Phenobarbital-induced cytoprotective mechanisms in menadione metabolism: the role of glutathione reductase and DT-diaphorase.

1. Treatment with N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU) (80 microM) led to decreases in cell viability in both naive and sodium phenobarbital (PB) induced hepatocytes. 2. Dicumarol (30 microM) selectively increased the cytotoxicity of menadione in hepatocytes isolated from naive vs PB-pretreated rats. 3. Inclusion of both BCNU and dicumarol to the incubation medium abolished the characteristic concentration-response curves of the hepatocytes for menadione. 4. A greater proportion of menadione was metabolized by DT-diaphorase in the hepatocytes isolated from PB-pretreated rats. 5. The role of glutathione reductase vs DT-diaphorase in mitigating menadione-cytotoxicity in the naive vs PB-induced hepatocyte is discussed.     

Endogenous defenses against the cytotoxicity of hydrogen peroxide in cultured rat hepatocytes

The catalase activity of cultured rat hepatocytes was inhibited by 90% pretreatment with 20 mM aminotriazole without effect on the activities of glutathione peroxidase or glutathione reductase, or on the viability of the cells over the subsequent 24 h. Glutathione reductase was inhibited by 85% by pretreatment with 300 microM 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) without effect on glutathione peroxidase, catalase, or on viability. Both pretreatments sensitized the hepatocytes to the cytotoxicity of H2O2 generated either by glucose oxidase (0.05-0.5 units/ml) or by the autoxidation of the one-electron-reduced state of menadione (50-250 microM). Aminotriazole pretreatment had no effect on the GSH content of the hepatocytes. BCNU reduced GSH levels by 50%. Depletion of GSH levels to less than 20% of control by treatment with diethyl maleate, however, did not sensitize the cells to either glucose oxidase or menadione, indicating that the effect of BCNU is related to inhibition of the GSH-GSSG redox cycle rather than to the depletion of GSH. With glucose oxidase, most of the cell killing in hepatocytes pretreated with either aminotriazole or BCNU occurred between 1 and 3 h. The antioxidant diphenylphenylenediamine (DPPD) had no effect on viability at 3 h. Catalase added to the culture medium 1 h after the addition of glucose oxidase prevented the cell killing measured at 3 h. The sulfhydryl reagents dithiothreitol (200 microM), N-acetyl-L-cysteine (4 mM), and alpha-mercaptopropionyl-L-glycine (2.5 mM) prevented the cell killing with exogenous H2O2 in hepatocytes sensitized by the inhibition of catalase or glutathione reductase. With menadione, there was no killing of nonpretreated hepatocytes at 1 h, and DPPD did not prevent the cell death after 3 h. Aminotriazole pretreatment enhanced the cell killing at 3 h but not at 1 h, and DPPD was not protective. Catalase added to the medium at 1 h inhibited the cell death measured at 3 h. In contrast, menadione killed hepatocytes pretreated with BCNU within 1 h. DPPD prevented cell death at 1 h, and there was evidence of lipid peroxidation in the accumulation of malondialdehyde in the culture medium. Catalase added with menadione did not prevent the cell killing at 1 h but did prevent it at 3 h. These data indicate that catalase and the GSH-GSSG cycle are active in the defense of hepatocytes against the toxicity of H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Redox cycling and sulphydryl arylation; their relative importance in the mechanism of quinone cytotoxicity to isolated hepatocytes

Quinones are believed to be toxic by a mechanism involving redox cycling and oxidative stress. In this study, we have used 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ), which redox cycles to the same degree as menadione, but does not react with free thiol groups, to distinguish between the importance of redox cycling and arylation of free thiol groups in the causation of toxicity to isolated hepatocytes. Menadione was significantly more toxic to isolated hepatocytes than 2,3-diOMe-1,4-NQ. Both menadione and 2,3-diOMe-1,4-NQ caused an extensive GSH depletion accompanied by GSSG formation, preceding loss of viability. Both compounds stimulated a similar increase in oxygen uptake in isolated hepatocytes and NADPH oxidation in microsomes suggesting they both redox cycle to similar extents. Further evidence for the redox cycling in intact hepatocytes was the detection of the semiquinone anion radicals with electron spin resonance spectroscopy. In addition we have, using the spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide), demonstrated for the first time the formation of superoxide anion radicals by intact hepatocytes. These radicals result from oxidation of the semiquinone by oxygen and further prove that both these quinones redox cycle in intact hepatocytes. We conclude that while oxidative processes may cause toxicity, the arylation of intracellular thiols or nucleophiles also contributes significantly to the cytotoxicity of compounds such as menadione.     

