Effect of pretreatment with some mixed-function oxidase enzyme inducers on the acute hepatotoxicity of coumarin in the rat

Male Sprague-Dawley rats were pretreated with saline, corn oil, sodium phenobarbitone (PB) (100 mg/kg body weight/day), 20-methylcholanthrene (20 MC) (20 mg/kg body weight/day) or Aroclor 1254 (ARO) (100 mg/kg body weight/day) by daily ip injections for 5 days. Animals were then given single oral doses of either 250 or 500 mg coumarin/kg body weight and hepatotoxicity was assessed after 24 hr. Coumarin produced hepatotoxicity, which comprised hepatocyte necrosis and elevation of plasma alanine aminotransferase and aspartate aminotransferase activities, in all pretreated groups. Hepatic microsomal cytochrome P-450 levels were reduced after coumarin administration. In rats pretreated with saline, corn oil or PB, coumarin produced centrilobular hepatic necrosis, whereas in rats pretreated with 20 MC or ARO, coumarin produced periportal hepatic necrosis. These results demonstrate that mixed-function oxidase enzyme inducers can modulate acute coumarin-induced hepatotoxicity in the rat. As coumarin is known to be bioactivated by cytochrome P-450-dependent enzymes, the change in the lobular distribution of toxicity after pretreatment with 20 MC or ARO is presumably due to the induction of particular cytochrome P-450 isoenzymes in periportal hepatocytes.     

In vivo and in vitro hepatotoxicity and glutathione interactions of N-methyldithiocarbamate and N,N-dimethyldithiocarbamate in the rat.

The ability of N-methyldithiocarbamate (NMDC) to generate methylisothiocyanate and HS(-) together with its greater acid stability suggest that NMDC may exert greater acute toxicity following oral exposure than its dialkyl analog,N,N-dimethyldithiocarbamate (DMDC). To assess this possibility, cell culture, perfused liver, and in vivo studies were performed to delineate differences in the hepatotoxicity and thiol interactions of NMDC and DMDC in the rat. The role of methylisothiocyanate and HS(-) in NMDC-induced hepatotoxicity was evaluated and glutathione interactions characterized through analysis of reduced glutathione (GSH), glutathione disulfide (GSSG), and S-methylthiocarbamoylglutathione (GSMITC) using HPLC and liquid chromatography tandem mass spectrometry (LC/MS/MS). Following oral administration, centrilobular hepatocyte necrosis and enzyme leakage was observed for NMDC but not for DMDC. Dose dependent decreases of intracellular GSH were produced by both dithiocarbamates in primary hepatocytes but DMDC appeared to deplete GSH through the generation of GSSG whereas NMDC produced GSMITC consistent with the generation of a methylisothiocyanate intermediate. In primary hepatocytes, both NMDC and DMDC cytotoxicity was increased by prior depletion of intracellular GSH and diminished by prior supplementation of GSH. The results obtained using perfused livers were similar for NMDC in that elevated levels of GSMITC were detected in the bile; however, DMDC produced only a modest increase of GSSG over controls that was not significantly different to that produced by NMDC. Results obtained from isolated liver mitochondria and primary hepatocytes were not consistent with NMDC producing HS(-)-mediated inhibition of mitochondrial respiration. These data support a greater potential for hepatotoxicity to result following oral exposure to NMDC relative to DMDC and that glutathione may play a role in cytoprotection for NMDC, presumably through detoxification of a methylisothiocyanate metabolite.     

Nitrofurantoin-mediated oxidative stress cytotoxicity in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to study the mechanism(s) of toxicity of the antimicrobial drug nitrofurantoin. This 5-nitrofuran derivative stimulated hepatocyte oxygen uptake in the presence of the mitochondrial respiration inhibitors KCN or antimycin A. This could indicate the formation of O2- and H2O2, following intracellular nitrofurantoin reduction. Addition of nitrofurantoin to suspensions of isolated rat hepatocytes produced a dose- and time-dependent decrease of cell viability. H2O2 probably plays a significant role in the cytotoxic effects of nitrofurantoin as the catalase inhibitors azide or aminotriazole markedly enhanced cytotoxicity. The loss of cell viability was preceded by glutathione (GSH) depletion and a concomitant and nearly stoichiometric formation of oxidised glutathione (GSSG) that did not occur in hepatocytes lacking glutathione peroxidase activity isolated from rats fed a low-selenium diet. This indicates that H2O2 and the seleno-enzyme glutathione peroxidase are responsible for GSH oxidation. Furthermore, addition of nitrofurantoin to isolated rat hepatocytes produced a reversible inactivation of hepatocyte glutathione reductase activity and explains the maintenance of high GSSG levels. The compromised hepatocytes were also highly susceptible to H2O2. The hepatocyte toxicity of nitrofurantoin may, therefore, be attributed to oxidative stress caused by redox-cycling mediated oxygen activation.     

