Cytotoxic effects of N-acetyl-p-benzoquinone imine,a common arylating intermediate of paracetamol and N-hydroxyparacetamol

The cytotoxic effects of N-acetyl-p-benzoquinone imine (NAPQI), a postulated ultimate reactive metabolite of paracetamol (pHAA), was studied in suspensions of isolated rat hepatocytes. Incubation of cells for 10–300 min with 0.1–0.5 mM NAPQI led to concentration dependent cell damage. as determined by increased trypan blue exclusion, lactate dehydrogenase release and glutathione (GSH) depletion. NAPQI and N-hydroxyparacetamol (N-OH-pHAA), a postulated proximate metabolite of pHAA, caused cytotoxic effects in the same concentration range. In contrast, no toxic effects of pHAA (? 20 mM) could be demonstrated. With the short half-life of NAPQI, less than 0.5% of the NAPQI added is expected to be left in the incubation medium after a 2 min incubated period. Nevertheless, 10–120 min (depending on the concentration of NAPQI) elapsed before the cells responded with increased membrane permeability. Clearly, the initial damage caused by NAPQI must be followed by subsequent cellular steps before toxicity becomes apparent. The addition of N-acetylcysteine, GSH or ascorbate during the NAPQI exposure period fully protected the hepatocytes from NAPQI damage. Lesser effects were demonstrated when these agents were added after the 5 min NAPQI exposure period. The results presented in this study further support the hypothesis that NAPQI is the ultimate reactive formed from pHAA.     

Increased toxicity of cocaine on human hepatocytes induced by ethanol: role of GSH.

Increased toxicity of cocaine to human hepatocytes is observed when cells are simultaneously incubated with ethanol. Ethanol might exacerbate cocaine hepatocyte toxicity by three different pathways: a) by increasing the oxidative metabolism of cocaine and hence the oxidative damage; b) by the formation of a more toxic metabolite, namely cocaethylene; or c) by decreasing the defence mechanisms of the cell (i.e. GSH). In the present study, experiments were conducted to investigate the feasibility of these hypotheses. In hepatocytes preincubated for 48 hr with ethanol, neither significant changes in cocaine metabolism nor cytotoxicity were found despite differences in hepatocyte p-nitrophenol hydroxylase (largely CYP2E1 activity). Cocaethylene, the transesterification product of cocaine and ethanol, was found to be more toxic than cocaine for human hepatocytes (3x). However, the small amount formed when human hepatocytes were incubated with cocaine and ethanol would hardly explain the increased toxicity observed. On the other hand, the simultaneous presence of cocaine and ethanol caused a sustained decline in the intracellular GSH content that was larger than that observed in cocaine- or ethanol-treated cultures. Parallel to this phenomenon, a significant increase in lipid peroxidation was observed, as compared to cells treated with equimolar amounts of cocaine, ethanol, or cocaethylene. Finally, depletion of hepatocyte GSH with diethylmaleate down to levels similar to those found in ethanol-treated cells made hepatocytes more susceptible to cocaine. Taken together, the results of this research suggest that by decreasing GSH levels, ethanol makes human hepatocytes more sensitive to cocaine-induced oxidative damage.     

Potentiation of heroin and methadone hepatotoxicity by ethanol: an in vitro study using cultured human hepatocytes.

1. The hepatotoxic effects of heroin and methadone, and the effect of ethanol on opioid-induced hepatotoxicity, have been investigated in human cultured hepatocytes. Hepatocytes pretreated with 50 and 100 mM ethanol were exposed to increasing concentrations of heroin and methadone. 2. Cytotoxicity was evaluated by measuring leakage of intracellular lactate dehydrogenase, and by assessment of hepatocyte mitochondrial succinate dehydrogenase. The half-maximal cytotoxic concentration of heroin for human hepatocytes (TC50) was decreased by 70-55% by pre-exposure to 50 mM ethanol, and that for methadone was decreased by 60-40%. 3. Metabolic functions of human hepatocytes were significantly impaired at concentrations of opioids that had shown little cytotoxicity. Ethanol potentiated opioid-induced hepatotoxicity; concentrations of heroin and methadone that had little or no effect on hepatocyte metabolism in the absence of ethanol caused a significant decrease in urea synthesis rate, metabolism of glycogen and depletion of the intracellular GSH pool after ethanol pretreatment. 4. The increase in toxicity of heroin and methadone produced by ethanol is concomitant with a 40% increase in cytochrome P-450 levels of the pretreated hepatocytes.     

