Protective effect of S-adenosylmethionine on cellular and mitochondrial membranes of rat hepatocytes against tert-butylhydroperoxide-induced injury in primary culture

Accumulating evidence that administration of S-adenosylmethionine (SAMe) protects hepatocytes against oxidative stress-mediated injury led us to evaluate the effect of SAMe on hepatocyte injury induced in culture by oxidant substance tert-butylhydroperoxide (1.5 mM tBHP) with regard to prevent mitochondrial injury. The pretreatment of hepatocyte culture with SAMe in doses of 0.25, 0.5, 1, 2.5, 5, 10, 25 and 50 mg/l for 30 min prevented the release of LDH from cells incubated for 30 min with tBHP in a dose dependent manner. The inhibitory effect of SAMe on lipid peroxidation paralleled the effect on cell viability. SAMe also moderated the decrease of the mitochondrial membrane potential induced by tBHP. Our results indicate that the inhibition of lipid peroxidation by SAMe can contribute to the prevention of disruption of both cellular and mitochondrial membranes. While the protective effect of SAMe against tBHP-induced GSH depletion was not confirmed, probably the most potent effect of SAMe on membranes by phospholipid methylation should be verified.     

S-adenosylmethionine (SAMe) protects against acute alcohol induced hepatotoxicity in mice small star, filled

Although S-Adenosylmethionine (SAMe) has beneficial effects in many hepatic disorders, the effects of SAMe on acute alcohol-induced liver injury are unknown. In the present study, we investigated effects of SAMe on liver injury in mice induced by acute alcohol administration. Male C57BL/6 mice received ethanol (5 g/kg BW) by gavage every 12 hrs for a total of 3 doses. SAMe (5 mg/kg BW) was administrated i.p. once a day for three days before ethanol administration. Subsequent serum ALT level, hepatic lipid peroxidation, enzymatic activity of CYP2E1 and hepatic mitochondrial glutathione levels were measured colorimetrically. Intracellular SAMe concentration was measured by high-performance liquid chromatography (HPLC). Histopathological changes were assessed by H&E staining. Our results showed that acute ethanol administration caused prominent microvesicular steatosis with mild necrosis and an elevation of serum ALT activity. SAMe treatment significantly attenuated the liver injury. In association with the hepatocyte injury, acute alcohol administration induced significant decreases in both hepatic SAMe and mitochondrial GSH levels along with enhanced lipid peroxidation. SAMe treatment attenuated hepatic SAMe and mitochondrial GSH depletion and lipid peroxidation following acute alcohol exposure. These results demonstrate that SAMe protects against the liver injury and attenuates the mitochondrial GSH depletion caused by acute alcohol administration. SAMe may prove to be an effective therapeutic agent in many toxin-induced liver injuries including those induced by alcohol.     

Extracellular calcium protects isolated rat hepatocytes from injury

The incubation of isolated rat hepatocytes in calcium-free medium resulted in a pronounced increase in lipid peroxidation, mitochondrial and cytoplasmic glutathione depletion, glutathione disulfide formation and efflux of reduced glutathione as compared with hepatocytes incubated in calcium containing medium. These data suggest that extracellular calcium ions serve a protective role in isolated rat hepatocytes against cell injury.     

A role of vitamin E in protection against cell injury. Maintenance of intracellular glutathione precursors and biosynthesis

The depletion of cell calcium from isolated rat hepatocytes results in stimulated lipid peroxidation, loss of intracellular and mitochondrial GSH (reduced glutathione), and enhancement of both efflux and oxidation of GSH. These events are followed by cell injury and enhance the susceptibility of the cells to toxic chemicals. It is shown herein that an initial event in the generation of such injury is the depletion of cellular alpha-tocopherol. alpha-Tocopheryl succinate addition (25 microM) to the calcium-depleted cells markedly elevated the alpha-tocopherol content of the cells, inhibited the associated lipid peroxidation, and maintained intracellular GSH levels without affecting its efflux or redox status. This resulted in an enhanced formation of total glutathione after a 5-h incubation, which correlated with the alpha-tocopherol content of the cells, and was greater than that expected by a direct sparing action of vitamin E. Inhibition of hepatocyte glutathione biosynthesis by buthionine sulfoximine (0.5 mM) eliminated the enhancement of GSH formation by vitamin E. Analysis of endogenous and 35S-labelled precursors of glutathione biosynthesis by high-performance liquid chromatography demonstrated that the depletion of cellular alpha-tocopherol resulted in the efflux of glutathione precursors. It is concluded that cell injury associated with alpha-tocopherol depletion is partly the result of the efflux of glutathione precursors, and hence diminished biosynthesis and intracellular levels of GSH. These losses and resultant cell injury are preventable by maintenance of cellular alpha-tocopherol levels.     

