Metabolism and cytotoxicity of bisphenol A and other bisphenols in isolated rat hepatocytes

The relation between the metabolism and the cytotoxic effects of bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane) has been studied in freshly isolated rat hepatocytes and isolated hepatic mitochondria. The incubation of hepatocytes with BPA (0.25–1.0 mM) elicited a concentration- and time-dependent cell death, accompanied by losses of intracellular ATP and total adenine nucleotide pools. BPA at a low-toxic level (0.25 mM) in the hepatocyte suspensions was rapidly converted to its major conjugate, BPA-glucuronide, and other minor products without marked loss of cell viability, although at a toxic level (0.5 mM), more than 65% of the compound presented in an unaltered form 2 h after the incubation. Addition of salicylamide (2 mM), non-toxic to hepatocytes during the incubation period, enhanced BPA-induced cytotoxicity and reduced the loss of BPA and the formation of BPA-glucuronide. The addition of BPA to isolated hepatic mitochondria caused a concentration (0–0.5 mM)-dependent increase in the rate of state 4 oxygen consumption in the presence of an FAD-linked substrate (succinate), indicating an uncoupling effect, whereas the rate of state 3 oxygen consumption was inhibited by BPA. Further, the addition of BPA (0.25 mM) reduced state 3 respiration with NAD⁺-linked substrates (pyruvate plus malate) and/or with the FAD-linked substrate, whereas state 3 respiration with ascorbate plus tetramethyl-p-phenylenediamine (cytochrome oxidase-linked respiration) was not significantly affected by BPA. A comparative study of the toxic effects of BPA and some bisphenols on cell viability (at 1.0 mM) and mitochondrial respiration (at 0.25 mM) revealed that 4,4′-(1,2-diethyl-1,2-ethenediyl)bisphenol (diethylstilbestrol) was more toxic than BPA, followed by 4,4′-methylenediphenol and 4,4′-biphenol. These results indicate that the onset of cytotoxicity caused by BPA may depend on the intracellular energy status and that mitochondria are important targets of the compound. The toxicity caused by the inhibition of ATP synthesis may be related to the concentration of unmetabolised free BPA remaining in the cell suspensions. In addition, the toxic potency of bisphenols to hepatocytes and mitochondria depends on the relative elongation and/or molecular size of the hydrocarbon bridge between the phenolic groups. Received: 25 October 1999 / Accepted: 31 January 2000     

Melatonin ameliorates bisphenol A-induced biochemical toxicity in testicular mitochondria of mouse

Bisphenol A (BPA) is a monomer of polycarbonate plastic used to manufacture plastic baby bottles and lining of food cans. It has endocrine-disrupting potential and exerts both toxic and estrogenic effects on mammalian cells. We studied BPA-induced perturbation of mitochondrial marker enzymes in testes of Swiss albino mice and its amelioration by melatonin. Mice exposed to standardized dose of BPA (10 mg/kg body weight) orally for 14 days showed decrease in activities of marker mitochondrial enzymes such as succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, monoamine oxidase and NADH dehydrogenase. Besides, it also affected activities of antioxidant enzymes such as superoxide dismutase, glutathione reductase and glutathione peroxidase. BPA also caused lipid peroxidation (LPO) and decrease in reduced glutathione (GSH) content of mitochondria. Concomitant melatonin administration (10 mg/kg body weight; intraperitoneally for 14 days) lowered mitochondrial lipid peroxidation. It also restored the activity of mitochondrial marker enzymes and ameliorated decreased enzymatic and non-enzymatic antioxidants of mitochondria. These results demonstrate that melatonin has a potential role in ameliorating BPA-induced mitochondrial toxicity and the protection is due to its antioxidant property or by the direct free radical scavenging activity.     