Effect of fructose on the biochemical toxicity of hydrazine in isolated rat hepatocytes

Rat hepatocyte suspensions were incubated with various concentrations of hydrazine (0, 8, 12, 16, 20 mM) for 1, 2 and 3 h. In some experiments fructose (10 mM) was added either during the preincubation period, or 1 h after the start of hydrazine treatment. In certain experiments in which fructose was added, the glycolytic inhibitor sodium fluoride (3 mM) was also added during the preincubation period. Hepatocytes incubated with hydrazine alone demonstrated both a concentration- and time-dependent loss of cell viability as measured by increased Trypan blue uptake and lactate dehydrogenase (LDH) leakage. These parameters were reduced and delayed by fructose when added either before or 1 h after hydrazine treatment. There was also both a concentration- and time-dependent loss of ATP and reduced glutathione (GSH) content with hydrazine treatment. Moreover, fructose caused an initial rapid depletion of ATP but thereafter ATP levels were increased in control hepatocytes. Fructose reduced both the depletion of ATP and GSH in hydrazine- treated hepatocytes. Urea synthesis was inhibited by all concentrations of hydrazine studied but fructose treatment after 1 h did not alter this. This study also demonstrated that fluoride, an enolase inhibitor, abolished the protection against depletion of ATP levels provided by fructose, without affecting cell viability or GSH levels. These findings suggest that the cytotoxicity of hydrazine and its effects on urea synthesis and GSH levels are not a direct result of ATP depletion. The protective effects of fructose against the cytotoxicity may be due to a direct interaction with hydrazine.     

Species differences in the effects of 1,1-di-(4-chlorophenyl)-2-chloroethylene (DDMU) and 1,1-di-(4-chlorophenyl)-2,2-dichloroethylene (DDE) on glutathione levels in isolated hepatocytes from Japanese quail and rat

1. Hepatocytes have been isolated from adult Japanese quail in high yields (26-40 X 10(6) cells/g) with 95% viability. Variation in the collagenase and hyaluronidase levels and incubation time used in the isolation procedure affected the yield and viability of the hepatocytes. Quail hepatocytes were more stable in a standard nutrient medium than those of the rat. 2 1,1-Di-(4-chlorophenyl)-2-chloroethylene (DDMU), a metabolite of DDT, depleted quail hepatic GSH levels both in vitro and in vivo, but had no effect on rat hepatocytes. DDMU (0.1 mM) depleted GSH levels in the quail to the same extent as diethyl maleate (a known GSH-depleting agent) (0.04 mM) but the latter acted more rapidly. 3. Pretreatment of quail in vivo with DDMU or phenobarbitone, known inducers of the hepatic mixed-function oxidases, resulted in faster depletion of GSH when the hepatocytes were incubated subsequently with DDMU in vitro. 4. In contrast, 1,1-di-(4-chlorophenyl)-2,2-dichloroethylene (DDE), another metabolite of DDT, did not deplete GSH levels in the quail but did cause some reduction in the rat. Phenobarbitone pretreatment had no effect on GSH depletion by DDE in vitro in quail hepatocytes but enhanced GSH depletion in rat hepatocytes.     

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

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

Protection of rat hepatocytes from tert-butyl hydroperoxide-induced injury by catechol

Metabolism of tert-butyl hydroperoxide (TBHP, 2.0 mM) by glutathione peroxidase within isolated rat hepatocytes caused a rapid oxidation of intracellular reduced glutathione and ultimately NADPH through glutathione reductase. TBHP also caused the formation of surface blebs in the hepatocyte plasma membrane followed by the leakage of cytosolic enzymes, such as lactate dehydrogenase, into the incubation medium. Catechol (0.1 mM) protected hepatocytes from the cytotoxic effects of TBHP but did not prevent the rapid oxidation of glutathione indicating normal metabolism of TBHP through glutathione reductase. In contrast, addition of catechol to the hepatocyte incubations prevented TBHP-induced depletion of intracellular NADPH and increased the total NADP+ + NADPH concentration without altering significantly the intracellular NADP+ content or the NADPH/NADP + NADPH ratio. Catechol did not alter TBHP stimulation of the pentose phosphate pathway. Hepatocytes incubated with sublethal concentrations of TBHP (1.0 mM) did not leak lactate dehydrogenase into the medium but did lose intracellular potassium. In these experiments, TBHP caused a sustained increase in phosphorylase alpha activity suggesting that TBHP metabolism may be associated with a sustained increase in cytosolic free Ca2+. In the presence of catechol, phosphorylase alpha activity was increased by 5 min but returned toward control by 20 min. These data suggest that catechol may be protecting hepatocytes from TBHP-induced injury by preventing a sustained rise in cytosolic free Ca2+ concentration.     