Paracetamol, 3-monoalkyl- and 3,5-dialkyl derivatives. Comparison of their microsomal cytochrome P-450 dependent oxidation and toxicity in freshly isolated hepatocytes

The effects of 3-monoalkyl- and 3,5-dialkyl-substitution on the cytotoxicity of paracetamol (PAR) in rat hepatocytes was studied. PAR is known to be bioactivated by the hepatic microsomal cytochrome P-450 containing a mixed-function oxidase system presumably to N-acetyl-para-benzoquinone imine (NAPQI), a reactive metabolite which upon overdosage of the drug causes depletion of cellular glutathione (GSH) and hepatotoxicity. The four 3-mono- and the four 3,5-di-alkyl-substituted derivatives of PAR investigated in this study (R = CH3, C2H5, C3H7, C4H9) interacted with cytochrome P-450 giving rise to reverse type I spectral changes. Like PAR, all derivatives underwent cytochrome P-450-mediated oxidation to NAPQIs. In contrast to induction by phenobarbital, induction of cytochrome P-450 by 3-methylcholanthrene enhanced the microsomal oxidation of PAR and its derivatives. The NAPQIs formed from PAR and the 3-mono-alkyl derivatives by microsomal oxidation were found to conjugate with GSH and to oxidise GSH to GSSG. The NAPQIs formed from the 3,5-dialkyl-substituted derivatives, however, only oxidized GSH to GSSG. PAR and the 3-monoalkyl derivatives were found to deplete cellular GSH to about the same extent and to be equally toxic in freshly isolated hepatocytes from 3-methylcholanthrene treated rats. In contrast, the 3,5-di-alkyl-substituted derivatives of PAR did not affect the GSH levels and were not toxic in the hepatocytes, even at higher concentrations. It is suggested that the difference between the way of reacting of 3,5-dialkyl-NAPQIs and NAPQIs from PAR and 3-monoalkyl derivatives with thiols of cellular GSH and protein could account for the observed difference between the toxicity of the 3,5-dialkyl- and the 3-monoalkyl-substituted derivatives of PAR.     

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.     

Effects of model traumatic injury on hepatic drug metabolism in the rat. VI. Major detoxification/toxification pathways

A previously validated small mammal trauma model, hindlimb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to investigate the effects of traumatic injury on two of the major hepatic enzymes of detoxification, glutathione S-transferase and epoxide hydrolase. Hepatic cytosolic glutathione S-transferase activity toward a variety of substrates showed a 26-34% decrease at 24 hr after model injury. Hepatic microsomal epoxide hydrolase activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane was diminished by 53% after model trauma. Both enzymatic activities toward styrene oxide were similarly depressed. The toxicological sequelae of these derangements were illustrated by administering a dose of styrene oxide (300 mg/kg, ip) which was below the threshold dose (350 mg/kg, ip) necessary to produce hepatotoxicity in control animals. Model trauma dramatically enhanced the hepatotoxic effects of the subthreshold dose, as well as the covalent binding of labeled styrene oxide to liver proteins. These findings indicate that traumatic injury renders the animal more susceptible to agents which are detoxified by glutathione S-transferase and epoxide hydrolase. Conversely, model trauma provided almost complete protection from the hepatotoxic effects of a standard dose (200 mg/kg, ip) of bromobenzene. This protection appeared to derive from a post-traumatic alteration of cytochrome P-450 subpopulations that decreased the formation of the potentially toxic 3,4-epoxide metabolite, despite an increase in the cytochrome P-448-mediated generation of the nontoxic 2,3-epoxide. For bromobenzene, the change in cytochrome P-450-mediated activation appeared quantitatively more significant in overall toxicity than the post-traumatic depression of detoxification pathways described above, leading to decreased toxicity in vivo. For other compounds, the combination of post-traumatic influences on cytochrome P-450/P-448 activity and epoxide hydrolase/glutathione S-transferase activities could lead to markedly enhanced toxicity.     

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.     