Protective effect of S-adenosylmethionine against galactosamine-induced injury of rat hepatocytes in primary culture

The protective effect of S-adenosylmethionine (SAMe) on D-galactosamine (GalN)-induced damage to rat hepatocytes was tested in primary cultures. SAMe at concentrations of 50 and 1000 mg/l significantly reduced lactate dehydrogenase release from cells injured by 40 mM GalN after 24 h of incubation. There were no significant changes in urea production after 24 h among tested groups, including control hepatocytes. Exposure of hepatocytes to GalN leads to 3.5-fold decrease in urea synthesis after 48 h in comparison with control cell cultures. Addition of the highest dose of SAMe (1000 mg/l) into the culture media attenuated this decrease by 180 %. None of the tested doses of SAMe (5, 25, 50 and 1000 mg/l) affected considerably the reduced activity of mitochondrial dehydrogenases. The content of reduced and oxidized glutathione in GalN-exposed cells was diminished to 1.5 % and 16 %, respectively, of the control values after 24 h. Using only the highest concentration SAMe increased significantly these contents. SAMe had no effect on dramatically decreased albumin synthesis. These findings indicate beneficial effect of SAMe, especially of the highest concentration, on GalN-induced toxicity to rat hepatocytes in primary culture. This action of SAMe seems to be associated with reduction of plasma membrane damage and increased synthesis of glutathione.     

The effect of capillarisin on glycochenodeoxycholic acid-induced apoptosis and heme oxygenase-1 in rat primary hepatocytes

The accumulation of hydrophobic bile acids plays a role in the induction of apoptosis and necrosis of hepatocytes during cholestasis. Glycochenodeoxycholate acid (GCDC) triggers a rapid oxidative stress response as an event of glutathione (GSH) depletion and nuclear factor kappa B (NF-κB) activation. We therefore investigated whether the bioactivity of the antioxidant capillarisin (Cap) prevents GCDC-induced hepatocyte damage. Isolated rat hepatocytes were co-incubated with 100 μM GCDC and 0.5 mg/ml Cap for 4 h. GSH depletion and thiobarbituric acid-reactive substances (TBARS, measure of lipid peroxidation) increased after GCDC exposure, but were markedly suppressed by Cap treatment. Cap protected hepatocytes from a GCDC-induced increase in reactive oxygen species (ROS) generation and mitochondrial membrane potential induction, as measured by flow cytometry analysis. In addition, Cap was shown to inhibit GCDC-mediated NF-κB activation by using electrophoretic mobility shift assays (EMSA). In contrast to GCDC, Cap not only significantly decreased cytochrome c release and caspase-3 enzyme activity, but also suppressed heme oxygenase-1 protein and mRNA expression in hepatocytes. These results demonstrate that Cap function as an antioxidant reduced hepatocyte injury caused by hydrophobic bile acids, perhaps by preventing generation of ROS and release of cytochrome c, thereby minimizing hepatocytes apoptosis.     

Mitochondrial adaptations to obesity-related oxidant stress

It is not known why viable hepatocytes in fatty livers are vulnerable to necrosis, but associated mitochondrial alterations suggest that reactive oxygen species (ROS) production may be increased. Although the mechanisms for ROS-mediated lethality are not well understood, increased mitochondrial ROS generation often precedes cell death, and hence, might promote hepatocyte necrosis. The aim of this study is to determine if liver mitochondria from obese mice with fatty hepatocytes actually produce increased ROS. Secondary objectives are to identify potential mechanisms for ROS increases and to evaluate whether ROS increase uncoupling protein (UCP)-2, a mitochondrial protein that promotes ATP depletion and necrosis. Compared to mitochondria from normal livers, fatty liver mitochondria have a 50% reduction in cytochrome c content and produce superoxide anion at a greater rate. They also contain 25% more GSH and demonstrate 70% greater manganese superoxide dismutase activity and a 35% reduction in glutathione peroxidase activity. Mitochondrial generation of H(2)O(2) is increased by 200% and the activities of enzymes that detoxify H(2)O(2) in other cellular compartments are abnormal. Cytosolic glutathione peroxidase and catalase activities are 42 and 153% of control values, respectively. These changes in the production and detoxification of mitochondrial ROS are associated with a 300% increase in the mitochondrial content of UCP-2, although the content of beta-1 ATP synthase, a constitutive mitochondrial membrane protein, is unaffected. Supporting the possibility that mitochondrial ROS induce UCP-2 in fatty hepatocytes, a mitochondrial redox cycling agent that increases mitochondrial ROS production upregulates UCP-2 mRNAs in primary cultures of normal rat hepatocytes by 300%. Thus, ROS production is increased in fatty liver mitochondria. This may result from chronic apoptotic stress and provoke adaptations, including increases in UCP-2, that potentiate necrosis.     

