Metabolism-dependent hepatotoxicity of amodiaquine in glutathione-depleted mice

We investigated the hepatotoxicity induced by AQ using a glutathione (GSH)-depleted mice model. Although sole administration of either AQ or l-buthionine-S,R-sulfoxinine (BSO), a well-known GSH synthesis inhibitor, produced no significant hepatotoxicity, combined administration of AQ with BSO induced hepatotoxicity characterized by centrilobular necrosis of the hepatocytes and an elevation of plasma alanine aminotransferase activity. Pretreatment of aminobenzotriazole, a nonspecific inhibitor for P450s, completely suppressed the above hepatotoxicity caused by AQ co-treatment with BSO. Administration of radiolabeled AQ in combination with BSO exhibited significantly higher covalent binding to mice liver proteins than that observed after sole dosing of radiolabeled AQ. The results obtained in this GSH-depleted animal model suggest that the reactive metabolite of AQ formed by hepatic P450 binds to liver proteins, and then finally leads to hepatotoxicity. These observations may help to understand the risk factors and the mechanism for idiosyncratic hepatotoxicity of AQ in humans.     

Sex difference in susceptibility to acetaminophen hepatotoxicity is reversed by buthionine sulfoximine

Gender is a factor that influences susceptibility of individuals to drug-induced liver injury in experimental animals and humans. In this study, we investigated the mechanisms underlying resistance of female mice to acetaminophen (APAP)-induced hepatotoxicity. Overnight-fasted male and female CD-1 mice were administered APAP intraperitoneally. A minor increase in serum alanine aminotransferase levels was observed in female mice after APAP administration at a dose that causes severe hepatotoxicity in males. Hepatic glutathione (GSH) depleted rapidly in the both genders prior to development of hepatotoxicity, whereas its recovery was more rapid in female than in male mice. This was consistent with higher induction of hepatic glutamate-cysteine ligase (GCL) in females. Pretreatment of mice with L-buthionine sulfoximine (BSO), an inhibitor of GCL, exaggerated APAP hepatotoxicity only in female mice, resulting in much higher hepatotoxicity in female than in male mice. In addition, hepatic GSH was markedly depleted in BSO-pretreated female mice compared with male mice, which supports severe hepatotoxicity in BSO-pretreated females. APAP treatment highly induced multidrug resistance-associated protein 4 (Mrp4) only in female mice. The resulting high Mrp4 expression could thus contribute to decreased hepatic GSH levels via sinusoidal efflux when GCL is inhibited. In conclusion, resistance to APAP hepatotoxicity in female mice and its reversal by pretreatment with BSO could be attributed to sex differences in disposition of hepatic GSH, which may generally determine susceptibility to drug-induced liver injury.     

Metabolism-dependent hepatotoxicity of methimazole in mice depleted of glutathione.

Methimazole (MMI) (>0.1 mmol kg(-1), p.o.) given in combination with DL-buthionine sulphoximine (BSO) (3 mmol kg(-1), i.p., 1 h before MMI administration), an inhibitor of glutathione (GSH) synthesis, caused liver injury in mice. The injury was characterized by centrilobular necrosis of hepatocytes and an increase in serum alanine transaminase (ALT) activity. Methionazole (2 mmol kg(-1)) alone resulted in only a marginal increase in serum ALT activity, but produced no histopathological changes in the liver. Pretreatment with hepatic cytochrome P-450 monooxygenase inhibitors--cobalt chloride, isosafrole, methoxsalen, metyrapone and piperonyl butoxide-prevented or tended to suppress the hepatotoxicity induced by MMI in combination with BSO. Treatment with N,N-dimethylaniline and ethyl methyl sulphide, competitive substrates of flavin-containing monooxygenases (FMO), also resulted in remarkable suppression of the hepatotoxicity caused by MMI in combination with BSO. These results suggest that MMI is activated by reactions mediated by both cytochrome P-450 monooxygenases and FMO, and that the inadequate rates of detoxification of the resulting metabolite are responsible for the hepatotoxicity in GSH-depleted mice.     

Effect of ethanol on hepatotoxicity and hepatic DNA-binding of aflatoxin B1 in rats

The hepatocarcinogen aflatoxin B1 is converted to reactive metabolites that bind covalently to cellular macromolecules. These metabolites may also react with glutathione, resulting in the formation of glutathione conjugates and detoxication of the reactive metabolite. When rats were pretreated with ethanol by gastric intubation at a dose of 100 mmol/kg, 6 hr (the time of maximal GSH depletion) before the administration of aflatoxin B1, the covalent binding of 8,9-epoxide-aflatoxin B1 to DNA in vivo was increased by 47% and the hepatotoxicity was also potentiated. However, the covalent binding was not increased by pretreatment with ethanol 18 hr (time with approximately normal GSH levels) before administration of the toxin, and no potentiation of hepatotoxicity was observed. Pretreatment with a non-toxic dose of ethanol had no effects on the activity of glutathione S-transferase and glutathione peroxidase. These results suggest that the depletion of GSH and the increased formation of DNA-adduct from the liver constitute an important mechanism for the potentiation of aflatoxin B1-induced hepatotoxicity by ethanol.     

