no effect on the covalent binding of [14C-ring]NAPQI to cellular proteins. Furthermore, dithiothreitol added to isolated hepatocytes after maximal covalent binding of [14C-ring]NAPQI but preceding cell death protects cells from cytotoxicity and regenerates protein thiols. Thus, the toxicity of NAPQI to isolated hepatocytes may result primarily from its oxidative effects on cellular proteins.     

Effects of oxygen on the reactivity of nitrogen oxide species including peroxynitrite

This paper describes the O(2)-dependent control of the reactivity of nitrogen oxide species for the production of biologically important nitrated and nitrosated compounds. In this study, the effects of O(2) on the reactivity of NO, NO(2), and ONOO(-)/ONOOH for nitration of tyrosine (Tyr) and nitrosation of glutathione (GSH) and morpholine (MOR) were examined. NO produced S-nitrosoglutathione (GSNO) and N-nitrosomorpholine (NMOR) through the formation of N(2)O(3) under aerobic conditions, and NO(2) produced 3-nitrotyrosine (3-NO(2)Tyr), GSNO, and NMOR. Transnitrosation from GSNO to MOR was observed only in the presence of O(2). Although preformed ONOO(-)/ONOOH produced all the products under aerobic conditions, the formation of 3-NO(2)Tyr and GSNO was markedly reduced and the formation of NMOR was enhanced under anaerobic conditions. The reactivity of the CO(2) adduct of ONOO(-) was similarly dependent on O(2). 3-NO(2)Tyr was produced effectively by reaction with ONOO(-)/ONOOH at the O(2) concentration of 270 microM and by reaction with its CO(2) adduct at O(2) concentrations greater than 5 microM. Generation of.OH from ONOO(-)/ONOOH was suppressed under anaerobic conditions. The reactivity of ONOO(-)/ONOOH and.OH generation from ONOO(-) were reversibly controlled by the O(2) concentration.     

Chemical instability and methods for measurement of cisplatin adducts formed by interactions with cysteine and glutathione

Reactions between cisplatin or its aquated species and L-cysteine (L-cys) or glutathione (GSH) were studied in vitro using liquid chromatography on-line with inductively coupled plasma mass spectrometry (LC-ICPMS) and/or electrospray ionization mass spectrometry (LC-MS) in order to obtain information on the mechanisms occurring in treated patients. Reaction between cisplatin and L-cys yielded initially 4 adducts of which only 2 were stable and detectable after 24 hours incubation; their structures corresponded to bis-platinum cysteinyl adducts. Reaction of cisplatin with GSH proceeded via the formation of at least 11 glutathione-platinum adducts (G1 - G11) which underwent parallel reactions within 24 hours of incubation, probably to form higher molecular weight species. Of the 11 adducts, only 2, G3 and G7, whose structures correspond to [Pt(NH3)2Cl]2(SG) and [Pt(NH3)2OH]2(SG) were still present in the reaction mixture after 24 hours incubation. This study shows that GSH, and to a lesser extent L-cys, incubated with cisplatin in vitro forms unstable and reactive platinum compounds and that LC-ICPMS and LC-MS are 2 complementary techniques suitable for the study of organometallic compounds.     

Protective effect of N-acetyl-L-cysteine against maneb induced oxidative and apoptotic injury in Chinese hamster V79 cells

The role of antioxidant N-acetyl-L-cysteine (NAC) in protection against cellular changes triggered by maneb during in vitro exposure was investigated in cultured Chinese hamster V79 cells. We observed high apoptotic activity and high oxidative stress induced by exposure to maneb evidenced by a statistically significant increase in lipid peroxidation (measured as TBARS - thiobarbituric acid reactive substances) as well as a decrease of glutathione (GSH) and glutathione disulfide (GSSG) ratio (GSH/GSSG). Maneb did not exhibit any effect on protein oxidation (measured by protein carbonyls content). NAC suppressed cellular changes induced by maneb in V79 cells. NAC pre-treatment prevented TBARS production and significantly decreased the number of apoptotic cells. However, protective effect of NAC on GSH and GSSG levels has been shown only in cells exposed to lower concentration of maneb (100 μM).     

Chrysotile-mediated imbalance in the glutathione redox system in the development of pulmonary injury.