The mechanism of prevention of paracetamol-induced hepatotoxicity by 3,5-dialkyl substitution. The roles of glutathione depletion and oxidative stress

Recently, we have reported that 3,5-dialkyl substitution of paracetamol, in contrast to 3-monoalkyl substitution, prevented the paracetamol-induced toxicity in freshly isolated rat hepatocytes without having any effect on its cytochrome P-450 mediated bioactivation to reactive N-acetyl-p-benzoquinone imines (NAPQI). In the present study the mechanism of this prevention of toxicity, with special emphasis on oxidative stress, was studied in more detail in freshly isolated rat hepatocytes, using paracetamol, 3-methyl-, 3,5-dimethyl-paracetamol, synthetic NAPQI and 3,5-dimethyl-NAPQI. 3-Methyl-paracetamol was found to induce glutathione (GSH) depletion, lipid-peroxidation and cytotoxicity in hepatocytes to the same extent as paracetamol. 3,5-Dimethyl-paracetamol, however, even when added in a ten-fold higher concentration when compared to paracetamol, did not induce any of these effects. Similar differences of toxicity were observed between NAPQI and 3,5-dimethyl-NAPQI; 3,5-dimethyl-NAPQI, in contrast to NAPQI, did not reduce protein thiol levels, did not induce GSH depletion, lipid-peroxidation nor cytotoxicity. Only after artificial depletion of GSH levels in the hepatocytes by DEM or BCNU, 3,5-dimethyl-NAPQI was cytotoxic. This effect was accompanied by depletion of protein thiol levels, but not by lipid-peroxidation. Addition of the disulfide reducing agent, dithiothreitol, prevented the artificially created cytotoxicity of 3,5-dimethyl-NAPQI. It is concluded that prevention of paracetamol-induced toxicity by 3,5-dialkyl substitution is primarily due to prevention of irreversible GSH-depletion, presumably caused by the inability of 3,5-dialkyl-NAPQI to conjugate with thiols. As a result, the GSH-dependent cellular defense mechanism against potential oxidative cellular injury by 3,5-dialkyl-NAPQI is left unimpaired. Our observations indicate that a compound, not capable of covalent binding to thiol groups of proteins, can induce toxicity solely as a result of protein thiol oxidation without inducing lipid-peroxidation.     

S-adenosylmethionine but not glutathione protects against galactosamine-induced cytotoxicity in rat hepatocyte cultures

A gradual but extensive depletion of hepatic GSH has long been known to accompany development of galactosamine-induced hepatotoxicity in rats, and some protection from liver injury has been observed after administration of sulfhydryl-donating compounds. Although these observations support a key role for GSH in the underlying mechanism, the impact of GSH depletion and repletion on the hepatotoxic response to galactosamine is unclear. To investigate the role of GSH in galactosamine-induced liver injury, we examined the effect of modulating GSH content on galactosamine toxicity in rat primary hepatocyte cultures. Galactosamine (4 mM) cytotoxicity was assessed by release of lactate dehydrogenase into the culture medium, and hepatocellular GSH content was measured by HPLC with electrochemical detection. The data indicated that prior depletion of GSH with either diethyl maleate or buthionine sulfoximine significantly enhanced galactosamine toxicity; however, addition of GSH-ester or alternate sulfur nucleophiles at various times during the incubation did not abrogate toxicity. In contrast, co-addition of S-adenosylmethionine (SAMe) with galactosamine exerted a marked protective effect without significantly altering hepatocyte GSH content. These data suggest that GSH depletion is not directly involved in the sequelae for galactosamine-induced hepatotoxicity, and raise the possibility that SAMe may have hepatoprotective effects that are not dependent on its ability to enhance GSH synthesis.     

