Influences of glutathione status on different cytocidal responses of monolayer rat hepatocytes exposed to aflatoxin B1 or acetaminophen

In short-term primary monolayer cultures of rat hepatocytes, aflatoxin B1 (AFB1) causes a characteristic prelethal cytomorphological response in which peripheral attached cytoplasm contracts segmentally to form finger-like blebs. This response precedes lethal injury as detected by release of lactate dehydrogenase (LDH) into culture medium. We compared the influences of various modifiers of cellular glutathione (GSH) status on cytocidal responses of Fischer 344 rats hepatocytes exposed to AFB1 or acetaminophen (AAP), a hepatotoxin which does not produce segmental cytoplasmic contraction. N-Acetylcysteine (4 mM) reduced the degree of LDH release by AAP (4 to 16 mM) but was not protective against cell killing by AFB1, although it slightly reduced the percentage of hepatocytes with segmental cytoplasmic contraction at 6 hr. BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) at 40 microM markedly inhibited glutathione reductase and also strongly potentiated cell killing by AAP but did not significantly influence segmental cytoplasmic contraction or LDH release in response to AFB1. Diethylmaleate (40 to 160 microM), a depletor of hepatocellular GSH, and buthionine-D,L-sulfoximine (4 mM), an inhibitor of GSH synthesis, each did not alter hepatocyte killing by AFB1 but were strong potentiators of toxicity of AAP. AAP inhibited glutathione reductase but AFB1 did not. Total GSH concentrations at 6 and 18 hr were reduced by AAP and to a lesser extent by AFB1 in comparison with control cultures. These findings demonstrate that, in contrast to AAP toxicity, the characteristic mode of hepatocyte killing by AFB1 in monolayer cultures is substantially independent of induced alterations in GSH. These results indicate that GSH-dependent detoxification mechanisms do not play a major role in removing necrogenic metabolites of AFB1 in Fischer 344 rat hepatocytes. They further suggest that prelethal responses of AFB1-injured hepatocytes are not affected by GSH-dependent cytoprotective mechanisms.     

The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity

Acetaminophen (AAP) overdose can cause severe hepatotoxicity and even liver failure in experimental animals and humans. Despite substantial efforts over the last 30 years, the mechanism of AAP-induced liver cell injury is still not completely understood. It is widely accepted that the injury process is initiated by the metabolism of AAP to a reactive metabolite, which first depletes glutathione and then binds to cellular proteins including a number of mitochondrial proteins. One consequence of this process may be the observed inhibition of mitochondrial respiration, ATP depletion and mitochondrial oxidant stress. In the presence of sufficient vitamin E, reactive oxygen formation does not induce severe lipid peroxidation but the superoxide reacts with nitric oxide to form peroxynitrite, a powerful oxidant and nitrating agent. Peroxynitrite can modify cellular macromolecules and may aggravate mitochondrial dysfunction and ATP depletion leading to cellular oncotic necrosis in hepatocytes and sinusoidal endothelial cells. Thus, we hypothesize that reactive metabolite formation and protein binding initiate the injury process, which may be then propagated and amplified by mitochondrial dysfunction and peroxynitrite formation. This concept also reconciles many of the controversial findings of the past and provides a viable hypothesis for the mechanism of hepatocellular injury after AAP overdose.     

Glutathione status of isolated rat hepatocytes affects bile acid-induced cellular necrosis but not apoptosis