Quinone toxicity in hepatocytes without oxidative stress

The toxicity of quinones is believed to be mediated via redox cycling involving formation of semiquinone radicals which autoxidize to form active oxygen species. However, when the cytotoxicity of benzoquinones was compared using freshly isolated rat hepatocytes, benzoquinones which did not mediate oxidative stress were highly toxic. Thus, the benzoquinone analogs in decreasing order of cytotoxicity were 2-CH3-, 2-Br-, unsubstituted, 2,6-(CH3)2-, 2,5-(CH3)2-, and 2,3,5-(CH3)3-benzoquinone. Cellular thiols were rapidly depleted and glutathione (GSH) was converted to a quinone conjugate without oxidation to glutathione disulfide. No increase in cyanide-resistant respiration was observed and benzoquinone-induced cytotoxicity was not enhanced by inactivation of catalase or glutathione reductase. In contrast, duroquinone [2,3,5,6-(CH3)4-benzoquinone], which stimulated cyanide-resistant respiration and GSH oxidation, was only cytotoxic when catalase or glutathione reductase was inactivated. These results suggest that alkylation and/or oxidative stress may be important mechanisms in the cytotoxicity of benzoquinone derivatives.     

Evidence for stimulated glutathione synthesis by phenobarbital pretreatment during an oxidative challenge in isolated hepatocytes

Hepatocytes isolated from phenobarbital-pretreated and naive male Sprague-Dawley rats were preincubated with 80 microM N, N-bis (2-chloroethyl)-N-nitrosourea and subsequently exposed to varying concentrations of menadione. We observed that the reduced glutathione levels of the hepatocytes isolated from the sodium phenobarbital(PB)-pretreated, but not the naive rats, recovered to near-control levels after exposure to 200 microM menadione. Since this recovery occurred in the presence of N, N-bis (2-chloroethyl)-N-nitrosourea (an inhibitor of glutathione reductase), we hypothesized that this represented a PB-mediated increase in de novo synthesis of glutathione. To test this hypothesis and to further assess the possible contribution of glutathione reductase in the recovery of the glutathione levels, we preincubated hepatocytes isolated from PB-pretreated and naive rats with 2 mM buthionine sulfoximine, with or without N, N-bis (2-chloroethyl)-N-nitrosourea. Following exposure to menadione, samples were periodically removed for glutathione assessment. Consistent with our hypothesis, the addition of buthionine sulfoximine abrogated the ability of the PB-pretreated hepatocytes to restore glutathione levels following a menadione challenge. Buthionine sulfoximine in combination with N, N-bis (2-chloroethyl)-N-nitrosourea completely abolished hepatocellular glutathione homeostasis for all of the concentrations of menadione employed. The findings from this investigation underscore the importance of phenobarbital-mediated increases in glutathione synthesis, as well as the enhanced levels of glutathione reductase, in maintaining the pool of reduced glutathione and ultimately mitigating the consequences of oxidative stress. In addition, these findings suggest that PB pretreatment increases the reserve capacity of the hepatocyte for glutathione synthesis via a hitherto undescribed hormetic mechanism, a reserve expressed fully only on an oxidative stress of sufficient magnitude.     