Propyl gallate-induced DNA fragmentation in isolated rat hepatocytes

Incubation of isolated rat hepatocytes with propyl gallate (PG) at concentrations of ≥1 mM induced cell killing, whereas PG at ≤0.5 mM did not cause cell death during a 3-h incubation. PG at ≥0.5 mM elicited the ladder formation of soluble low-molecular weight DNA fragments with integer multiples of approximately 180 bp and specific nuclear DNA cleavages detected cytopathologically by labeling of a digoxigenin-nucleotide complex to new 3′-OH ends. Both of these PG-induced changes observed in hepatocytes are characteristic features of apoptosis. In contrast, the pretreatment of N-acetylcysteine (4 mM), a precursor of intracellular glutathione (GSH) and antioxidant, prevented PG (0.5 mM)-induced formation of soluble DNA fragments and loss of cellular GSH, ATP, and formation of blebbing. These results suggest that when the concentration of PG is decreased, the effects of PG on hepatocytes change from acute necrotic to apoptotic mode, and that the onset of DNA fragmentation is associated with GSH depletion. Received: 23 June 1997 / Accepted: 18 August 1997     

Dissociation of cell death from covalent binding of paracetamol by flavones in a hepatocyte system

Paracetamol metabolism and toxicity were studied in isolated rat hepatocytes. Cell damage, due to paracetamol, was shown to be dose dependent and was worse in cells from animals pre-treated with phenobarbitone. Exposure to 10 mM paracetamol for 1 hr caused a loss of intracellular reduced glutathione (GSH) and a later progressive leakage of isocitrate dehydrogenase (ICD). Treatment with (+)catechin, 3-O-methyl(+)catechin and promethazine reduced or prevented the paracetamol-induced ICD leakage. Similarly, studies on covalent binding of paracetamol showed that 3-O-methyl(+)catechin, which “protected” the cells, did so without affecting the amount of material bound covalently to cellular protein. Incubation in tissue culture for 24 hr, after prior treatment with paracetamol ± the protective agent, showed that the protected cells remained viable and attached to tissue culture plates much better than did the “unprotected” cells. These results suggest that the protective effect is much more than just a temporarily delayed cell death. GSH loss and covalent binding of paracetamol metabolites to cell protein are not sufficient causes of cell death, although they may act as starting points in the chain of events leading to cell death.     

Protective role of the glutathione redox cycle against adriamycin-mediated toxicity in isolated hepatocytes

Incubation of isolated rat hepatocytes with 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) resulted in the selective and extensive (> 90 per cent) inactivation of glutathione reductase. BCNU also depleted intracellular glutathione by 70 per cent but had no significant effect on Cell viability or lipid peroxidation. Incubation of BCNU-treated hepatocytes with adriamycin (ADR) resulted in a decrease in Cell viability concurrent with an increase in lipid peroxidation. These effects were not observed with untreated hepatocytes incubated with ADR. Glutathione depletion with diethylmaleate and incubation with ADR did not result in a significant decrease in Cell viability or increase in lipid peroxidation. Incubation of BCNU-treated hepatocytes with ADR in the presence of exogenous α-tocopherol resulted in a significant amount of protection from ADR-mediated damage.     

Effects of dicoumarol on cytotoxicity caused by tert-butylhydroquinone in isolated rat hepatocytes

The effects of dicoumarol, an inhibitor of DT-diaphorase, on the cytotoxicity of tert-butylhydroquinone (tBHQ) were studied in freshly isolated rat hepatocytes. Addition of tBHQ (0.5 mM) to hepatocytes resulted in a time-dependent cell death accompanied by depletion of intracellular ATP, glutathione (GSH), and protein thiols. Pretreatment of hepatocytes with dicoumarol (30 μM) did not affect cell viability or cellular levels of ATP, GSH, or protein thiols during the incubation period; however, dicoumarol did promote the appearance of cell blebs and the depletion of ATP and protein thiols induced by tBHQ and ultimately enhanced the cytotoxicity of tBHQ.     

On the mechanisms of glutathione depletion in hepatocytes exposed to morphine and ethylmorphine

Incubation of isolated rat hepatocytes with either morphine or ethylmorphine resulted in glutathione (GSH) depletion followed by loss of cell viability. Pretreatment of cells with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inactivate glutathione reductase did not markedly affect the rates of GSH depletion seen in untreated cells. In contrast, hexobarbital stimulated H2O2 production in isolated liver microsomes, incubated aerobically with NADPH, whereas the effects of morphine and ethylmorphine on microsomal H2O2 production were minimal. Finally, incubation of hepatocytes with radioactively labeled morphine resulted in formation of 2 glutathione conjugates, one of which was tentatively identified as formyl glutathione. We conclude that GSH consumption during the metabolism of morphine or ethylmorphine by hepatocytes is due mainly to formation of glutathione conjugates.     