An accumulation of hydrophobic bile acids is implicated in the pathogenesis of cholestatic liver diseases. In the present study, we determined if hydrophobic bile acid-induced cellular injury compromised hepatocyte glutathione (GSH) status, and if modulating intracellular GSH levels prevented or facilitated bile acid-induced cellular cytotoxicities. Freshly isolated rat hepatocytes incubated with >/=125 microM of the hydrophobic bile acid, glycochenodeoxycholic acid (GCDC), underwent a time- and dose-dependent decrease of intracellular GSH levels by 4-h incubation. This loss of intracellular GSH was not associated with an increase of intracellular GSH disulfide (GSSG). Rather, GCDC stimulated the dose-dependent accumulation of extracellular GSSG. The mechanism for extracellular GSSG accumulation by GCDC was through increased efflux of reduced GSH from hepatocytes into the media, where it subsequently oxidized to GSSG. Treatment of hepatocytes with GCDC (0-750 microM) did not directly alter GSH-dependent enzyme activities. The reduction of intracellular GSH with 125 microM GCDC correlated with extensive apoptosis at this concentration as determined by fluorescence microscopy of DAPI (4, 6-diamindino-2-phenylindole hydrochloride)-stained nuclei. Higher concentrations of GCDC (>/=500 microM) favored cellular necrosis and lipid peroxidation. Depleting GSH by treating hepatocytes with 1-bromoheptane increased their sensitivity toward GCDC-induced cellular necrosis, but not apoptosis. However, enhancing the hepatocyte GSH content by supplementation with GSH-ethylester (GSH-EE) failed to protect hepatocytes against either mode of cellular death. In conclusion, while GCDC-induced cytotoxicities were associated with an increased efflux of GSH from rat hepatocytes, GSH status modulated GCDC-induced necrosis, but not apoptosis.     

Oltipraz-induced amelioration of acetaminophen hepatotoxicity in hamsters. I. Lack of dependence on glutathione

These studies were designed to test the hypothesis that oltipraz (OTP) provided protection against AAP intoxication in a sensitive species, the hamster; and further, to show that the sparing effect was related to the marked increase in hepatic reduced glutathione (GSH) levels. Dose-response and time-course experiments demonstrated that maximal increases in liver GSH occurred at 48 hr after an oltipraz dose of approximately 2.0 mmol/kg (po). Accompanying greater GSH levels were increased glutathione disulfide (GSSG) levels. Decreased indices of the oxidation state of glutathione and of hepatic pyridine nucleotides indicated a greater share of glutathione existed as GSH and that increased reducing equivalents were present, respectively. Additionally, glutathione disulfide reductase activity was greater in OTP-treated groups. Glutathione S-transferase activities were only marginally increased. OTP treatment did not elicit observable hepatotoxicity, whereas AAP (2.6 mmol/kg, ip) resulted in a reproducible model of liver damage. OTP-treated groups were protected from AAP-induced toxicity, as shown by decreased plasma appearance of liver enzymes and unremarkable histopathology. However, the degree of liver GSH depletion by AAP was fourfold greater in non-OTP treated groups compared to those which had received the dithiolthione. To test the importance of increased hepatic GSH, the biosynthesis of glutathione was interrupted. Buthionine sulfoximine (BSO) treatment decreased hepatic GSH, the biosynthesis of glutathione was interrupted. Buthionine sulfoximine (BSO) treatment decreased hepatic GSH content to 50% of control in hamsters which either had or had not received OTP. The groups receiving BSO and AAP incurred 83% lethality, while no lethality, unremarkable liver histopathology, and plasma enzyme levels consistent with control were found in the group receiving OTP, BSO, and AAP. Treatment with BSO only had no influence on hepatotoxicity parameters. These results indicate that the increased GSH levels in the OTP-treated hamster are coincidental to the sparing effect of OTP and are not central to the protection scheme in AAP-induced hepatotoxicity.     

Chemoprotective effect of insulin-like growth factor I against acetaminophen-induced cell death in Chang liver cells via ERK1/2 activation