Mitochondrial dysfunction is an early event in ochratoxin A but not oosporein toxicity to rat renal proximal tubules

Ochratoxin A (OA) and oosporein (OSN) are two mycotoxins that may cause nephrotoxicity through either mitochondrial dysfunction or lipid peroxidation. Using isolated rat renal proximal tubules in suspension, the cellular events preceding OA- or OSN-induced cytotoxicity were investigated. OA and OSN decreased tubule viability in a concentration (0-1 mM)- and time (0-4 hr)-dependent manner, with initial decreases occurring 1 hr after exposure. Tubule basal and nystatin-stimulated oxygen consumption decreased before cell death after OA (0.5 and 1 mM) and 0.25 mM t-butyl hydroperoxide (TBHP) exposure, but did not decrease after OSN exposure (0.25-1 mM). The oxidant TBHP was used as a positive control in these studies. Direct probing of mitochondrial function within proximal tubules confirmed the toxicity of OA to mitochondria. Respiration was reduced in the absence and presence of a phosphate acceptor using site I (glutamate/malate) and site II (succinate) respiratory substrates 15 and 30 min after exposure to 1 mM OA. Lipid peroxidation preceded cell death after exposure to 1 mM OA and 0.25 mM TBHP, but did not occur after exposure to 1 mM OSN. Deferoxamine (1 mM) pretreatment before the addition of 1 mM OA or OSN prevented OA-induced lipid peroxidation, but did not prevent OA- or OSN-induced cytotoxicity. In contrast, deferoxamine pretreatment prevented lipid peroxidation, mitochondrial dysfunction, and the loss of tubule viability after exposure to 0.25 mM TBHP. This study shows that mitochondrial dysfunction is an early event during the development of OA toxicity, but not in OSN-induced toxicity. Furthermore, iron-mediated lipid peroxidation does not contribute to OA- or OSN-induced proximal tubule cell death.     

Ameliorative Effects of Taurine Against Methimazole-Induced Cytotoxicity in Isolated Rat Hepatocytes

Methimazole is used as an antithyroid drug to control the symptoms of hyperthyroidism and maintain patients in a euthyroid state. Administration of this drug is associated with agranulocytosis and hepatotoxicity, which are the two most significant adverse effects. The present investigation was conducted to study the protective role of taurine against cytotoxicity induced by methimazole and its proposed reactive intermediary metabolite, N-methylthiourea, in an in vitro model of isolated rat hepatocytes.At different points in time, markers such as cell viability, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and hepatocyte glutathione content were evaluated.Treating hepatocytes with methimazole resulted in cytotoxicity characterized by the reduction in cell viability, an increase in ROS formation and lipid peroxidation, mitochondrial membrane potential collapse, and a reduction in cellular glutathione content. Furthermore, a significant amount of oxidized glutathione (GSSG) was formed when rat hepatocytes were treated with methimazole. N-methylthiourea toxicity was accompanied by a reduction in cellular GSH content, but no significant changes in lipid peroxidation, ROS formation, GSSG production, or changes in mitochondrial membrane potential were detected. Administration of taurine (200 μM) effectively reduced the toxic effects of methimazole or its metabolite in isolated rat hepatocytes.     

N-Nitrosofenfluramine induces cytotoxicity via mitochondrial dysfunction and oxidative stress in isolated rat hepatocytes

The cytotoxic effects of fenfluramine, an appetite suppressant, and its N-nitroso derivative, N-nitrosofenfluramine, have been studied in freshly isolated rat hepatocytes and isolated hepatic mitochondria. Exposure of hepatocytes to N-nitrosofenfluramine caused not only concentration (0.25–1.0 mmol L^–1) and time (0–3 h)-dependent cell death accompanied by the loss of cellular ATP, adenine nucleotide pools, reduced glutathione (GSH), and protein thiols, but also the accumulation of oxidized glutathione and malondialdehyde (MDA), indicating lipid peroxidation. There was a time lag for the onset of the accumulation of MDA after the rapid depletion of ATP. Supplementation of the hepatocyte suspensions with N-acetylcysteine (4 mmol L^–1), a precursor of intracellular GSH, partially inhibited N-nitrosofenfluramine (1 mmol L^–1)-induced cytotoxicity. In comparative effects based on cell viability and rhodamine 123 retention, an index of mitochondrial membrane potential, fenfluramine was less toxic than N-nitrosofenfluramine. In mitochondria isolated from rat liver, N-nitrosofenfluramine caused an increase in the rate of state-4 oxygen consumption, indicating an uncoupling effect, and a decrease in the rate of state-3 oxygen consumption in a concentration-dependent manner. These results indicate that (a) mitochondria are target organelles for N-nitrosofenfluramine, which elicits cytotoxicity through mitochondrial dysfunction related to membrane potential and/or oxidative phosphorylation at an early stage and subsequently lipid peroxidation at a later stage; and (b) the toxicity of N-nitrosofenfluramine is greater than that of fenfluramine, suggesting participation of the nitroso group in the toxicity.     