Species differences in the induction of hepatocellular DNA synthesis by diethanolamine.

Diethanolamine increased the incidence and multiplicity of liver tumors in the mouse following chronic exposure. Diethanolamine is known to inhibit cellular choline uptake. Since choline deficiency produces tumors in rodents, diethanolamine, through choline depletion, may result in tumor development in rodents. The potential for diethanolamine to function through this mode of action in humans is not known. The present studies examined the effect of diethanolamine (0-500 mug/ml) and choline depletion on DNA synthesis and changes in expression of genes involved in cell growth pathways in primary cultures of mouse, rat, and human hepatocytes. In mouse and rat hepatocytes DNA synthesis was increased following treatment with 10 mug/ml diethanolamine and higher (3- to 4-fold over control). In contrast, diethanolamine failed to increase DNA synthesis in human hepatocytes. Incubation of hepatocytes in medium containing reduced choline (1/10 to 1/100 of normal medium; 0.898 to 0.0898 mg/l vs. 8.98 mg/l) increased DNA synthesis (1.6- and 1.8-fold of control in mouse and rat hepatocytes, respectively); however, choline depletion did not induce DNA synthesis in human hepatocytes. Mouse and rat hepatocytes incubated in medium supplemented with 2- to 50-fold excess choline reduced diethanolamine-induced DNA synthesis to control levels or below. Gene expression analysis of mouse and rat hepatocytes following diethanolamine treatment showed increases in genes associated with cell growth and decreases in expression of genes involved in apoptotic pathways. These results support the hypothesis that choline depletion is central to the mode of action for the induction of rodent hepatic neoplasia by diethanolamine. Furthermore, since diethanolamine treatment or choline depletion failed to induce DNA synthesis in human hepatocytes, these results suggest that humans may not be at risk from the carcinogenic effects of diethanolamine.     

S-Adenosylmethionine protects against acetaminophen-induced hepatotoxicity in mice

An overdose of acetaminophen (APAP) is the most frequent cause of fulminant liver failure in the United States. Increasing evidence demonstrates that oxidative stress plays an important etiologic role in APAP-induced liver injury. S-Adenosylmethionine (SAMe) is a key intermediate in the hepatic trans-sulfuration pathway and serves as a precursor for glutathione (GSH) as well as the methyl donor in most transmethylation reactions. In the present study, we investigated effects of SAMe on liver injury induced by APAP administration in male C57BL/6 mice. Two related studies were performed. In the first experiment, SAMe (1g/kg BW) was injected intraperitoneally 4 h before APAP (600 mg/kg BW) administration. In the second experiment, SAMe was injected intraperitoneally 1 h after APAP administration. Our results showed that APAP administration induced changes typical of confluent centrilobular necrosis by histological examination and a marked elevation in serum alanine aminotransferase (ALT) activity. APAP administration induced significant decreases in both hepatic and blood SAMe concentrations. In addition, APAP decreased intracellular (both cytosolic and mitochondrial) GSH concentrations along with increased lipid peroxidation in conjunction with mitochondrial dysfunction as documented by Ca2+-induced mitochondrial permeability transition. SAMe treatment (both before and after APAP) significantly attenuated the liver injury. Exogenous SAMe prevented the decrease in liver and blood SAMe concentrations. Moreover, SAMe treatment attenuated both cytosolic and mitochondrial GSH depletion as well as mitochondrial dysfunction. We conclude that SAMe at least in part protects the liver from APAP-induced injury by preventing intracellular GSH depletion and mitochondrial dysfunction.     