The insulin-like growth factor (IGF) system and type-I IGF receptor (IGF-IR) signaling are involved in protecting against chemotherapeutic drug-induced cell death in human hepatoma cells. Acetaminophen (AAP) hepatotoxicity is the leading cause of liver failure, and the prevention of AAP-induced cell death has been the focus of many studies. We determined whether IGF-I could protect against AAP-induced cell death in Chang liver cells and investigated the protective mechanism. Based on the results of MTS assays, LDH release assays, Hoechst 33342 cell staining, and DNA fragmentation experiments, AAP induced cell death in a dose-dependent manner. According to Western blot analysis, treatment with AAP increased the level of poly(ADP-ribose) polymerase (PARP) fragments in cells compared with that in control cells; however, caspase-3, a critical signaling molecule in apoptosis, was not activated after AAP overdose. Moreover, combined treatment with AAP and IGF-I inhibited PARP cleavage, which was consistent with the ability of IGF-I to restore the level of glutathione (GSH) and cell viability in GSH and MTS assays, respectively. We investigated whether the protective effect of IGF-I against AAP cytotoxicity is related to the extracellular signal-related kinase ERK1/2, which is generally activated by mitogenic and proliferative stimuli such as growth factors. Compared with AAP treatment alone, IGF-I and AAP co-treatment increased ERK1/2 phosphorylation but inhibited PARP cleavage. Thus ERK1/2 activation is instrumental in the protective effect of IGF-I against AAP-induced cell death in Chang liver cells.     

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

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

1,2-Dichlorobenzene-mediated hepatocellular oxidative stress in Fischer-344 and Sprague-Dawley rats

1,2-Dichlorobenzene (1,2-DCB) is a potent hepatotoxicant in male Fischer 344 (F-344) rats but not in Sprague-Dawley (SD) rats. While Kupffer cell-dependent oxidative stress plays a role in the progression of 1,2-DCB-mediated liver injury, we hypothesize that initiation of liver injury is due to oxidative events within the hepatocyte. This study compared hepatocellular oxidative stress marked by glutathione disulfide (GSSG) and glutathione (GSH) production in either bile, liver, or isolated hepatocytes of F-344 and SD rats following 1,2-DCB administration. Hepatic GSH concentrations were depleted at a greater rate in F-344 than in SD rats within 12 h of 1,2-DCB administration (3.6 mmol/kg ip). In bile, GSSG concentrations were threefold greater in F-344 rats compared to SD rats by 9 h of 1,2-DCB treatment. Moreover, 1-aminobenzotriazole but not gadolinium chloride pretreatment blocked the rise in biliary GSSG concentrations following 1,2-DCB administration. In in vitro studies, isolated hepatocytes of F-344 rats had a 15% increase in cellular GSSG concentrations following 1 h of 1,2-DCB (3.55 nmol) exposure, while GSH decreased 22% by 6.5 h compared to controls. In contrast, isolated SD hepatocytes exposed to 1,2-DCB had no increase in GSSG and only an 8% reduction in GSH. Furthermore, parameters of lipid peroxidation were increased in F-344 rats and not in SD rats. Collectively, these data suggest that hepatocellular oxidative stress is dependent upon bioactivation and the enhanced oxidative stress in the F-344 rat may explain its susceptibility to 1,2-DCB compared to the SD rat.     

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.     

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).     

Intracellular glutathione plays important roles in pyrrolizidine alkaloids-induced growth inhibition on hepatocytes

Pyrrolizidine alkaloids (PAs) are well-known natural hepatotoxins distributed widely in thousands of plants in the world. Adonifoline (Adon), senecionine (Sene) and monocrotaline (Mono) are retronecine-type PAs, and the present study is designed to observe the effects of intracellular glutathione on toxicity of these three PAs in human normal liver L-02 cells. The ratio of cellular reduced glutathione (GSH) and oxidized glutathione (GSSG) was assayed after L-02 cells were incubated with these three PAs for various times. Results showed that Adon, Sene and Mono all significantly decreased the ratio of GSH/GSSG in L-02 cells in the time- and concentration-dependent manner. The results of 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) and trypan blue staining assay showed that these three PAs all significantly decreased cell viability in L-02 cells when pretreated with 10 μM BSO (L-Buthionine-S-R-Sulfoximine) for 24 h to deplete intracellular GSH. Further results showed that anti-oxidant compounds such as NAC (N-Acetyl-Cysteine) and GSH could rescue the cytotoxicity caused by these three PAs with BSO pretreatment. Taken together, those results suggest that intracellular GSH plays important roles in regulating the cytotoxicity induced by PAs.     