A significant depletion in the content of glutathione (GSH) and alteration in GSH redox system enzymes were observed in the lung of chrysotile-exposed animals (5 mg) during different developmental stages of asbestosis. In the alveolar macrophages (AM) of exposed animals, the depletion in GSH started from day 1 and reached a maximum at day 16, whereas in lung tissue the maximum depletion was observed when fibrosis has matured. It appears that cellular GSH depletion triggers oxidative stress in the system as observed from increased thiobarbituric acid reactive substance (TBARS) production and alteration in the activities of glutathione peroxidase (GPx), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PD) and glutathione S-transferase (GST), the enzymes regulating oxidative tone. The depletion in GSH was also observed in red blood cells (RBC) of the exposed animals reaching a maximum when fibrosis matured. Thus the observed depletion in GSH, ascorbic acid and alteration in GSH redox system enzymes may be involved in fibrosis and carcinogenesis induced by chrysotile.     

H(2)O(2) mediates oxidative stress-induced epidermal growth factor receptor phosphorylation

We used a well-established thiol-alkylating agent, N-ethylmaleimide (NEM), to oxidatively stress human keratinocytes. Time course studies revealed that NEM rapidly depleted keratinocytes of reduced glutathione (GSH), which was followed by rapidly increasing levels of intracellular reactive oxygen species (ROS) and subsequently by phosphorylation of epidermal growth factor receptor (EGFR). Pretreatment with antioxidants or enhanced catalase activity in keratinocytes inhibited ROS/H(2)O(2) accumulation and EGFR phosphorylation, demonstrating that H(2)O(2) production is a mediator required for EGFR phosphorylation. Collectively, these results suggest a sequence of events leading to EGFR phosphorylation which is likely shared by oxidative stress-inducing agents, namely: (1) GSH depletion; (2) H(2)O(2) accumulation; and (3) EGFR phosphorylation. We propose that depletion of GSH and accumulation of H(2)O(2) are upstream events and critical mediators required for ligand-independent phosphorylation of growth factor receptors in response to oxidative stress.     

Glutathione level regulates HNE-induced genotoxicity in human erythroleukemia cells

4-hydroxy-trans-2-nonenal (HNE) is one of the most abundant and toxic lipid aldehydes formed during lipid peroxidation by reactive oxygen species. We have investigated the genotoxic effects of HNE and its regulation by cellular glutathione (GSH) levels in human erythroleukemia (K562) cells. Incubation of K562 cells with HNE (5-10 microM) significantly elicited a 3- to 5-fold increased DNA damage in a time- and dose-dependent manner as measured by comet assay. Depletion of GSH in cells by L-buthionine-[S,R]-sulfoximine (BSO) significantly increased HNE-induced DNA damage, whereas supplementation of GSH by incubating the cells with GSH-ethyl ester significantly decreased HNE-induced genotoxicity. Further, overexpression of mGSTA4-4, a HNE-detoxifying GST isozyme, significantly prevented HNE-induced DNA damage in cells, and ablation of GSTA4-4 and aldose reductase with respective siRNAs further augmented HNE-induced DNA damage. These results suggest that the genotoxicity of HNE is highly dependent on cellular GSH/GST/AR levels and favorable modulation of the aldehyde detoxification system may help in controlling the oxidative stress-induced complications.     

Dansyl glutathione as a trapping agent for the quantitative estimation and identification of reactive metabolites

A sensitive and quantitative method was developed for the estimation of reactive metabolite formation in vitro. The method utilizes reduced glutathione (GSH) labeled with a fluorescence tag as a trapping agent and fluorescent detection for quantitation. The derivatization of GSH was accomplished by reaction of oxidized glutathione (GSSG) with dansyl chloride to form dansylated GSSG. Subsequent reduction of the disulfide bond yielded dansylated GSH (dGSH). Test compounds were incubated with human liver microsomes in the presence of dGSH and NADPH, and the resulting mixtures were analyzed by HPLC coupled with a fluorescence detector and a mass spectrometer for the quantitation and mass determination of the resulting dGSH adducts. The comparative chemical reactivity of dGSH vs GSH was investigated by monitoring the reaction of each with 1-chloro-2,4-dinitrobenzene or R-(+)-pulegone after bioactivation. dGSH was found to be equivalent to GSH in chemical reactivity toward both thiol reactive molecules. dGSH did not serve as a cofactor for glutathione S-transferase (GST)-mediated conjugation of 3,4-dichloronitrobenzene in incubations with either human liver S9 fractions or a recombinant GST, GSTM1-1. Reference compounds were tested in this assay, including seven compounds that have been reported to form GSH adducts along with seven drugs that are among the most prescribed in the current U.S. market and have not been reported to form GSH adducts. dGSH adducts were detected and quantitated in incubations with all seven positive reference compounds; however, there were no dGSH adducts observed with any of the widely prescribed drugs. In comparison with existing methods, this method is sensitive, quantitative, cost effective, and easy to implement.     