Cytotoxicity of ortho-phenylphenol in isolated rat hepatocytes.

The effects of ortho-phenylphenol (OPP) and its metabolites, phenyl-hydroquinol (PHQ) and phenyl-benzoquinone (PBQ), on isolated rat hepatocytes were investigated. Addition of OPP (0.5-1.0 mM) to cells caused a dose-dependent cell death accompanied by the depletion of intracellular levels of ATP, glutathione (GSH) and protein thiols. GSH loss correlated with the formation of oxidized GSH. In addition, PHQ and especially PBQ (both at 0.5 mM) resulted in acute cell death with rapid depletion of ATP, GSH and protein thiols, and further low doses of PBQ (10-50 microM) elicited serious impairment of mitochondrial functions related to oxidative phosphorylation and Ca fluxes in isolated liver mitochondria. These results indicate that mitochondria are a target for these compounds and that OPP is itself toxic to hepatocytes even when metabolism is inhibited. The loss of cellular GSH and protein thiols accompanied by the impairment of mitochondrial function may be the main mechanisms of cytotoxicity induced by OPP and its metabolites.     

Chlorpropham induces mitochondrial dysfunction in rat hepatocytes

The metabolism and action of chlorpropham (isopropyl N-(3-chlorophenyl)carbamate; CIPC, a post-harvest agent) and its metabolites were studied in freshly isolated rat hepatocytes and isolated rat hepatic mitochondria, respectively. The exposure of hepatocytes to CIPC caused a concentration (0.25–1.0 mM)- and time (0–3 h)-dependent cell death accompanied by loss of cellular ATP and adenine nucleotides. CIPC at a weakly toxic level (0.5 mM) was metabolized to isopropyl N-(3-chloro-4-hydroxyphenyl)carbamate (4OH-CIPC) and subsequently to its glucuronide and sulfate conjugates (major metabolites) or alternatively to a minor metabolite 3-chloroaniline (3CA). The addition of SKF-525A (50 μM), an inhibitor of microsomal monooxygenase, enhanced the CIPC (0.5 mM)-induced cytotoxicity accompanied by loss of ATP and 4OH-CIPC and inhibited the decrease in the concentration of the parent compound. CIPC led to a strong decrease in cellular ATP content compared to its metabolites, 4OH-CIPC and 3CA. On the other hand, the exposure of isolated hepatic mitochondria to CIPC reduced State 3 respiration with a FAD-linked substrate (succinate plus rotenone) and/or with a NAD⁺-linked substrate (pyruvate plus malate), whereas State 3 respiration with ascorbate plus tetramethyl-p-phenylendiamine (cytochrome oxidase-linked respiration) was not affected markedly by CIPC. Further, the addition of CIPC caused an increase in the rate of State 4 oxygen consumption, indicating an uncoupling effect, and a decrease in the rate of State 3 oxygen consumption in a concentration-dependent manner, respectively. In contrast, the addition of neither 4OH-CIPC nor 3CA markedly affected the rate of states 3 and/or 4 oxygen consumption. These results indicate that CIPC-induced cytotoxicity is mediated by the parent compound rather than by its metabolites such as 4OH-CIPC and 3CA, and that the toxicity is associated with a rapid depletion of ATP via impairment of mitochondrial function related to oxidative phosphorylation.     