Nephrotoxicity and covalent binding of 1,1-dichloroethylene in buthionine sulphoximine-treated mice

Autoradiography of mice injected i.p. with¹⁴C-labelled 1,1-dichloroethylene (vinylidene chloride, VDC) in C57Bl/6 mice revealed a selective covalent binding of radioactivity in the proximal tubules, in the midzonal parts of the liver lobules and in the mucosa of the upper and lower respiratory tract. Since VDC is a renal carcinogen in male mice the effects of compounds modulating biotransformation and glutathione (GSH) levels on the renal covalent binding were examined following a single i.p. dose of¹⁴C-VDC. Most pretreatments did not influence the level of binding but treatment with buthionine sulphoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase and glutathione (GSH)-depleting agent, increased the renal covalent binding of VDC three-fold. Histopathological examination of kidneys in BSO-pretreated male mice given single i.p. injections of subtoxic doses of VDC (25 and 50 mg/kg) showed necrosis in the proximal tubules (S₁ and S₂ segments) 24 h following administration. In mice given VDC only, no significant lesions in the kidneys were observed. The severe renal toxicity of VDC in BSO-pretreated mice is suggested to be related to metabolic activation of VDC in the proximal tubules, resulting in further GSH depletion and covalent binding.     

Dichlorovinyl cysteine (DCVC) in the mouse kidney: tissue-binding and toxicity after glutathione depletion and probenecid treatment

The kidney binding of dichloro[¹⁴C]vinyl cysteine (¹⁴C-DCVC, 8 mg/kg body wt) and the kidney histopathology of DCVC (5 mg/kg body wt) were examined and compared in female C57BL mice subjected to various treatments. To evaluate the roles of organic anion transport and glutathione (GSH) status, mice were pretreated with probenecid (inhibitor of organic anion transport), l-buthionine-S,R-sulfoximine (BSO; inhibitor of GSH synthesis) or with diethyl maleate (DEM; GSH-depleting agent). In addition, the sites of ¹⁴C-DCVC binding in BSO-treated and control mice were monitored by microautoradiography. Probenecid was found to inhibit both kidney binding and toxicity of DCVC. In BSO-treated mice, DCVC binding remained roughly unchanged, whereas nephrotoxicity was severely increased and topographically extended to the subcapsular region. Microautoradiography showed that the site of DCVC binding in the straight portion of the proximal tubule was not changed by BSO. In DEM-treated mice, a clearly decreased DCVC binding was observed, while the effect on nephrotoxicity was minute. The effects of probenecid on DCVC binding and toxicity support a role for carriermediated transport of DCVC equivalents into the target cells. The BSO result suggests a protective function of GSH towards the nephrotoxicity of DCVC. Moreover, they support our previous contention that a primary lesion occurs at the site of DCVC binding, followed by a secondary, dose-dependent lesion localized outside the DCVC-binding region. In the case of DEM it is proposed that a DEM-GSH conjugate might compete for the uptake and/or activation of DCVC in the target cells.Part of this study was presented at the 10th European Drug Metabolism Workshop, Guildford, England, 6–11 July, 1986     

The role of glutathione in L-2-chloropropionic acid induced cerebellar granule cell necrosis in the rat

 The role of glutathione (GSH) in the neurotoxicity produced following a single oral dose of 750 mg/kg L-2-chloropropionic acid (L-CPA) has been investigated in rats. L-CPA-induced neurotoxicity was characterised by up to 80–90% loss in cerebellar granule cells and cerebellar oedema leading to locomotor dysfunction. Neurochemically, L-CPA-induced neurotoxicity produced a reduction in the concentration of aspartate and glutamate in the cerebellum and a reduction in the density of NMDA receptors in the cerebellar cortex, whilst there was an increase in cerebellar glycine, glutamine and GABA concentrations. Treatment of rats with buthionine sulfoximine (BSO) at 1 g/kg, i.p., an inhibitor of GSH synthesis, potentiated the toxicity of L-CPA, such that many of the neurochemical markers were significantly different from controls at earlier time points, compared to animals which had received L-CPA alone, and toxicity was also seen in the kidney of BSO plus L-CPA treated rats. In contrast, supplementing GSH concentrations by administration of the isopropyl ester of glutathione (ip-GSH) at 1 g/kg, s.c., was able to protect rats against L-CPA neurotoxicity and prevent many of the neurochemical changes. In order to assess whether the depletion of GSH in the rat cerebellum following L-CPA treatment was related to the delivery of cysteine or cystine, the accumulation of [¹⁴C] cystine into cerebellar slices was characterised and found to be energy dependent, Na⁺ independent and obey saturation kinetics with an apparent K_m of 77 μM and an apparent V_max of 450 nmol/g wet weight per h. The accumulation of cystine into cerebellar slices was non-competitively inhibited by the cysteine conjugate of L-CPA with an apparent K_i of approximately 60 μM, whilst glutamate only inhibited cystine accumulation at doses which were cytotoxic to cerebellar slices. Hence the depletion of GSH in the rat cerebellum, following L-CPA administration, may be due to a reduction in the delivery to the brain of cysteine or cystine, one of the components required for GSH synthesis, by the cysteine conjugate of L-CPA. Our studies show the pivotal role GSH plays in cerebellar granule cell necrosis induced by L-CPA in the rat, indicating that a marked and sustained reduction in cerebellar GSH content by L-CPA may leave granule cells vulnerable to cytotoxic free radical damage leading to cell death, possibly mediated through excitatory amino acids. Received: 24 October 1995/Accepted: 24 January 1996     