Toxic consequence of the abrupt depletion of glutathione in cultured rat hepatocytes

Cultured hepatocytes were exposed to two chemicals, dinitrofluorobenzene (DNFB) and diethyl maleate (DEM), that abruptly deplete cellular stores of glutathione. Upon the loss of GSH, lipid peroxidation was evidenced by an accumulation of malondialdehyde in the cultures followed by the death of the hepatocytes. Pretreatment of the hepatocytes with a ferric iron chelator, deferoxamine, or the addition of an antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD), to the culture medium prevented both the lipid peroxidation and the cell death produced by either DNFB or DEM. However, neither deferoxamine nor DPPD prevented the depletion of GSH caused by either agent. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or inhibition of catalase by aminotriazole sensitized the hepatocytes to the cytotoxicity of DNFB. In a similar manner, pretreatment with BCNU potentiated the cell killing by DEM. DPPD and deferoxamine protected hepatocytes pretreated with BCNU and then exposed to DNFB or DEM. These data indicate that an abrupt depletion of GSH leads to lipid peroxidation and cell death in cultured hepatocytes. It is proposed that GSH depletion sensitizes the hepatocyte to its constitutive flux of partially reduced oxygen species. Such an oxidative stress is normally detoxified by GSH-dependent mechanisms. However, with GSH depletion these activated oxygen species are toxic as a result of the iron-dependent formation of a potent oxidizing species.     

Alterations in energy status by menadione metabolism in hepatocytes isolated from fasted and fed rats

The biochemical mechanism of cytotoxicity, induced by the quinoid compound 2-methyl 1,4-naphthoquinone (menadione), was investigated in hepatocytes freshly isolated from fasted and fed rats. Hepatocytes from fasted rats were significantly more vulnerable to the toxicity of menadione than hepatocytes from fed rats. Menadione (150 microM) induced a 50% loss of viability of cells (LT50) from fasted rats after 55 min of incubation, whereas a LT50 of 80 min was observed after exposure of hepatocytes from fed rats to menadione. Glutathione and NADPH levels were rapidly depleted by menadione metabolism. This depletion was sustained during the incubation period. No significant differences were found in the time course and extent of the menadione-induced glutathione and NADPH depletion in hepatocytes of both nutritional states. Menadione also affected the energy status of the hepatocytes. The ATP content of cells from fasted rats decreased to 50% (AT50) within 18 min of exposure to menadione, whereas a 50% loss of ATP content of hepatocytes from fed rats was reached at 65 min. In contrast to depletion of glutathione and NADPH, the time course and extent of menadione-induced ATP depletion correlated well with the time of onset and rate of cell killing. Our results suggest that menadione metabolism may interfere with both mitochondrial and glycolytic ATP production. Depletion of ATP might be a critical step in menadione-induced cytotoxicity.     

Studies on the mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine cytotoxicity in isolated hepatocytes

Oxidative stress and covalent binding have been proposed as possible mechanisms involved in the cytotoxic effects of the parkinsonism-causing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the toxicity induced by MPTP in isolated rat hepatocytes seems to be relatively independent of oxygen radical-induced oxidative stress. Here we demonstrate that MPTP cytotoxicity is not potentiated by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase, nor prevented by the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) or the iron-chelating agent desferrioxamine. Moreover, preincubation of hepatocytes with diethylmaleate to lower the level of intracellular reduced glutathione (to 20% of the initial value) did not affect either the rate or extent of MPTP cytotoxicity. Thus, nucleophilic soluble thiols do not seem to play a protective role against MPTP-induced cell damage, in contrast to what one would have expected if covalent protein binding and oxidative stress were involved as toxic mechanisms. On the other hand, MPTP cytotoxicity was potentiated by pretreatment of hepatocytes with cytochrome P-450 inhibitors (e.g., SKF 525A and metyrapone) and a more rapid depletion of ATP was observed in these experimental conditions. We conclude that mitochondrial damage and subsequent ATP depletion are likely to play a critical role in the toxicity of MPTP to isolated hepatocytes and that the metabolism of MPTP via the cytochrome P-450 monooxygenase system can be considered to be a detoxifying pathway.     