Bromobenzene metabolism in vivo and in vitro. The mechanism of 4-bromocatechol formation

The metabolism of bromobenzene has been examined in isolated hepatocytes and liver microsomes from phenobarbital-induced rats and in phenobarbital-induced rats in vivo. The metabolite profile produced upon incubation of isolated rat hepatocytes with bromobenzene differed with the hepatocyte concentration. At a low hepatocyte concentration (0.5 x 10(6) cells/ml), 4-bromophenol was the major metabolite, while at higher hepatocyte concentrations (2.0 and 5.0 x 10(6) cells/ml) bromobenzene-3,4-dihydrodiol was the major metabolite. 4-Bromophenol was the primary metabolite in incubations with rat liver microsomes. In vivo, 3- and 4-bromophenol were more predominant, with very little dihydrodiol formed. 4-Bromocatechol, a potentially toxic metabolite of bromobenzene, was formed in vivo as well as in isolated hepatocytes and microsomes. However, the mechanism of catechol formation differed, as determined by the retention of a deuterium label at the para position of bromobenzene. In microsomes, 4-bromophenol was the predominant precursor metabolite of 4-bromocatechol. In isolated hepatocytes, although the relative contribution of 4-bromophenol as the bromocatechol precursor differed with hepatocyte concentration, bromobenzene-3,4-dihydrodiol was the predominant precursor at all concentrations. In vivo, as in isolated hepatocytes, 4-bromocatechol was formed primarily via bromobenzene-3,4-dihydrodiol.     

Protective effects of selenium on acetaminophen-induced hepatotoxicity in the rat

Experiments were undertaken to examine the ability of selenium to protect against acetaminophen-induced hepatotoxicity and to examine possible mechanisms for this protective effect. Pretreatment of male, Sprague-Dawley rats with sodium selenite (12.5 mumol Se/kg, ip) 24 hr prior to acetaminophen administration produced a significant protection against the hepatotoxic effects of acetaminophen as assessed by a decrease in the plasma appearance of alanine aminotransferase and aspartate aminotransferase activities following acetaminophen. This was accompanied by an increase in the hepatic glutathione levels in selenium-treated animals and an inhibition in the decrease in hepatic glutathione content observed in animals receiving hepatotoxic doses of acetaminophen. Selenium pretreatment decreased the in vivo covalent binding of acetaminophen metabolites to hepatic protein, but did not alter hepatic microsomal cytochrome P-450 content or NADPH cytochrome c reductase activity, suggesting that selenium does not significantly alter the metabolism of acetaminophen to reactive electrophilic metabolites by the cytochrome P-450-dependent mixed-function oxidase enzyme system. Selenium produced an increase in the activity of gamma-glutamylcysteine synthetase which may account for the increased glutathione availability in selenium-treated animals and increased the activities of glutathione S-transferase and glucose-6-phosphate dehydrogenase. Examination of the urinary metabolite profile in selenium-treated animals revealed that the urinary excretion of acetaminophen and its metabolites was significantly increased over a 72-hr period. The increase occurred in the AAP-glucuronide metabolite while parent AAP and AAP-sulfate were actually decreased in selenium-treated rats. No change in recovery was observed in the AAP-glutathione or AAP-mercapturate urinary metabolites. While the glutathione conjugating system is enhanced by selenium treatment, amelioration of acetaminophen toxicity is most likely the result of enhanced glucuronidation which effectively diverts the amount of acetaminophen to be converted by the cytochrome P-450 system to the toxic metabolite.     

Hepatic metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rat and guinea pig