Formation and reduction of glutathione-protein mixed disulfides during oxidative stress. A study with isolated hepatocytes and menadione (2-methyl-1,4-naphthoquinone)

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) resulted in a dose-dependent depletion of intracellular reduced glutathione (GSH), most of which was oxidized to glutathione disulfide (GSSG). Menadione metabolism was also associated with a dose- and time-dependent inhibition of glutathione reductase, impairing the regeneration of GSH from GSSG produced during menadione-induced oxidative stress. Inhibition of glutathione reductase by pretreatment of hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) greatly potentiated both GSH depletion and GSSG formation during the metabolism of low concentrations of menadione. Concomitant with GSH oxidation, mixed disulfides between glutathione and protein thiols were formed. The amount of mixed disulfides produced and the kinetics of their formation were dependent on both the intracellular GSH/GSSG ratio and the activity of glutathione reductase. The mixed disulfides were mainly recovered in the cytosolic fraction and, to a lesser extent, in the microsomal and mitochondrial fractions. The removal of glutathione from protein mixed disulfides formed in hepatocytes exposed to oxidative stress was dependent on GSH and/or cysteine and appeared to occur predominantly via a thiol-disulfide exchange mechanism. However, incubation of the microsomal fraction from menadione-treated hepatocytes with purified glutathione reductase in the presence of NADPH also resulted in the reduction of a significant portion of the glutathione-protein mixed disulfides present in this fraction. Our results suggest that the formation of glutathione-protein mixed disulfides occurs as a result of increased GSSG formation and inhibition of glutathione reductase activity during menadione metabolism in hepatocytes.     

In vivo effects of fenthion on oxidative processes by the modulation of glutathione metabolism in the brain of Oreochromis niloticus

The present study was designed to understand the oxidative stress potential of fenthion, an organophosphate (OP) pesticide and its involvement in glutathione metabolism modulated buthionine sulfoximine (BSO, 50 mg/kg) and N-acetylcysteine (NAC, 100 mg/kg) in the brain of fish, Oreochromis niloticus. A sublethal fenthion concentration (0.45 mg/L) was applied for 24, 48, and 96 h together with injection with BSO or NAC; following treatment, recovery periods for 24, 48, and 96 h were allowed. Total glutathione (tGSH), oxidized glutathione (GSSG), lipid peroxidation, protein level, and GSH-related enzyme activities were analyzed by using spectrophotometric methods. Fenthion in applied concentration did not change GSH levels, but increased GSSG levels. BSO application in fenthion exposure caused a depletion in GSH, while increasing the GSSG levels. Glutathione peroxidase (GPx; EC 1.11.1.9) specific activity increased in fenthion-applied groups at 24-h treatment. gamma-Glutamylcysteinyl synthetase (gamma-GCS; EC 6.3.2.2) was not detected in the brain. NAC injection in fenthion treatment decreased GSH and increased GSSG levels and GST activity. In conclusion, fenthion in sublethal concentration induced an oxidative stress processes in brain. BSO application provided an evidence for the involvement of fenthion in GSH metabolism. NAC elevated the fenthion-induced effects in spite of its antioxidant properties. Recovery period for 96 h was not adequate to eliminate the fenthion-induced changes.     