Schisandrin B-induced increase in cellular glutathione level and protection against oxidant injury are mediated by the enhancement of glutathione synthesis and regeneration in AML12 and H9c2 cells

To define the relative role of reduced glutathione (GSH) synthesis and regeneration in schisandrin B (Sch B)-induced increase in cellular GSH level and the associated cytoprotection against oxidative challenge, the effects of L-buthionine-[S,R]-sulfoximine (BSO, a specific inhibitor of gamma-glutamate cysteine ligase (GCL)) and 1,3-bis(2-chloroethyl)-1-nitrourea (BCNU, a specific inhibitor of glutathione reductase (GR)) treatments or their combined treatment were examined in control and Sch B-treated AML12 and H9c2 cells, without and/or with menadione intoxication. Both BSO and BCNU treatments reduced cellular GSH level in AML12 and H9c2 cells, with the effect of BSO being more prominent. The GSH-enhancing effect of Sch B was also suppressed by BSO and BCNU treatments, with the effect of the combined treatment with BSO and BCNU being semi-additive. While Sch B treatment increased the GR but not GCL activity in AML12 and H9c2 cells, it increased the cellular cysteine level. BSO treatment also suppressed the Sch B-induced increase in GR activity. BSO or BCNU treatment per se did not cause any detectable cytotoxic effect, as assessed by lactate dehydrogenase leakage, but the combined treatment with BSO and BCNU was cytotoxic, particularly in H9c2 cells. The cytotoxic effect of BSO and BCNU became more apparent following the menadione challenge. The cytoprotection afforded by Sch B pretreatment was partly suppressed by BSO or BCNU treatment, or completely abrogated by the combined treatment with BSO and BCNU. In conclusion, the results indicate that the cytoprotective action of Sch B is causally related to the increase in cellular GSH level, which is likely mediated by the enhancement of GSH synthesis and regeneration.     

Comparative studies on the mechanisms of paraquat and 1-methyl-4-phenylpyridine (MPP+) cytotoxicity

1-methyl-4-phenylpyridine (MPP+) is the putative toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is structurally similar to the herbicide paraquat (PQ++). We have therefore compared the effects of MPP+ and PQ++ on a well characterized experimental model, namely isolated rat hepatocytes. PQ++ generates reactive oxygen species within cells by redox cycling and its toxicity to hepatocytes was potentiated by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. In BCNU-treated cells, PQ++ caused GSH depletion, lipid peroxidation and cell death. These cytotoxic effects were prevented by the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) and the iron-chelating agent desferrioxamine. MPP+ also caused GSH depletion in BCNU-treated hepatocytes but its cytotoxicity was not markedly affected by BCNU, nor was it accompanied by significant lipid peroxidation. DPPD and desferrioxamine also failed to prevent MPP+-induced cell death. We conclude that the production of active oxygen species is likely to play a major role in PQ++ cytotoxicity, while MPP+-induced cell damage may involve additional, more important toxic mechanisms.     

Autoxidation of extracellular hydroquinones is a causative event for the cytotoxicity of menadione and DMNQ in A549-S cells

Cytotoxicity of 1,4-naphthoquinones has been attributed to intracellular reactive oxygen species (ROS) generation through one-electron-reductase-mediated redox cycling and to arylation of cellular nucleophiles. Here, however, we report that in a subclone of lung epithelial A549 cells (A549-S previously called A549-G4S (Watanabe, et al., Am. J. Physiol. 283 (2002) L726-736), the mechanism of ROS generation by menadione and by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and therefore that of cytotoxicity, differs from the paradigm. Ninety percent of H(2)O(2) generation by both the quinones can be prevented by dicumarol, an inhibitor of NAD(P)H quinone oxidoreductase (NQO1), at the submicromolar level, regardless of the quinone concentrations. Exogenous SOD also inhibits H(2)O(2) production at low but not high concentrations of the quinones, especially DMNQ. Thus, at low quinone concentrations, superoxide-driven hydroquinone autoxidation accounts for more than half of H(2)O(2) generation by both quinones, whereas at high quinone concentrations, especially for DMNQ, comproportionation-driven hydroquinone autoxidation becomes the predominant mechanism. Hydroquinone autoxidation appears to occur predominantly in the extracellular environment than in the cytosol as extracellular catalase can dramatically attenuate quinone-induced cytotoxicity throughout the range of quinone concentrations, whereas complete inactivation of endogenous catalase or complete depletion of intracellular glutathione has only a marginal effect on their cytotoxicity. Finally, we show evidence that ROS production is a consequence of the compensatory defensive role of NQO1 against quinone arylation.     