Marked interspecies variability exists in the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), with the rat having an LD50 about 25-fold greater than the guinea pig. The metabolism of TCDD was examined by incubating hepatocytes isolated from these animals with purified [14C]TCDD (2.2 microM) for 8 hr. Over the 8-hr incubation, cytochrome P-450 content and ethoxyresorufin O-deethylase and benzphetamine N-demethylase activities were well maintained, indicating the functional viability of the hepatocytes. Quantitative differences were observed in the rate of [14C]TCDD metabolism, with hepatocytes from control rats metabolizing TCDD at a rate 2.8-fold greater than hepatocytes from control guinea pigs. The role of the hepatic cytochrome P-450-448-dependent monooxygenase system in the metabolism of TCDD was examined through the use of hepatocytes isolated from animals pretreated with either TCDD (5 micrograms/kg, ip; 72 hr prior to hepatocyte isolation) or phenobarbital (80 mg/kg, ip X 3 days; 24 hr prior to isolation). The rate of [14C]TCDD metabolite formation in hepatocytes from TCDD pretreated guinea pigs (0.26 +/- 0.14 pmol mg cell protein-1 hr-1) was unchanged from the control rate (0.25 +/- 0.07), while the rate in hepatocytes from TCDD pretreated rats (2.26 +/- 0.43 pmol mg-1 hr-1) was 3.2-fold greater than control (0.70 +/- 0.10) and nine times greater than in hepatocytes from TCDD-pretreated guinea pigs. In addition, significant differences were observed in the profiles of the metabolites formed by hepatocytes from TCDD-pretreated rats and guinea pigs. On the other hand, phenobarbital pretreatment produced little change in the rate of [14C]TCDD metabolism in rat hepatocytes (0.98 +/- 0.13 pmol mg-1 hr-1). These results suggest that TCDD may be metabolized by a TCDD inducible form of cytochrome P-448 which is expressed in the rat but not in the guinea pig. Furthermore, the differences in the hepatic metabolism of TCDD in the rat and guinea pig and in the ability of TCDD to induce its own rate of metabolism may play a major role in explaining the varying susceptibility of these species to the acute toxicity of TCDD.     

Role of glutathione reductase during menadione-induced NADPH oxidation in isolated rat hepatocytes

Metabolism of menadione (2-methyl-1,4-naphthoquinone) results in the rapid oxidation of NADPH within isolated rat hepatocytes. The glutathione redox cycle is thought to play a major role in the consumption of NADPH during menadione metabolism, chiefly through glutathione reductase (GSSG-reductase). This enzyme reduces oxidized glutathione (GSSG), formed via the glutathione-peroxidase reaction, with the concomitant oxidation of NADPH. To explore the relationship between GSSG-reductase and the consumption of NADPH during menadione metabolism, isolated rat hepatocyte suspensions were exposed to non-lethal and lethal menadione concentrations (100 and 300 microM respectively) following the inhibition of GSSG-reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Menadione produced a concentration-related depletion of GSH (measured as non-protein sulfhydryl content) which was potentiated markedly by BCNU. Menadione toxicity was potentiated at either concentration by BCNU based on lactate dehydrogenase leakage at 2 hr. In addition, the NADPH content of isolated hepatocytes rapidly declined following exposure to either concentration of menadione. However, at the lower menadione concentration (100 microM), the NADPH content returned to control values or above by 60 min, whereas the NADPH content of cells exposed to 300 microM menadione with or without BCNU remained depressed for the duration of the incubation. These data suggest that, although NADPH is required by GSSG-reductase for the reduction of GSSG to GSH during quinone-induced oxidative stress, this pathway does not appear to be the major route by which NADPH is consumed during the metabolism of menadione in isolated hepatocytes.     

Sequential changes in hepatic and renal glutathione and development of renal karyomegaly in 1-cyano-3,4-epithiobutane toxicity in rats

The effect of 1-cyano-3,4-epithiobutane (CEB) on glutathione (GSH) metabolism was investigated in rat liver, kidney and pancreas. Male Fischer 344 rats were gavaged with a single dose (125 mg/kg body weight or 50 mg/kg body weight) of CEB. Tissue samples were taken for histological examination, determination of GSH and oxidized glutathione (GSSG) concentrations and gamma-glutamyl transpeptidase (GGT) and glutathione S-transferase (GST) activities. Urine samples were analysed for non-protein thiol (NP-RSH) content. The high dose of CEB induced hepatic GSH depletion followed by increased GSH. The low dose of CEB induced elevated hepatic GSH by 12 hr without depletion. Renal GSH was increased with both doses without an observed depletion phase. Renal tubule epithelial cell death was observed only with the high dose of CEB, but both doses caused renal proximal tubule karyomegaly. Pancreatic GSH content was unaffected. No alterations of GSSG were observed. GST activity was unaffected in any tissue. Renal GGT activity was decreased at 12 hr with both doses and at 24 and 48 hr with the high dose. Urinary NP-RSH excretion was increased with both doses. Depletion of hepatic GSH concurrent with increased urinary NP-RSH excretion suggests that conjugation with GSH is a significant pathway in CEB metabolism.     