Enhancement of reactive oxygen-dependent mitochondrial membrane lipid peroxidation by the anticancer drug adriamycin

Mitochondrial degeneration is a consistently prominent morphological alteration associated with adriamycin toxicity which may be the consequence of adriamycin-enhanced peroxidative damage to unsaturated mitochondrial membrane lipids. Using isolated rat liver mitochondria as an in vitro model system to study the effects of the anticancer drug adriamycin on lipid peroxidation, we found that NADH-dependent mitochondrial peroxidation--measured by the 2-thiobarbituric acid method--was stimulated by adriamycin as much as 4-fold. Marker enzyme analysis indicated that the mitochondria were substantially free of contaminating microsomes (less than 5%). Lipid peroxidation in mitochondria incubated in KCl-Tris-HCl buffer (pH 7.4) under an oxygen atmosphere was optimal at 1-2 mg of mitochondrial protein/ml and with NADH at 2.5 mM. Malonaldehyde production was linear with time to beyond 60 min, and the maximum enhancement of peroxidation was observed with adriamycin at 50-100 microM. Interestingly, in contrast to its stimulatory effect on NADH-supported mitochondrial peroxidation, adriamycin markedly diminished ascorbate-promoted lipid peroxidation in mitochondria. Superoxide dismutase, catalase, 1,3-dimethylurea, reduced glutathione, alpha-tocopherol and EDTA added to incubation mixtures inhibited endogenous and adriamycin-augmented NADH-dependent peroxidation of mitochondrial lipids, indicating that multiple species of reactive oxygen (superoxide anion radical, hydrogen peroxide and hydroxyl radical) and possibly trace amounts of endogenous ferric iron participated in the peroxidation reactions. In submitochondrial particles freed of endogenous defenses against oxyradicals, lipid peroxidation was increased 7-fold by adriamycin. These observations suggest that some of the effects of adriamycin on mitochondrial morphology and biochemical function may be mediated by adriamycin-enhanced reactive oxygen-dependent mitochondrial lipid peroxidation.     

Mitochondrial oxidative stress and dysfunction induced by isoniazid: study on isolated rat liver and brain mitochondria

Isoniazid (INH or isonicotinic hydrazide) is used for the treatment and prophylaxis of tuberculosis. Liver and brain are two important target organs in INH toxicity. However, the exact mechanisms behind the INH hepatotoxicity or neurotoxicity have not yet been completely understood. Considering the mitochondria as one of the possible molecular targets for INH toxicity, the aim of this study was to evaluate the mechanisms of INH mitochondrial toxicity on isolated mitochondria. Mitochondria were isolated by differential ultracentrifugation from male Sprague–Dawley rats and incubated with different concentrations of INH (25–2000?μM) for the investigation of mitochondrial parameters. The results indicated that INH could interact with mitochondrial respiratory chain and inhibit its activity. Our results showed an elevation in mitochondrial reactive oxygen species (ROS) formation, lipid peroxidation and mitochondrial membrane potential collapse after exposure of isolated liver mitochondria in INH. However, different results were obtained in brain mitochondria. Noteworthy, significant glutathione oxidation, adenosine triphosphate (ATP) depletion and lipid peroxidation were observed in higher concentration of INH, as compared to liver mitochondria. In conclusion, our results suggest that INH may initiate its toxicity in liver mitochondria through interaction with electron transfer chain, lipid peroxidation, mitochondrial membrane potential decline and cytochrome c expulsion which ultimately lead to cell death signaling.     

Comparative study of the endocrine-disrupting activity of bisphenol A and 19 related compounds.

The endocrine-disrupting activities of bisphenol A (BPA) and 19 related compounds were comparatively examined by means of different in vitro and in vivo reporter assays. BPA and some related compounds exhibited estrogenic activity in human breast cancer cell line MCF-7, but there were remarkable differences in activity. Tetrachlorobisphenol A (TCBPA) showed the highest activity, followed by bisphenol B, BPA, and tetramethylbisphenol A (TMBPA); 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,1-bis(4-hydroxyphenyl)propionic acid and 2,2-diphenylpropane showed little or no activity. Anti-estrogenic activity against 17beta-estradiol was observed with TMBPA and tetrabromobisphenol A (TBBPA). TCBPA, TBBPA, and BPA gave positive responses in the in vivo uterotrophic assay using ovariectomized mice. In contrast, BPA and some related compounds showed significant inhibitory effects on the androgenic activity of 5alpha-dihydrotestosterone in mouse fibroblast cell line NIH3T3. TMBPA showed the highest antagonistic activity, followed by bisphenol AF, bisphenol AD, bisphenol B, and BPA. However, TBBPA, TCBPA, and 2,2-diphenylpropane were inactive. TBBPA, TCBPA, TMBPA, and 3,3'-dimethylbisphenol A exhibited significant thyroid hormonal activity towards rat pituitary cell line GH3, which releases growth hormone in a thyroid hormone-dependent manner. However, BPA and other derivatives did not show such activity. The results suggest that the 4-hydroxyl group of the A-phenyl ring and the B-phenyl ring of BPA derivatives are required for these hormonal activities, and substituents at the 3,5-positions of the phenyl rings and the bridging alkyl moiety markedly influence the activities.     