Prooxidant cytotoxicity of chromate in mammalian cells: the opposite roles of DT-diaphorase and glutathione reductase

The geno- and cytotoxicity of chromate, an important environmental pollutant, is partly attributed to the flavoenzyme-catalyzed reduction with the concomitant formation of reactive oxygen species. The aim of this work was to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) and glutathione reductase (GR, EC 1.6.4.2) in the mammalian cell cytotoxicity of chromate, which was evidenced controversially so far. The chromate reductase activity of NQO1 was higher than that of GR, but lower than that of lipoamide dehydrogenase (EC 1.6.4.3), ferredoxin:NADP+ reductase (EC 1.18.1.2), and NADPH: cytochrome P-450 reductase (EC 1.6.2.4). The reduction of chromate by NQO1 was accompanied by the formation of reactive oxygen species. The concentration of chromate for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (22 +/- 4) microM. The cytotoxicity was partly prevented by desferrioxamine, the antioxidant N,N'-diphenyl-p-phenylene diamine and by an inhibitor of NQO1, dicumarol, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which inactivates GR. The NADPH-dependent chromate reduction by digitonin-permeabilized FLK cells was partly inhibited by dicumarol and not affected by BCNU. Taken together, these data indicate that the oxidative stress-type cytotoxicity of chromate in FLK cells may be partly attributed to its reduction by NQO1, but not by GR. The effect of BCNU on the chromate cytotoxicity may indicate that the general antioxidant action of reduced glutathione is more important than its prooxidant activities arising from the reactions with chromate.     

Epidermal growth factor receptor is a common mediator of quinone-induced signaling leading to phosphorylation of connexin-43: role of glutathione and tyrosine phosphatases

Rat liver epithelial cells were exposed to three quinones with different properties: menadione (2-methyl-1,4-naphthoquinone, vitamin K3), an alkylating as well as redox-cycling quinone, the strongly alkylating p-benzoquinone (BQ), and the non-arylating redox-cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). All three quinones induced the activation of extracellular signal-regulated kinase (ERK) 1 and ERK 2 via the activation of epidermal growth factor receptor (EGFR) and MAPK/ERK kinases (MEK) 1/2. ERK activation resulted in phosphorylation at Ser-279 and Ser-282 of the gap junctional protein, connexin-43, known to result in the loss of gap junctional intercellular communication. Another EGFR-dependent pathway was stimulated, leading to the activation of the antiapoptotic kinase Akt via phosphoinositide 3-kinase. The activation of EGFR-dependent signaling by these quinones was by different mechanisms: (i) menadione, but not BQ or DMNQ, inhibited a protein-tyrosine phosphatase regulating the EGFR, as concluded from an EGFR dephosphorylation assay; (ii) although menadione-induced activation of ERK was unimpaired by pretreatment of cells with N-acetyl cysteine, activation by BQ and DMNQ was prevented; (iii) cellular glutathione (GSH) levels were strongly depleted by BQ. The mere depletion of GSH by application of diethyl maleate EGFR-dependently activated ERK and Akt, thus mimicking BQ effects. GSH levels were only moderately decreased by menadione and not affected by DMNQ. In summary, EGFR-dependent signaling was mediated by protein-tyrosine phosphatase inactivation (menadione), GSH depletion (BQ), and redox-cycling (DMNQ), funneling into the same signaling pathway.     

Evaluation of reactive oxygen species involvement in amiodarone pulmonary toxicity in vivo and in vitro