Re-evaluation of tacrine hepatotoxicity using gel entrapped hepatocytes

Controversial results about the involvement of CYP 1A2 and oxidative stress in tacrine-induced hepatotoxicity have been described by the different research groups. We suggested that different expression levels of CYP 1A2 in cell lines and primary hepatocytes in vitro may be the cause of the controversial results above. Therefore, this paper re-evaluated the toxicity of tacrine by using gel entrapment culture of rat hepatocytes. The toxic effect of tacrine on hepatocytes was assayed by the reduction of methyl thiazolyl tetrazolium (MTT) and intracellular glutathione (GSH), as well as by albumin synthesis. It was found that the detectable hepatotoxic dose of tacrine is much lower in hepatocytes entrapped in gel than in those in monolayer cultures. The fact that fluvoxamine, a potent cytochrome P450 (CYP) 1A2 inhibitor, reduced tacrine toxicity and the expression of the CYP 1A2 gene was maintained in gel entrapped hepatocytes, but not in hepatocyte monolayers, could illustrate a close association between CYP 1A2 expression levels and tacrine toxicity. Glycyrrhetinic acid (GA), a free radical scavenger, protected gel entrapped hepatocytes from tacrine toxicity, but was ineffective in hepatocyte monolayers. Hence, gel entrapped hepatocytes could reflect higher tacrine hepatotoxicity in vivo than hepatocyte monolayers.     

Effect of inducers of cytochrome P-450 on the metabolism of [3-14C]coumarin by rat hepatic microsomes

1. The metabolism of [3-14C]coumarin has been studied in rat hepatic microsomes and with two purified cytochrome P-450 isoenzymes. 2. [3-14C]Coumarin was converted by liver microsomes to several polar products including 3- and/or 5-hydroxycoumarin, omicron-hydroxyphenylacetic acid and a major unidentified novel coumarin metabolite. 3. [3-14C]Coumarin was also converted to reactive metabolite(s) as indicated by covalent binding to proteins, and by the depletion of reduced glutathione added to the microsomal incubations. 4. [3-14C]Coumarin metabolism to polar and covalently bound metabolites by rat liver microsomes was induced by pretreatment with phenobarbitone, 3-methylcholanthrene, beta-naphthoflavone, Aroclor 1254 and isosafrole; but not by dexamethasone or nafenopin. 5. The profile of [3-14C]coumarin metabolism to polar products was similar in control and pretreated liver microsomes and in incubations with purified cytochrome P450 IA1 and P450 IIB1 isoenzymes. 6. The results indicate that coumarin is a substrate for isoenzymes of the cytochrome P450 IA and P450 IIB subfamilies. The bioactivation of coumarin by rat hepatic microsomes is postulated to result in the formation of a coumarin 3,4-epoxide intermediate which may rearrange to 3-hydroxycoumarin, be further metabolized to a coumarin 3,4-dihydrodiol, or form a glutathione conjugate.     

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

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

Toxicologic enhancement by a combination of drugs which deplete hepatic glutathione: Acetaminophen and doxorubicin (Adriamycin)

A number of chemicals are metabolized to reactive and toxic intermediates. Some reactive metabolites are detoxified by conjugation with glutathione (GSH). When endogenous GSH levels are low, or when excessive quantities of reactive metabolites are produced, the metabolite can bind to essential cellular macromolecules causing toxicity. Since both acetaminophen and doxorubicin (Adriamycin) deplete hepatic GSH, it was hypothesized that under certain conditions, doxorubicin might potentiate the hepatic centralobular necrosis characteristically induced by high doses or chronic administration of acetaminophen. Such an interaction could have clinical significance since acetaminophen is used in high doses as an analgesic for cancer patients being treated with doxorubicin. In male Swiss ICR mice, doxorubicin 20 mg/kg ip enhanced the toxicity of acetaminophen in doses of 250 to 500 mg/kg. Compared with acetaminophen alone, combination treatment produced a 15-fold increase in lethality, a dose-dependent, 90-fold increase in SGPT concentration, and an increase in the incidence and severity of hepatic centralobular necrosis. Dororubicin pretreatment caused both a 66% increase in the covalent binding of acetaminophen to hepatic protein at 4 hr, and a further depletion of hepatic GSH, 18% below that induced by acetaminophen alone. The timing of treatments to allow congruence of peak hepatic GSH depletion was necessary for toxicologic enhancement, suggesting a crucial protective role for GSH, although contributions from other toxicologic mechanisms cannot be excluded. Under certain clinical circumstances, this interaction could occur in cancer patients being treated with doxorubicin and acetaminophen.     