Metabolic alterations in hepatocytes promoted by the herbicides paraquat,dinoseb and 2,4-D

The cytotoxic effects of the herbicides paraquat (1,1′-dimethyl-4,4′-bipyridylium dichloride), dinoseb (2-sec-butyl-4,6-dinitrophenol) and 2,4-D (2,4-dichlorophenoxyacetic acid) on freshly isolated rat hepatocytes were investigated. Paraquat and 2,4-D (1–10 mM) caused a dose and time dependent cell death accompanied by depletion of intracellular glutathione (GSH) and mirroring increase of oxidized glutathione (GSSG). Dinoseb, the most effective cytotoxic compound under study (used in concentrations 1000 fold lower than paraquat and 2,4-D), exhibited moderate effects upon the level of GSH and GSSG. These limited effects are at variance with significant effects upon the adenine and pyridine nucleotide contents. ATP and NADH levels are rapidly depleted by herbicide metabolism. This depletion is observed in the millimolar range for paraquat and 2,4-D and in the micromolar range for dinoseb. 2,4-D completely depletes cellular ATP, with subsequent cell death, as detected by LDH leakage. Paraquat rapidly depletes NADH, according to the redox cycling of the herbicide metabolism. The most effective compound is dinoseb since it exerts similar effects as described for paraquat and 2,4-D at concentrations 1000 fold lower. Simultaneously with NADH and ATP depletion, the levels of ADP, AMP and NAd+ increase in hepatocytes incubated in the presence of the herbicides. In contrast to NADH, the time course and extent of ATP depletion and fall in energy charge correlate reasonably with the time of onset and rate of cell death. It is concluded that the herbicides, paraquat and 2,4-D are hepatotoxic and initiate the process of cell death by decreasing cellular GSH. As a consequence of this primary disturbance, alteration of adenine and pyridine nucleotides contents is a critical event in the induction of irreversible cell injury.     

Lipid peroxidation and protein thiol depletion are not involved in the cytotoxicity of N-hydroxy-2-acetylaminofluorene in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to study the mechanism of cell death induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Exposure to 1.0 mM N-OH-AAF resulted in more than 90% cell death (as measured by LDH leakage) of hepatocytes isolated from male rats within 6 hr. Only 36% of the hepatocytes isolated from female rats died within this period. When inorganic sulfate was omitted from the incubation medium, a 6 hr exposure to 1.0 mM N-OH-AAF resulted in only 40% cell death of male hepatocytes. These findings are in accordance with the sex difference and sulfation dependence of N-OH-AAF hepatotoxicity observed in the rat in vivo. N-OH-AAF decreased glutathione (GSH) in male hepatocytes in a concentration-dependent manner. This GSH consumption was only partly dependent on the presence of inorganic sulfate. No lipid peroxidation was observed during N-OH-AAF exposure; N-OH-AAF even prevented endogenous and diethyl maleate (DEM)-induced lipid peroxidation. No reduction of free protein thiol groups was found after exposure to N-OH-AAF, even after 75% cell death had occurred. A reduction of protein thiols after N-OH-AAF exposure was observed in GSH depleted hepatocytes (obtained by DEM plus vitamin E pretreatment). Under these conditions N-OH-AAF-induced cell death occurred earlier. Therefore, GSH protects against protein thiol depletion by N-OH-AAF in control cells. N-OH-AAF-induced cell death was preceded by a loss of intracellular ATP. It is concluded, therefore, that neither lipid peroxidation nor depletion of protein thiols, but possibly loss of intracellular ATP, is involved in the sulfation-dependent cytotoxic mechanism of N-OH-AAF in isolated rat hepatocytes.     

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.     

Epigallocatechin-3-gallate(-)protects Chang liver cells against ethanol-induced cytotoxicity and apoptosis

The objective of the study was to investigate the effect of epigallocatechin-3-gallate (EGCG) on ethanol (EtOH)-induced cytotoxicity in human Chang liver cells. Cells were incubated with either 30 mM EtOH alone or together in presence of (25 microM) EGCG for 24 hr. Assays were performed in treated cells to evaluate the ability of EGCG to prevent the toxic effects of EtOH. EtOH exposure suppressed the growth of Chang liver cells and induced lactate dehydrogenase leakage, oxygen radical formation, peroxidation of lipids, mitochondrial dysfunction and apoptosis. Reduced glutathione (GSH) concentration was significantly decreased (P < 0.05) while oxidized glutathione (GSSG) concentration was significantly elevated in EtOH-treated cells as compared to normal cells. Incubation of EGCG along with EtOH significantly prevented EtOH-dependent cell loss and lactate dehydrogenase leakage. This was associated with a reduction in oxidative damage as reflected by a reduction in the generation of reactive oxygen species, and in lipid peroxidation and maintenance of intracellular GSH/GSSG ratio. EGCG decreased the accumulation of sub-G(1) phase cells and reduced apoptosis. The findings suggest that EGCG exerts a protective action during EtOH-induced liver cell damage.