Amiodarone (AM) is an effective antidysrhythmic agent, restricted in use by the development of adverse effects, including potentially fatal AM-induced pulmonary toxicity (AIPT). Although the pathogenesis of AIPT is unknown, an oxidant mechanism has been proposed. The present study evaluated the role of reactive oxygen species (ROS) in AM-induced toxicity. The effect of inhibiting lung antioxidant defense on in vivo development of AIPT was evaluated in hamsters. Lung glutathione reductase activity was inhibited by 66%, 6 hours following administration of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (20 mg/kg i.p.). When AM (1.83 μmol) was administered intratracheally 6 hours after BCNU, toxicity was enhanced, as indicated by lung hydroxyproline content and histological evaluation 21 days later. However, BCNU treatment did not affect AM-induced alterations in lung glutathione, suggesting that the increased toxicity was not due to decreased antioxidant capacity following BCNU. The effect of BCNU on AM cytotoxicity in vitro was evaluated using rabbit lung alveolar macrophages. Incubation with 5 μM BCNU for 2 hours caused greater than 95% inhibition of glutathione reductase activity. However, BCNU treatment had no effect on 146 μM AM-induced cytotoxicity, as assessed by lactate dehydrogenase latency following 12 hours of incubation. Rabbit macrophages loaded with 2′,7′-dichlorofluorescin, which is oxidized by ROS to fluorescent 2′,7′-dichlorofluorescein (DCF), were used to evaluate ROS generation by AM. Incubation of macrophages with AM (73 or 146 μM) for 1 hour, with or without the catalase inhibitor sodium azide (1 mM), did not result in DCF formation. Overall, these results do not support the hypothesis that AIPT is due to ROS action. © 1997 John Wiley & Sons, Inc.     

The toxicity of menadione (2-methyl-1,4-naphthoquinone) and two thioether conjugates studied with isolated renal epithelial cells

Menadione (2-methyl-1,4-naphthoquinone) was used as a model compound to test the hypothesis that thioether conjugates of quinones can be toxic to tissues associated with their elimination through a mechanism involving oxidative stress. Unlike menadione, the glutathione (2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone; MGNQ) and N-acetyl-L-cysteine (2-methyl-3-(N-acetylcysteine-S-yl)-1,4-naphthoquinone; M(NAC)NQ) thioether conjugates were not able to arylate protein thiols but were still able to redox cycle with cytochrome c reductase/NADH and rat kidney microsomes and mitochondria. Interestingly, menadione and M(NAC)NQ were equally toxic to isolated rat renal epithelial cells (IREC) while MGNQ was nontoxic. The toxicity of both menadione and M(NAC)NQ was preceded by a rapid depletion of soluble thiols and was associated with a depletion of soluble thiols and was associated with a depletion of protein thiols. Treatment of IREC with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1-nitrosourea, potentiated the thiol depletion and toxicity observed with menadione and M(NAC)NQ indicating the involvement of oxidative stress in this model of renal cell toxicity. The lack of MGNQ toxicity can be attributed to an intramolecular cyclization reaction which destroys the quinone nucleus and therefore eliminates its ability to redox cycle. These findings have important implications with regard to our understanding of the toxic potential of quinone thioether conjugates and of quinone toxicity in general.     

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 microM 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.     

Potentiation in the intact rat of the hepatotoxicity of acetaminophen by 1,3-bis(2-chloroethyl)-1-nitrosourea

Studies of the killing of cultured hepatocytes by acetaminophen indicate that the cells are injured by an oxidative stress that accompanies the metabolism of the toxin (J. L. Farber et al. (1988) Arch. Biochem. Biophys. 267, 640-650). The present report documents that the essential features of the killing of cultured hepatocytes by acetaminophen are reproduced in the intact animal. Male rats had no evidence of liver necrosis 24 h after administration of up to 1000 mg/kg of acetaminophen. Induction of mixed function oxidase activity by 3-methylcholanthrene increased the hepatotoxicity of acetaminophen. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) potentiated the hepatotoxicity of acetaminophen in male rats induced with 3-methylcholanthrene. Whereas the pretreatment with BCNU reduced the GSH content by 40%, a comparable depletion of GSH by diethylmaleate did not potentiate the toxicity of acetaminophen. The antioxidant diphenylphenylenediamine (25 mg/kg) and the ferric iron chelator deferoxamine (1000 mg/kg) prevented the liver necrosis produced by 500 mg/kg acetaminophen in rats pretreated with BCNU. Neither protective agent prevented the fall in GSH produced by acetaminophen. It is concluded the conditions of the irreversible injury of cultured hepatocytes by acetaminophen previously reported are not necessarily different from those that obtain in the intact rat with this toxin.     