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.     

ATP-generating glycolytic substrates prevent N-nitrosofenfluramine-induced cytotoxicity in isolated rat hepatocytes

The relationship between cytotoxicity induced by N-nitrosofenfluramine and mitochondrial or glycolytic adenosine triphosphate (ATP) synthesis-dependent intracellular bioenergetics was studied in isolated rat hepatocytes. The supplementation of fructose, an ATP-generating glycolytic substrate, to hepatocyte suspensions prevented N-nitrosofenfluramine-induced cell injury accompanied by the formation of cell blebs, abrupt loss of intracellular ATP and reduced glutathione and mitochondrial membrane potential (DeltaPsi), and the accumulation of oxidized glutathione and malondialdehyde, indicating lipid peroxidation, during a 2h incubation period. Fructose (1-20mM) resulted in concentration-dependent protection against the cytotoxicity of N-nitrosofenfluramine at a concentration of 0.6mM, a low toxic dose. Pretreatment with xylitol, another glycolytic substrate, at concentration of 15mM also prevented the cytotoxicity caused by the nitroso compound, but neither glucose nor sucrose exhibited protective effects. In addition, fructose inhibited N-nitrosofenfluramine (0.5 and 0.6mM)-induced DNA damage, as evaluated in the comet assay, indicating that nuclei as well as mitochondria are target sites of the compound. These results indicate that (a) the onset of N-nitrosofenfluramine-induced cytotoxicity in rat hepatocytes is linked to mitochondrial failure, and that (b) the insufficient supply of ATP in turn limits the activities of all energy-requiring reactions and consequently leads to acute cell death.     

Mechanisms of Trazodone‐Induced Cytotoxicity and the Protective Effects of Melatonin and/or Taurine toward Freshly Isolated Rat Hepatocytes

It has been reported that the bioactive intermediate metabolites of trazodone might cause hepatotoxicity. This study was designed to investigate the exact mechanism of hepatocellular injury induced by trazodone as well as the protective effects of taurine and/or melatonin against this toxicity. Freshly isolated rat hepatocytes were used. Trazodone was cytotoxic and caused cell death with LC₅₀ of 300 µm within 2 h. Trazodone caused an increase in reactive oxygen species (ROS) formation, malondialdehyde accumulation, depletion of intracellular reduced glutathione (GSH), rise of oxidized glutathione disulfide (GSSG), and a decrease in mitochondrial membrane potential, which confirms the role of oxidative stress in trazodone‐induced cytotoxicity. Preincubation of hepatocytes with taurine prevented ROS formation, lipid peroxidation, depletion of intracellular reduced GSH, and increase of oxidized GSSG. Taurine could also protect mitochondria against trazodone‐induced toxicity. Administration of melatonin reduced the toxic effects of trazodone in isolated rat hepatocytes. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:457‐462, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21509     

Effect of benzophenones from Hypericum annulatum on carbon tetrachloride-induced toxicity in freshly isolated rat hepatocytes