Role of mitochondrial membrane permeability transition in N-nitrosofenfluramine-induced cell injury in rat hepatocytes

The role of mitochondrial membrane permeability transition in N-nitrosofenfluramine-induced cell injury was studied in mitochondria and hepatocytes isolated from rat liver. Mitochondrial permeability transition has been proposed as a common final pathway in acute cell death through mitochondrial dysfunction. In isolated mitochondria, N-nitrosofenfluramine (0.25 to 1.0 mM) in the presence of Ca(2+) (50 microM) elicited a concentration-dependent induction of mitochondrial swelling dependent on mitochondrial permeability transition and the release of cytochrome c, both of which were prevented by pretreatment with a specific inhibitor of mitochondrial permeability transition, cyclosporin A (0.2 microM). The effects of N-nitrosofenfluramine on mitochondria were more potent than those of fenfluramine, which is a sympathomimetic amine with anorectic action. The pretreatment of isolated hepatocytes with cyclosporin A (2 microM) partially but not completely prevented N-nitrosofenfluramine (0.6 mM; a low toxic dose)-induced cell death, loss of cellular ATP, formation of cell blebs and decrease in mitochondrial membrane potential. These results suggest that the onset of N-nitrosofenfluramine-induced cytotoxicity is linked to mitochondrial failure dependent upon induction of mitochondrial permeability transition accompanied by mitochondrial depolarization, the release of cytochrome c and depletion of intracellular ATP through uncoupling of oxidative phosphorylation.     

Depletion in vitro of mitochondrial glutathione in rat hepatocytes and enhancement of lipid peroxidation by adriamycin and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)

Treatment of isolated rat hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and adriamycin (ADR) produced a complete depletion of cellular glutathione accompanied by a significant increase in lactate dehydrogenase (LDH) leakage. Separation of the mitochondrial and cytoplasmic pools of glutathione by digitonin disruption showed that, although BCNU, a specific inhibitor of glutathione, completely depleted the cytoplasmic pool of glutathione, the mitochondrial supply was not entirely expended and LDH leakage was only moderately stimulated. Only after depletion of the mitochondrial supply of glutathione by ADR and BCNU did LDH leakage increase markedly. Measurement of lipid peroxidation, by monitoring malondialdehyde through the thiobarbituric acid procedure, showed that malondialdehyde accumulated more extensively and at a rate mirroring release of LDH from ADR/BCNU treated cells. The time of increase in LDH leakage and malondialdehyde production corresponded to the time of depletion of mitochondrial glutathione to less than 10% of the initial pool size. No such increase in LDH leakage was observed with BCNU or ADU treatment alone or when aminopyrine, an inhibitor of lipid peroxidation, was included. Aminopyrine was found to prevent, in a dose-dependent manner, both LDH leakage and malondialdehyde production stimulated by ADR/BCNU treatment. The protective effect peaked at 5 mM aminopyrine, and higher concentrations produced significant LDH leakage exhibiting LDH release kinetics different than those observed with ADR/BCNU. Although aminopyrine had no effect on the rate or extent of cytoplasmic glutathione depletion by ADR/BCNU treatment, the mitochondrial pool was conserved significantly in those cells protected by aminopyrine. These data suggest that enhanced hepatocyte damage observed after treatment with a combination of ADR and BCNU versus BCNU or ADR alone is due to the extensive depletion of mitochondrial glutathione supported by ADR after glutathione reductase inhibition. Further, enhancement of lipid peroxidation is strongly implicated in the mechanism of adriamycin toxicity.     