Cytotoxic effects of thioxanthone derivatives as photoinitiators on isolated rat hepatocytes

Thioxanthone and its analogues, 2- or 4-isopropylthioxanthone, 2-chlorothioxanthone , 2,4-diethylthioxanthone (DETX) and xanthone, are used as photoinitiators of ultraviolet (UV) light-initiated curable inks. As these photoinitiators were found in numerous food/beverage products packaged in cartons printed with UV-cured inks, the cytotoxic effects and mechanisms of these compounds were studied in freshly isolated rat hepatocytes. The toxicity of DETX was greater than that of other compounds. DETX elicited not only concentration (0–2.0 mm )- and time (0–3 hours)-dependent cell death accompanied by the depletion of cellular adenosine triphosphate (ATP), and reduced glutathione (GSH) and protein thiol levels, but also the accumulation of GSH disulfide and malondialdehyde. Pretreatment of hepatocytes with either fructose at a concentration of 10 mm or N-acetyl-l -cysteine (NAC) at a concentration of 5.0 mm ameliorated DETX (1 mm )-induced cytotoxicity. Further, the exposure of hepatocytes to DETX resulted in the induction of reactive oxygen species (ROS) and loss of mitochondrial membrane potential, both of which were partially prevented by the addition of NAC. These results indicate that: (1) DETX-induced cytotoxicity is linked to mitochondrial failure and depletion of cellular GSH; (2) insufficient cellular ATP levels derived from mitochondrial dysfunction were, at least in part, ameliorated by the addition of fructose; and (3) GSH loss and/or ROS formation was prevented by NAC. Taken collectively, these results suggest that the onset of toxic effects caused by DETX may be partially attributable to cellular energy stress as well as oxidative stress.     

Mitochondrial oxidative stress in female and male rat brain after ex vivo carbon monoxide treatment

Carbon monoxide (CO) is the most common cause of fatal poisoning all over the world. At the cellular level, a combination of tissue hypoxia and direct cellular damage underlie the pathophysiology of CO toxicity. The purpose of this study was to determine the effect of CO treatment on oxidative stress parameters in mitochondria isolated from male and female rat brains. Mitochondria prepared from frontal cortex, hippocampus and corpus striatum were treated with 0.1% CO at 37 degrees C for 30 minutes; control samples were not exposed to CO. Cytochrome c oxidase activity (COX), lipid peroxidation (thiobarbituric acid reactive species = TBARS), protein oxidation (protein carbonyls) and glutathione (GSH) levels were measured in CO treated and control samples. Our results confirmed previous studies reporting the inhibition of cytochrome c oxidase activity by CO in rat brain. Additionally, protein carbonyl levels in the hippocampus and striatum significantly increased after CO treatment in male rats. While CO treatment caused a significant decrease in GSH levels in the cortex and striatum in male rats, reduced GSH levels were observed in the cortex and hippocampus in female rats following CO exposure. Taken together, our data suggest a role for mitochondrial oxidative stress in CO toxicity at the cellular level during CO poisoning.     

Thiram-induced cytotoxicity is accompanied by a rapid and drastic oxidation of reduced glutathione with consecutive lipid peroxidation and cell death

The toxic effect of thiram, a widely used dithiocarbamate fungicide, was investigated in cultured human skin fibroblasts. Cell survival assays demonstrated that thiram induced a dose-dependent decrease in the viable cell recovery. Thiram exposure resulted in a rapid depletion of intracellular reduced glutathione (GSH) content with a concomitant increase in oxidized glutathione (GSSG) concentration. Alteration of glutathione levels was accompanied by a dose-dependent decrease in the activity of glutathione reductase (GR), a key enzyme for the regeneration of GSH from GSSG. Thiram-exposed cells exhibited increased lipid peroxidation reflected by enhanced thiobarbituric acid reactive substances (TBARS) production, suggesting that GSH depletion and the lower GR activity gave rise to increased oxidative processes. To investigate the role of decreased GSH content in the toxicity of thiram, GSH levels were modulated prior to exposure. Pretreatment of fibroblasts with N-acetyl-L-cysteine (NAC), a GSH biosynthesis precursor, prevented both lipid peroxidation and cell death induced by thiram exposure. In contrast, thiram cytotoxicity was exacerbated by the previous depletion of cellular GSH by L-buthionine-(S,R)-sulfoximine (BSO). Taken together, these results strongly suggest that thiram induces GSH depletion, leading to oxidative stress and finally cell death.