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

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

Effect of sodium selenite upon bromobenzene toxicity in rats. I. Hepatotoxicity

The effects of sodium selenite on bromobenzene hepatotoxicity were examined in male rats. Rats pretreated with sodium selenite (12.5 or 30 mumol/kg, ip) 72 hr prior to injection of bromobenzene (7.5 mmol/kg, ip) showed a marked reduction in bromobenzene-induced liver injury as evidenced by decreased plasma alanine and aspartate transaminase values, sorbitol dehydrogenase activity, and reduced histologic damage. Administration of bromobenzene did not affect the selenium content of blood or liver. At 72 hr after treatment with selenite, hepatic reduced (GSH) and oxidized (GSSG) glutathione values or GSH synthetic and degradation enzyme activities were not altered. However, from 3 to 12 hr following bromobenzene administration, hepatic GSH and cysteine amounts declined less rapidly in selenite-treated rats compared to control. Thus, acute selenite treatment ameliorated bromobenzene hepatotoxicity in a manner suggesting facilitation of hepatic GSH production by selenite for use in bromobenzene detoxication.     

Protection of rats against 3-butene-1,2-diol-induced hepatotoxicity and hypoglycemia by N-acetyl-l-cysteine

3-Butene-1,2-diol (BDD), an allylic alcohol and major metabolite of 1,3-butadiene, has previously been shown to cause hepatotoxicity and hypoglycemia in male Sprague-Dawley rats, but the mechanisms of toxicity were unclear. In this study, rats were administered BDD (250 mg/kg) or saline, ip, and serum insulin levels, hepatic lactate levels, and hepatic cellular and mitochondrial GSH, GSSG, ATP, and ADP levels were measured 1 or 4 h after treatment. The results show that serum insulin levels were not causing the hypoglycemia and that the hypoglycemia was not caused by an enhancement of the metabolism of pyruvate to lactate because hepatic lactate levels were either similar (1 h) or lower (4 h) than controls. However, both hepatic cellular and mitochondrial GSH and GSSG levels were severely depleted 1 and 4 h after treatment and the mitochondrial ATP/ADP ratio was also lowered 4 h after treatment relative to controls. Because these results suggested a role for hepatic cellular and mitochondrial GSH in BDD toxicity, additional rats were administered N-acetyl-l-cysteine (NAC; 200 mg/kg) 15 min after BDD administration. NAC treatment partially prevented depletion of hepatic cellular and mitochondrial GSH and preserved the mitochondrial ATP/ADP ratio. NAC also prevented the severe depletion of serum glucose concentration and the elevation of serum alanine aminotransferase activity after BDD treatment without affecting the plasma concentration of BDD. Thus, depletion of hepatic cellular and mitochondrial GSH followed by the decrease in the mitochondrial ATP/ADP ratio was likely contributing to the mechanisms of hepatotoxicity and hypoglycemia in the rat.     

Mechanisms of N-acetyl-p-benzoquinone imine cytotoxicity

N-Acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, rapidly reacts at physiological pH with glutathione (GSH) forming an acetaminophen-glutathione conjugate and stoichiometric amounts of acetaminophen and glutathione disulfide (GSSG). The same reaction products are formed in isolated hepatocytes incubated with NAPQI. In hepatocytes which have been treated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) in order to inhibit glutathione reductase, the initial rise in GSSG concentration in the presence of NAPQI is maintained, whereas GSSG is rapidly reduced back to GSH in untreated hepatocytes. Oxidation by NAPQI of GSH to GSSG and the reduction of GSSG back to GSH by the NADPH-dependent glutathione reductase appear to be responsible for the rapid oxidation of NADPH that occurs in hepatocytes incubated with NAPQI in that the effect is blocked by pretreatment of cells with BCNU. When added to hepatocytes, NAPQI not only reacts with GSH but also causes a loss in protein thiol groups. The loss in protein thiols occurs more rapidly in cells pretreated with BCNU or diethylmaleate. Whereas both of these treatments enhance cytotoxicity caused by NAPQI, BCNU pretreatment has no effect on the covalent binding of [14C-ring]NAPQI to cellular proteins. Furthermore, dithiothreitol added to isolated hepatocytes after maximal covalent binding of [14C-ring]NAPQI but preceding cell death protects cells from cytotoxicity and regenerates protein thiols. Thus, the toxicity of NAPQI to isolated hepatocytes may result primarily from its oxidative effects on cellular proteins.