Cytotoxic effects of hydroxylated fullerenes on isolated rat hepatocytes via mitochondrial dysfunction

The cytotoxic effects of hydroxylated fullerenes, also termed fullerenols or fullerols [C₆₀(OH) _n ], which are known nanomaterials and water-soluble fullerene derivatives, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to C₆₀(OH)₂₄ caused not only concentration (0–0.25 mM)- and time (0–3 h)-dependent cell death accompanied by the formation of cell blebs, loss of cellular ATP, reduced glutathione (GSH), and protein thiol levels, but also the accumulation of glutathione disulfide and malondialdehyde, indicating lipid peroxidation. Of the other analogues examined, the cytotoxic effects of C₆₀(OH)₁₂ and fullerene C₆₀ at a concentration of 0.125 mM were less than those of C₆₀(OH)₂₄. The loss of mitochondrial membrane potential and generation of oxygen radical species in hepatocytes incubated with C₆₀(OH)₂₄ were greater than those with C₆₀(OH)₁₂ and fullerene C₆₀. In the oxygen consumption of mitochondria isolated from rat liver, the ratios of state-3/state-4 respiration were more markedly decreased by C₆₀(OH)₂₄ and C₆₀(OH)₁₂ compared with C₆₀. In addition, C₆₀(OH)₂₄ and C₆₀(OH)₁₂ resulted in the induction of the mitochondrial permeability transition (MPT), and the effects of C₆₀(OH)₁₂ were less than those of C₆₀(OH)₂₄. Taken collectively, these results indicate that (a) mitochondria are target organelles for fullerenols, which elicit cytotoxicity through mitochondrial failure related to the induction of the MPT, mitochondrial depolarization, and inhibition of ATP synthesis in the early stage and subsequently oxidation of GSH and protein thiols, and lipid peroxidation through oxidative stress at a later stage; and (b) the toxic effects of fullerenols may depend on the number of hydroxyl groups participating in fullerene in rat hepatocytes.     

Induction of hepatotoxicity by sanguinarine is associated with oxidation of protein thiols and disturbance of mitochondrial respiration

Sanguinarine (SANG) has been suggested to be one of the principle constituents responsible for the toxicity of Argemone mexicana seed oil. In this study, we focused on the possible mechanism of SANG-induced hepatotoxicity. The serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) activities, hepatic vacuolization, lipid accumulation and lipid peroxidation of the liver were increased, and triglyceride (TG) was decreased in SANG-treated mice (10 mg kg(-1) i.p.), indicating damage to the liver. SANG induced cell death and DNA fragmentation, in a concentration- (0-30 microm) and time-dependent (0-24 h) manner, and the cytotoxicity of SANG (15 microm) was accompanied by an increase in reactive oxygen species and a lessening in protein thiol content; these outcomes were reversed by glutathione, N-acetyl-l-cysteine and 1,4-dithiothretol, and slightly improved by other antioxidants in hepatocytes. SANG can affect the function of mitochondria, leading to the depletion of the mitochondrial membrane potential and adenosine 5'-triphosphate content of hepatocytes. SANG caused an uncoupling effect of the respiratory chain at lower concentrations, but inhibited the respiratory chain at higher concentrations in mitochondria isolated from rat liver. In conclusion, the data suggest that SANG is a liver toxin that induces cytotoxicity in liver cells, possibly through oxidation of protein thiols, resulting in oxidative stress on the cells and disturbance of mitochondrial function.     