Five benzophenones and a xanthone, isolated from Hypericum annulatum Moris, were investigated for their protective effect against carbon tetrachloride toxicity in isolated rat hepatocytes. The benzophenones and the xanthone gentisein were administered alone (100 microM) and in combination with CCl4 (86 microM). CCl4 undergoes dehalogenation in the liver endoplasmic reticulum. This process leads to trichlormethyl radical (*CCl3) formation, initiation of lipid peroxidation, and measurable toxic effects on the hepatocytes. The levels of thiobarbituric acid reactive substances (TBARS) were assayed as an index of lipid peroxidation (LPO). Lactate dehydrogenase (LDH) leakage, cell viability and reduced glutathione (GSH) depletion were used as signs of cytotoxicity. CCl4 significantly decreased hepatocyte viability, GSH level and increased TBARS level and LDH leakage as compared to the control. Our data indicate that 2,3',5',6-tetrahydroxy-4-methoxybenzophenone, 2-O-alpha-L-arabinofuranosyl-3',5',6-trihydroxy-4-methoxybenzophenone and 2-O-alpha-L-3'-acetylarabinofuranosyl-3',5',6-trihydroxy-4-methoxybenzophenone showed weaker toxic effects compared to CCl4 and in combination showed statistically significant protection against the toxic agent.     

Ca(2+)-dependent and independent mitochondrial damage in hepatocellular injury

The alterations of mitochondrial membrane potential during the development of irreversible cell damage were investigated by measuring rhodamine-123 uptake and distribution in primary cultures as well as in suspensions of rat hepatocytes exposed to different toxic agents. Direct and indirect mechanisms of mitochondrial damage have been identified and a role for Ca2+ in the development of this type of injury by selected compounds was assessed by using extracellular as well as intracellular Ca2+ chelators. In addition, mitochondrial uncoupling by carbonylcyanide-m-chloro-phenylhydrazone (CCCP) resulted in a marked depletion of cellular ATP that was followed by an increase in cytosolic Ca2+ concentration, immediately preceding cell death. These results support the existence of a close relationship linking, in a sort of reverberating circuit, the occurrence of mitochondrial dysfunction and the alterations in cellular Ca2+ homeostasis during hepatocyte injury.     

Nitric oxide ameliorates hydrophobic bile acid-induced apoptosis in isolated rat hepatocytes by non-mitochondrial pathways

Hydrophobic bile acids are toxic to isolated rat hepatocytes by mechanisms involving mitochondrial dysfunction and oxidative stress. In the current study we examined the role of nitric oxide (NO), a potential mediator of apoptosis, during bile acid-induced apoptosis. Freshly isolated rat hepatocytes and hepatic mitochondria generated NO and peroxynitrite (ONOO(-)) in a concentration- and time-dependent manner when exposed to the toxic bile salt glycochenodeoxycholate (GCDC) (25-500 microm), which was prevented by the nitric-oxide synthase (NOS) inhibitors N(G)-monomethyl-N-arginine monoacetate (l-NMMA) and 1400W. Relationships between hepatocyte NO production and apoptosis were examined by comparing the effects of NOS inhibitors with other inhibitors of GCDC-induced apoptosis. Inhibitors of caspases 8 and 9, the mitochondrial permeability transition blocker cyclosporin A, and the antioxidant idebenone reduced NO generation and apoptosis in GCDC-treated hepatocytes. In contrast, NOS inhibitors had no effect on GCDC-induced apoptosis despite marked reduction of NO and ONOO(-). However, treatment with the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate [N-(-aminoethyl)N-(2-hydroxy-2-nitrohydrazino)-1,2-ethylenediamine) inhibited apoptosis and caspase 3 activity while significantly elevating NO levels above GCDC-stimulated levels. Neither NO donors nor NOS inhibitors affected GCDC-induced mitochondrial permeability transition or cytochrome c release from liver mitochondria or GCDC-induced mitochondrial depolarization from isolated hepatocytes, suggesting that NO inhibits bile acid-induced hepatocyte apoptosis by a non-mitochondrial-dependent pathway. In conclusion, whereas NO produced from GCDC-treated hepatocytes neither mediates nor protects against bile acid-induced apoptosis, higher levels of NO inhibit GCDC-induced hepatocyte apoptosis by caspase-dependent pathways.     

Toxicity of 1,2-dibromoethane in isolated hepatocytes: Role of lipid peroxidation

Treatment of isolated hepatocytes with 1,2-dibromoethane (DBE) caused a concentration dependent depletion of cellular glutathione (GSH) content and a parallel increase in the covalent binding of reactive intermediates to cell proteins, as a consequence of the haloalkane activation. The reduction of the hepatocyte GSH content, induced by DBE, stimulated the onset of lipid peroxidation, as measured by malondialdehyde (MDA) accumulation. N-Acetylcysteine (1 mM) was found to partially prevent GSH loss and to inhibit MDA formation, whereas equal concentrations of cysteine and methionine were ineffective on these respects. The stimulation of the peroxidative reactions appeared to be also associated with an increase in the leakage of lactate dehydrogenase (LDH) from the cells, indicative of a severe hepatocyte injury. Antioxidants such as -tocopherol, N,N′-phenyl-phenylenediamine (DPPD) and promethazine, as well as N-acetylcysteine reduced MDA formation to various extents and also protect against LDH release, yet without interfering with the covalent binding of DBE reactive intermediates to hepatocyte proteins. These results suggest the involvement of lipid peroxidation, consequent to GSH depletion, in the pathogenesis of liver cell necrosis due to DBE.     