2-Amino-5-chlorophenol toxicity in renal cortical slices from Fischer 344 rats: effect of antioxidants and sulfhydryl agents

A rapid inhibition of protein synthesis is observed when isolated rat hepatocytes are incubated in the presence of 0.25-0.5 mM of tert-butyl hydroperoxide (tBOOH). Such an inhibition occurs in the absence of a cytolytic effect by tBOOH. Iron chelators (o-phenanthroline and desferrioxiamine), protected against oxidative cell death, but they did not modify the inhibition of protein synthesis caused by tBOOH (0.5 mM), suggesting that free radicals are less implicated in such an impairment. Electron micrographs of hepatocytes under oxidative stress show disaggregation of polyribosomes but not oxidative alterations, such as blebs or mitochondrial swelling. Protein synthesis inhibition is accompanied by a decrease in reduced glutathione (GSH) and an increase in glutathione disulfide (GSSG) and the level of protein S-thiolation (protein mixed disulfides formation). Such an increase of GSSG appears as a critical event since diethylmaleate (DEM) at 0.2 mM reduced GSH content by more than 50% but did not affect either GSSG content or protein synthesis. The addition of exogenous GSH and N-acetylcysteine (NAC) to tBOOH-treated hepatocytes significantly reduced the formation of protein mixed disulfides and restored the depressed protein synthesis either completely or partially. We suggest that S-thiolation of some key proteins may be involved in protein synthesis inhibition by tBOOH.     

Pathophysiological role of poly(ADP-ribose) polymerase (PARP) activation during acetaminophen-induced liver cell necrosis in mice.

DNA fragmentation in hepatocytes occurs early after acetaminophen (AAP) overdose in mice. DNA strandbreaks can induce excessive activation of poly(ADP-ribose) polymerases (PARP), which may lead to oncotic necrosis. Based on controversial findings with chemical PARP inhibitors, the role of PARP-1 activation in AAP hepatotoxicity remains unclear. To investigate PARP-1 activation and evaluate a pathophysiological role of PARP-1, we used both PARP inhibitors (3-aminobenzamide; 5-aminoisoquinolinone) and PARP gene knockout mice (PARP-/-). Treatment of C3Heb/FeJ mice with 300 mg/kg AAP resulted in DNA fragmentation and alanine aminotransferase (ALT) release as early as 3 h, with further increase of these parameters up to 12 h. Few nuclei of hepatocytes stained positive for poly-ADP-ribosylated nuclear proteins (PAR) as indicator for PARP-1 activation at 4.5 h. However, the number of PAR-positive cells and staining intensity increased substantially at 6 and 12 h. Pretreatment with 500 mg/kg 3-aminobenzamide before AAP attenuated hepatic glutathione depletion and completely eliminated DNA fragmentation and liver injury. Delayed treatment several hours after AAP was still partially protective. On the other hand, liver injury was not attenuated in PARP-/- mice compared to wild-type animals. Similarly, the specific PARP-1 inhibitor 5-aminoisoquinolinone (5 mg/kg) was not protective. However, 3-aminobenzamide attenuated liver injury in WT and PARP-/- mice. In summary, PARP-1 activation is a consequence of DNA fragmentation after AAP overdose. However, PARP-1 activation is not a relevant event for AAP-induced oncotic necrosis. The protection of 3-aminobenzamide against AAP-induced liver injury was due to reduced metabolic activation and potentially its antioxidant effect but independent of PARP-1 inhibition.     

GSSG/GSH ratios in cryopreserved rat and human hepatocytes as a biomarker for drug induced oxidative stress

The formation of reactive oxygen species (ROS) could cause cellular damage and eventually lead to apoptosis and necrosis. The ratio between oxidized glutathione and reduced glutathione (GSSG-to-GSH ratio) has been used as an important in vitro and in vivo biomarker of the redox balance in the cell and consequently of cellular oxidative stress. This paper optimizes a LC–MS/MS method for the simultaneous determination of GSH and GSSG. The proposed method is based on the derivatization of reduced GSH using iodoacetic acid (IAA) in order to prevent its rapid oxidation to GSSG during sample preparation. The optimized analytical method was applied to evaluate the effect of different pharmaceutical agents on GSSG-to-GSH ratio in cryopreserved rat and human hepatocytes in culture. Hepatocyte viabilities were also determined at the same time by using the WST-1 assay as a direct measurement of cell mitochondrial respiration. The results obtained demonstrate that cryopreserved rat and human hepatocytes in culture are reliable in vitro models for the evaluation of cellular oxidative stress. In addition, the GSSG-to-GSH ratio measurements could be a biomarker of hepatotoxicity providing similar results to those of cytotoxicity assay.     