Relationships between ascorbic acid and alpha-tocopherol during diquat-induced redox cycling in isolated rat hepatocytes

The effects of diquat-induced redox cycling on the levels of cellular ascorbic acid and alpha-tocopherol were investigated in isolated rat hepatocytes. In untreated hepatocytes, the metabolism of 1 or 2 mM diquat resulted in the depletion of cellular ascorbic acid and glutathione, but not of alpha-tocopherol, in association with the induction of cell death during the experimental period. In 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) pretreated cells, 1 mM diquat induced cell death accompanied by glutathione was rapid (to 9% of controls by 15 min) and cell ascorbate was completely consumed by 2 hr of incubation. In contrast, cellular alpha-tocopherol levels were stable for the first 30 min, but were depleted in association with the onset of lipid peroxidation. Supplementation of 0.1 or 1.0 mM ascorbic acid in the incubation medium delayed the onset of diquat-induced alpha-tocopherol loss, lipid peroxidation and cytotoxicity. When the concentration of exogenous cellular ascorbic acid was consumed to below that of endogenous ascorbic acid, alpha-tocopherol loss and lipid peroxidation were initiated. The results indicate that untreated hepatocytes have an effective multicomponent antioxidant system against diquat-induced oxidative stress. However, when glutathione is depleted from hepatocytes by treatment with BCNU and diquat, ascorbic acid plays a vital role in maintaining cellular alpha-tocopherol levels and survival of the cell.     

Decrease of reduced glutathione in isolated rat hepatocytes caused by acrolein,acrylonitrile, and the thermal degradation products of styrene copolymers

Decrease of reduced glutathione (GSH) was induced in isolated rat hepatocytes by incubation with acrolein or acrylonitrile for 120 min or exposure to the products of oxidative thermal degradation of acrylonitrile-butadiene-styrene copolymer (ABS), styrene-acrylonitrile copolymer (SAN), and high impact polystyrene (SB). The decrease of GSH by acrolein was rapid but the cells soon recovered at acrolein concentrations of 0.025–0.25 mM. 0.5 mM acrolein depleted the cells of GSH and they were uncapable of further GSH synthesis. At concentrations of 0.25–0.5 mM concomitant lipid peroxidation impaired the integrity of the cell membranes. Also acrylonitrile induced a dose dependent GSH decrease at concentrations of 0.05–1 mM. Neither membrane damage nor lipid peroxidation was detected during 120-min incubations at these acrylonitrile concentrations.The thermal degradation products of ABS, SAN and SB caused a decrease of GSH in hepatocytes. The extent of the decrease depended on the degradation temperature and the type of the plastic. The membrane integrity was impaired in the cases where GSH was depleted almost completely; ABS degraded at 350°C and SB at 250°C. The measurements of lipid peroxidation by the thiobarbituric acid and the diene conjugation methods were impossible because the degradation products contained compounds which interfered with these tests.     

Cytotoxic effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) herbicide on hepatocytes and mitochondria isolated from male rats

The cytotoxic effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) and its major metabolites were investigated in freshly isolated rat hepatocytes. Terbutol and its metabolite, especially 2,6-di-tert-butyl-4-methylphenyl carbamate (N-demethylterbutol), at a concentration of 1.0 mM resulted in a time dependent cell killing accompanied by losses of intracellular ATP, protein thiols, and glutathione (GSH) and the accumulation of oxidized GSH. Supplementation of the hepatocyte suspension with 5 mM N-acetylcysteine, a precursor of intracellular GSH, inhibited the cytotoxicity of N-demethylterbutol. In mitochondria isolated from rat liver, terbutol and its metabolites impaired respiration related to oxidative phosphorylation and the potency of their toxicity is associated with impairment of mitochondrial respiration. These results indicate that N-demethylterbutol is the most cytotoxic followed by terbutol and other metabolites, and that both the mitochondrial respiratory system and protein thiols are important targets for these compounds.     

Effects of silver in isolated rat hepatocytes

Addition of silver nitrate or silver lactate to freshly isolated hepatocytes caused dose-dependent loss of cell viability, measured by trypan blue exclusion, at concentrations within 30-70 microM. Silver cytotoxicity was accompanied by a decrease in hepatic thiol concentration and an increase in lipid peroxidation. Treatment of hepatocytes with the reduced glutathione (GSH)-depleting agent diethylmaleate markedly increased their vulnerability to silver toxicity whereas protective effects were produced by the thiol-reducing agent, dithiothreitol. Both alpha-tocopherol, which protected from the onset of silver-associated lipid peroxidation, and the iron chelator agent, deferoxamine failed to prevent loss of cell viability. These data suggest that perturbation of intracellular thiol homeostasis may play a critical role in the mechanism underlying silver-induced lethal damage to isolated rat hepatocytes.     