Mitochondrial damage and its role in causing hepatocyte injury during stimulation of lipid peroxidation by iron nitriloacetate.

Incubation of isolated rat hepatocytes with 0.1 mM iron nitrilotriacetic acid (FeNTA) caused a rapid rise in lipid peroxidation followed by a substantial increase in trypan blue staining and lactate dehydrogenase release, but did not affect the protein and non-protein thiol content of the cells. Hepatocyte death was preceded by the decline of mitochondrial membrane potential, as assayed by rhodamine 123 uptake, and by the depletion of cellular ATP. Chelation of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid or inhibition of Ca2+ cycling within the mitochondria by LaCl3 or cyclosporin A did not prevent the decline of rhodamine 123 uptake. On the other hand, a dramatic increase in the conjugated diene content was observed in mitochondria isolated from FeNTA-treated hepatocytes. Oxidative damage of mitochondria was accompanied by the leakage of matrix enzymes glutamic oxalacetic aminotransferase (GOT) and glutamate dehydrogenase (GLDH). The addition of the antioxidant N,N'-diphenylphenylene diamine (DPPD) completely prevented GOT and GLDH leakage, inhibition of rhodamine 123 uptake, and ATP depletion induced by FeNTA, indicating that Ca(2+)-independent alterations of mitochondrial membrane permeability consequent to lipid peroxidation were responsible for the loss of mitochondrial membrane potential. DPPD addition also protected against hepatocyte death. Similarly hepatocytes prepared from fed rats were found to be more resistant than those obtained from starved rats toward ATP depletion and cell death caused by FeNTA, in spite of undergoing a comparable mitochondrial injury. A similar protection was also observed following fructose supplementation of hepatocytes isolated from starved rats, indicating that the decline of ATP was critical for the development of FeNTA toxicity. From these results it was concluded that FeNTA-induced peroxidation of mitochondrial membranes impaired the electrochemical potential of these organelles and led to ATP depletion which was critical for the development of irreversible cell injury.     

Mitochondrial electron transport inhibitors cause lipid peroxidation-dependent and -independent cell death: protective role of antioxidants

Mitochondrial electron transport inhibitors induced two distinct pathways for acute cell death: lipid peroxidation-dependent and -independent in isolated rat hepatocytes. The toxic effects of mitochondrial complex I and II inhibitors, rotenone (ROT) and thenoyltrifluoroacetone (TTFA), respectively, were dependent on oxidative stress and lipid peroxidation, while cell death induced by inhibitors of complexes III and IV, antimycin A (AA) and cyanide (CN), respectively, was caused by MMP collapse and loss of cellular ATP. Accordingly, cellular and mitochondrial antioxidant depletion or supplementation, in general, resulted in a dramatic potentiation or prevention, respectively, of toxic injury induced by complex I and II inhibitors, with little or no effect on complex III and IV inhibitor-induced toxicity. ROT-induced oxidative stress was prevented by the addition of d-alpha-tocopheryl succinate (TS) but surprisingly TS did not afford hepatocytes protection against TTFA-induced oxidative damage. TS treatment prevented ROT-induced mitochondrial lipid hydroperoxide formation but had no effect on the loss of mitochondrial GSH or cellular ATP, suggesting a mitochondrial lipid peroxidation-mediated mechanism for ROT-induced acute cell death. In contrast, only fructose treatment provided excellent cytoprotection against AA- and CN-induced toxicity. Our findings indicate that complex III and IV inhibitors cause a rapid and severe depletion of cellular ATP content resulting in acute cell death that is dependent on cellular energy impairment but not lipid peroxidation. In contrast, inhibitors of mitochondrial complex I or II moderately deplete cellular ATP levels and thus cause acute cell death via a lipid peroxidation pathway.     