Fish tolerance to organophosphate-induced oxidative stress is dependent on the glutathione metabolism and enhanced by N-acetylcysteine

Dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP) is an organophosphorus (OP) insecticide and acaricide extensively used to treat external parasitic infections of farmed fish. In previous studies we have demonstrated the importance of the glutathione (GSH) metabolism in the resistance of the European eel (Anguilla anguilla L.) to thiocarbamate herbicides. The present work studied the effects of the antioxidant and glutathione pro-drug N-acetyl-L-cysteine (NAC) on the survival of a natural population of A. anguilla exposed to a lethal concentration of dichlorvos, focusing on the glutathione metabolism and the enzyme activities of acetylcholinesterase (AChE) and caspase-3 as biomarkers of neurotoxicity and induction of apoptosis, respectively. Fish pre-treated with NAC (1 mmol kg(-1), i.p.) and exposed to 1.5 mg l(-1) (the 96-h LC85) of dichlorvos for 96 h in a static-renewal system achieved an increase of the GSH content, GSH/GSSG ratio, hepatic glutathione reductase (GR), glutathione S-transferase (GST), glutamate:cysteine ligase (GCL), and gamma-glutamyl transferase (gammaGT) activities, which ameliorated the glutathione loss and oxidation, and enzyme inactivation, caused by the OP pesticide. Although NAC-treated fish presented a higher survival and were two-fold less likely to die within the study period of 96 h, Cox proportional hazard models showed that hepatic GSH/GSSG ratio was the best explanatory variable related to survival. Hence, tolerance to a lethal concentration of dichlorvos can be explained by the individual capacity to maintain and improve the hepatic glutathione redox status. Impairment of the GSH/GSSG ratio can lead to excessive oxidative stress and inhibition of caspase-3-like activity, inducing cell death by necrosis, and, ultimately, resulting in the death of the organism. We therefore propose a reconsideration of the individual effective dose or individual tolerance concept postulated by Gaddum 50 years ago for the log-normal dose-response relationship. In addition, as NAC increased the tolerance to dichlorvos, it could be a potential antidote for OP poisoning, complementary to current treatments.     

N-acetylcysteine protects against fluoride-induced oxidative damage in primary rat hepatocytes

Fluoride induces the overproduction of free radicals, which might in turn affect various biochemical parameters. Therefore, the aim of this study was to elucidate the role of N-acetylcysteine (NAC) in decreasing fluoride-induced oxidative stress. The fluoride intoxicated (0.002; 0.082; 0.164 mmol/l) rat hepatocytes was pre-treated (60 min) and simultaneously treated with NAC (1 mmol/l). The resulting levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and intracellular reduced glutathione (GSH) were measured along with the total antioxidant status (TAS) to determine whether NAC treatment reduced cell damage and/or the antioxidant state. These results suggest that NAC pre-treatment provides protection against fluoride-induced oxidative stress in hepatocytes.     

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.     

Enalapril cytotoxicity in primary cultures of rat hepatocytes. II. Role of glutathione

The cytotoxicity of enalapril maleate (EN) in primary cultures of rat hepatocytes, at concentrations of 0.5 mM or greater, was measured by the release of lactate dehydrogenase (LDH) into the culture medium. Pretreatment of the hepatocytes with L-buthionine-(S,R)-sulfoximine (BSO) and diethyl maleate (DEM) potentiated the toxicity whereas N-acetyl-L-cysteine (NAC) provided protection. EN produced a dose-dependent reduction in intracellular glutathione (GSH) concentration. This was an early effect, apparent after only 1 h of exposure to the drug, whereas loss of cell viability occurred after 6-18 h. These results suggest that the mechanism of EN cytotoxicity involves a GSH-dependent detoxification pathway.