Oxidative cell injury in the killing of cultured hepatocytes by allyl alcohol

The killing of cultured hepatocytes by allyl alcohol depended on the metabolism of this hepatotoxin by alcohol dehydrogenase to the reactive electrophile, acrolein. An inhibitor of alcohol dehydrogenase, pyrazole, prevented both the toxicity of allyl alcohol and the rapid depletion of GSH. Treatment of the hepatocytes with a ferric iron chelator, deferoxamine, or an antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD), prevented the cell killing but not the metabolism of allyl alcohol and the resulting depletion of GSH. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) sensitized the hepatocytes to allyl alcohol, an effect that was not attributable to the reduction in GSH with BCNU. The cell killing with allyl alcohol was preceded by the peroxidation of cellular lipids as evidence by an accumulation of malondialdehyde in the cultures. Deferoxamine and DPPD prevented the lipid peroxidation in parallel with their protection from the cell killing. These data indicate that acrolein produces an abrupt depletion of GSH that is followed by lipid peroxidation and cell death. Such oxidative cell injury is suggested to result from the inability to detoxify endogenous hydrogen peroxide and the ensuing iron-dependent formation of a potent oxidizing species. Oxidative cell injury more consistently accounts for the hepatotoxicity of allyl alcohol than does the covalent binding of acrolein to cellular macromolecules.     

Effects of Oxidative Stress Caused by Hyperoxia and Diquat. A Study in Isolated Hepatocytes

The effects of oxidative stress caused by hyperoxia or administration of the redox active compound diquat were studied in isolated hepatocytes, and the relative contribution of lipid peroxidation, glutathione (GSH) depletion, and NADPH oxidation to the cytotoxicity of active oxygen species was investigated.

The redox cycling of diquat occurred primarily in the microsomal fraction since diquat was found not ' to penetrate into the mitochondria. Depletion of intracellular GSH by pretreatment of the animals with diethyl maleate promoted lipid peroxidation and sensitized the cells to oxidative stress. Diquat toxicity was also greatly enhanced when glutathione reductase was inhibited by pretreatment of the cells with 1,3-bis(2-chloroethyI)-1-nitrosourea. Despite extensive lipid peroxidation, loss of cell viability was not observed, with either hyperoxia or diquat, until the GSH level had fallen below ≈ 6 nmol/10⁶ cells.

The iron chelator desferrioxamine provided complete protection against both diquat-induced lipid peroxidation and loss of cell viability. In contrast, the antioxidant a-tocopherol inhibited lipid peroxidation but provided only partial protection from toxicity. The hydroxy! radical scavenger α-keto-γ-methiol butyric acid, finally, also provided partial protection against diquat toxicity but had no effect on lipid peroxidation.

The results indicate that there is a critical GSH level above which cell death due to oxidative stress is not observed. As long as the glutathione peroxidase – glutathione reductase system is unaffected, even relatively low amounts of GSH can protect the cells by supporting glutathione peroxidase-mediated metabolism of H₂O₂ and lipid hydroperoxides.     

Superoxide potently induces ceramide formation in glomerular endothelial cells

Recent evidence suggests that the sphingolipid-derived second messenger ceramide and oxidative stress are intimately involved in apoptosis induction. Here we report that exposure of microcapillary glomerular endothelial cells to superoxide-generating substances, including hypoxanthine/xanthine oxidase and the redox cyclers DMNQ and menadione results in a dose-dependent and delayed increase in the lipid signaling molecule ceramide. Long-term incubation of endothelial cells for 2-30 h with either DMNQ or hypoxanthine/xanthine oxidase leads to a continuous increase in ceramide levels. In contrast, short-term stimulation for 1 min up to 1 h had no effect on ceramide formation. The DMNQ-induced delayed ceramide formation is dose-dependently inhibited by reduced glutathione, whereas oxidized glutathione was without effect. Furthermore, N-acetylcysteine completely blocks DMNQ-induced ceramide formation. All superoxide-generating substances were found to dose-dependently trigger endothelial cell apoptosis. In addition, glutathione and N-acetylcysteine also prevented superoxide-induced apoptosis and implied that ceramide represents an important mediator of superoxide-triggered cell responses like apoptosis.