Zinc supplementation attenuates thioacetamide-induced liver injury and hyperglycemia in mice

Zinc supplementation has been shown to improve not only liver dysfunction but also glucose intolerance in subjects with liver cirrhosis. In this study, we investigated the effects of zinc supplementation on the changes in circulating levels of tumor necrosis factor-α and total antioxidant capacity in mice with thioacetamide-induced liver injury. The protective effect of concurrent zinc administration for thioacetamide-induced hepatotoxicity was also examined. The results showed that zinc treatment significantly attenuated thioacetamide-induced liver injury and hyperglycemia. Furthermore, thioacetamide-induced hepatotoxicity was markedly weakened by the simultaneous zinc administration. These effects might be attributed to reduced tumor necrosis factor-α production and elevated total antioxidant capacity induced by the mineral. Our data suggest that zinc supplementation might be beneficial for the subjects with a high susceptibility to liver injury.     

Combining ursodeoxycholic acid or its NO-releasing derivative NCX-1000 with lipophilic antioxidants better protects mouse hepatocytes against amiodarone toxicity

Nonalcoholic steatohepatitis (NASH) is a common and potentially severe form of liver disease. This study aimed to determine the effect of ursodeoxycholic acid and its NO-releasing derivative NCX-1000 alone or in combination with antioxidants on cultured mouse hepatocytes treated with amiodarone to mimic certain aspects of hepatocyte injury found in NASH. Isolated mouse hepatocytes were incubated with ursodeoxycholic acid or NCX-1000 (0-100 micromol/L) combined or not combined with the hydrophilic antioxidants butylated hydroxytoluene and ascorbic acid (0-100 micromol/L) or with the lipophilic antioxidant alpha-tocopherol (0-100 micromol/L) 15 min before adding amiodarone (50 micromol/L) to the culture medium. Twenty hours later, necrosis, apoptosis, superoxide anion production, and malondialdehyde levels were assessed in cultured cells. Amiodarone led to a dose-dependent decrease in cell viability with an LD50 of 50 micromol/L and increased production of superoxide anion and lipid peroxidation. NCX-1000 showed a better protective potential than ursodeoxycholic acid against the toxic effects of amiodarone. The hydrophilic antioxidants had no effect on the toxicity of amiodarone, whereas alpha-tocopherol at a concentration >100 micromol/L almost completely suppressed it. Ursodeoxycholic acid and NCX-1000 protection was additive only when they were combined with alpha-tocopherol, not with butylated hydroxytoluene or ascorbic acid. In addition, all the antioxidants tested reduced the superoxide anion detected, but only alpha-tocopherol prevented lipid peroxidation induced by amiodarone. The combination of lipophilic antioxidants with ursodeoxycholic acid or NCX-1000 enhances their protective potential and could represent an interesting therapeutic approach to explore for the treatment of NASH.     

Oxygen toxicity: unique cytoprotective properties of vitamin E succinate in hepatocytes

Freshly isolated rat hepatocytes suspensions were incubated under an atmosphere of 95% O2/5% CO2 or 95% air/5% CO2 for 10 h. Cell injury and death were observed between the 6th and 10th hour of incubation, only in 95% O2-treated hepatocytes. Oxygen-induced injury was preceded by marked lipid peroxidation and rapid depletion of cellular alpha tocopherol content. The exogenous administration of unesterified alpha tocopherol (T, 25 microM) resulted in a 20-fold increase in cellular T levels (4.2 nmol/10(6) cells) but failed to protect these hepatocytes from the toxic effects of oxygen. In contrast, hepatocytes incubated with 25 microM of the succinate ester of alpha tocopherol (TS) contained both TS (3.0 nmol/10(6) cells) and T (1.4 nmol/10(6) cells) and were completely protected from the toxic effects of oxygen, including the induction of lipid peroxidation. These findings suggest that TS cytoprotection results not from the cellular accumulation of T but rather, from cellular TS accumulation. The data also indicate that the depletion of cellular T is not the critical cellular event that is responsible for hyperoxia (reactive oxygen intermediate)-induced injury. Instead, it appears that TS possesses unique cytoprotective properties that intervene in the critical cellular events that lead to oxygen toxicity. Thus, vitamin E succinate and our hyperoxic hepatocyte preparation provide a promising new model system for the study and prevention of tissue damage resulting from the toxic effects of hyperoxia and reactive oxygen intermediates.