Effects of culture duration on hydrogen peroxide-induced hepatocyte toxicity

The effects of culture duration on primary cultured mouse hepatocyte antioxidant levels (superoxide dismutase, catalase, glutathione peroxidase, vitamin E, and glutathione) and susceptibility to glucose oxidase (GO)- and hydrogen peroxide (H2O2)-induced cell killing and lipid peroxidation were examined. Membrane fatty acid composition was also evaluated. Adult male B6C3F1/CrlBR mouse hepatocytes were isolated by collagenase perfusion of the liver and cultured on 60-mm plastic dishes in Leibovitz's L-15 medium supplemented with glucose (1 mg/ml), dexamethasone (1 microM), fetal bovine serum (10%, v/v), and gentamicin sulfate (50 micrograms/ml) for 0 hr (freshly isolated cells) to 96 hr. Hepatocyte toxicity (determined by lactate dehydrogenase release and lipid peroxidation) after a 2-hr exposure to GO (0.8-80 micrograms/ml) or H2O2 (1-5 mM) decreased with increased time in culture. This decreased hepatocyte sensitivity to GO and H2O2 toxicity was not related to antioxidant enzyme activity since superoxide dismutase, catalase, and glutathione peroxidase declined during the 96-hr culture period. In contrast, glutathione and vitamin E levels in the cultured hepatocytes rose to 274.9 +/- 8.3% and 220.6 +/- 18.6% of the levels in freshly isolated cells (129.6 +/- 11.5 nmol and 0.10 +/- 0.01 nmol per 10(6) hepatocytes, respectively). The percentage of polyunsaturated fatty acids in hepatocyte phospholipids and triglycerides decreased with culture duration while the percentage of oleic acid increased in esterified and free fatty acid pools after 2 hr in culture. Total fatty acids were not affected by time in culture. These results suggest that the decreased hepatocyte susceptibility to the toxic effects of hydrogen peroxide may have been due to elevations in cellular GSH and vitamin E levels and decreases in membrane polyunsaturated fatty acids. The data also indicate that hepatocytes in primary culture undergo changes in antioxidant levels and fatty acid composition that may affect free radical toxicity at different times in culture.     

Is hyperthermia the triggering factor for hepatotoxicity induced by 3,4-methylenedioxymethamphetamine (ecstasy)? An in vitro study using freshly isolated mouse hepatocytes

The consumption of 3,4-methylenedioxymethamphetamine (ecstasy; MDMA) may cause hepatocellular damage in humans, a toxic effect that has been increasing in frequency in the last few years, although the underlying mechanisms are still unknown. The metabolism of MDMA involves the production of reactive metabolites which form adducts with intracellular nucleophilic sites, as is the case with glutathione (GSH). Also, MDMA administration elicits hyperthermia, a potentially deleterious condition that may aggravate its direct toxic effects. Thus, the objective of this study was to evaluate the extent of MDMA-induced depletion of GSH, induction of lipid peroxidation and loss of cell viability in freshly isolated mouse hepatocytes under normothermic conditions (37 degrees C) and to compare the results with the effects obtained under hyperthermic conditions (41 degrees C). By itself, hyperthermia was an important cause of cell toxicity. A rise in incubation temperature from 37 degrees C to 41 degrees C caused oxidative stress in freshly isolated mouse hepatocytes, reflected as a time-dependent induction of lipid peroxidation and consequent loss of cell viability (up to 40-45%), although the variations in GSH and GSSG levels were similar to those under normothermic conditions. MDMA (100, 200, 400, 800 and 1600 microM) induced a concentration- and time-dependent GSH depletion at 37 degrees C but had a negligible effect on lipid peroxidation and cell viability at this temperature. It is particularly noteworthy that hyperthermia (41 degrees C) potentiated MDMA-induced depletion of GSH, production of lipid peroxidation and loss of cell viability (up to 90-100%). It is therefore concluded that hyperthermia potentiates MDMA-induced toxicity in freshly isolated mouse hepatocytes.