Cellular glutathione as a determinant of sensitivity to mercuric chloride toxicity. Prevention of toxicity by giving glutathione monoester

Depletion of glutathione (GSH) by treatment of mice with buthionine sulfoximine (BSO), an effective inhibitor of gamma-glutamylcysteine synthetase, markedly enhanced (about 10-fold) the lethal and renal toxicity of mercuric chloride. The lethal toxicity of HgCl2 was prevented by administration of GSH monoester; this was observed in mice pretreated with BSO and given a low dose of HgCl2, and also in untreated mice that were given a much higher dose of HgCl2. In contrast, administration of GSH did not protect. Since administered GSH is not transported effectively into cells, whereas GSH monoester is transported and split intracellularly to GSH, the findings indicate that protection against HgCl2 requires intracellular GSH. The experimental approaches used here suggest that cellular GSH is a major determinant of sensitivity to HgCl2 toxicity, and also that administration of GSH esters may be useful for prevention of HgCl2 toxicity.     

Hepatotoxicity of precocene I in rats. Role of metabolic activation in vivo

The mechanism of the hepatotoxicity of precocene I has been investigated in male, Sprague-Dawley rats. Administration of a single dose of precocene I caused a large depletion of liver glutathione (GSH) levels that was both time and dose dependent. Concomitant with the decrease of liver GSH, there was an increase in serum glutamic pyruvic transaminase (GPT) levels which was also time and dose dependent. Administration of a single dose of [4-3H]precocene I resulted in extensive covalent binding of the radiolabel to liver proteins and DNA in the liver; the extent of binding increased with increasing dose. Treatment of the rats with the mixed-function oxidase inhibitor piperonyl butoxide, before administration of precocene I, significantly decreased the proportion of the radiolabel bound covalently to proteins and DNA, although the total radioactivity (bound and unbound) in the liver remained the same. Piperonyl butoxide pretreatment limited both the liver GSH depletion and the hepatic necrosis normally caused by precocene I. These results are consistent with the view that the hepatotoxicity of precocene I is due to reactive metabolites formed through cytochrome P-450 mediated metabolism of precocene I.     

The effects of buthionine sulphoximine (BSO) on glutathione depletion and xenobiotic biotransformation

Buthionine sulphoximine (BSO) is an inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS) and, consequently lowers tissue glutathione (GSH) concentrations. In fed male C3H mice, liver and kidney GSH levels were depleted by BSO in a dose dependent manner with maximum effect (35% of initial levels) occurring with doses between 0.8 and 1.6 g/kg, i.p. At these doses maximum effects on gamma-GCS and GSH were observed 2-4 hr after BSO administration; initial gamma-GCS activity and GSH content were restored approximately 16 hr post BSO. BSO, either in vivo or in vitro, had no effect on hepatic microsomal cytochrome P-450 levels, a range of cytochrome P-450 dependent enzyme activities or p-nitrophenol glucuronyl transferase activity. Similarly, BSO had no effect on phenol sulphotransferase and two GSH-transferase activities in the 105,000 g supernatant fraction. BSO had no effect on the duration of hexobarbitone induced narcosis in mice. Consistent with specific inhibition of GSH synthesis, BSO pretreatment of mice decreased the proportion of a 50 mg/kg dose of paracetamol excreted in the urine as GSH-derived conjugates but did not affect paracetamol clearance through the glucuronidation or sulphation pathways. Since BSO does not affect cytochrome P-450 or conjugating enzyme activity, its use as a specific depletor of tissue GSH in the investigation of mechanisms of xenobiotic-induced toxicities is preferable to the standard GSH-depleting agents as these have other enzymic effects.     

Enalapril hepatotoxicity in the rat. Effects of modulators of cytochrome P450 and glutathione.

The effects of modulators of cytochrome P450 and reduced glutathione (GSH) on the hepatotoxicity of enalapril maleate (EN) were investigated in Fischer 344 rats. Twenty-four hours following the administration of EN (1.5 to 1.8 g/kg), increased serum transaminases (ALT and AST) and hepatic necrosis were observed. Pretreatment of the animals with pregnenolone-16 alpha-carbonitrile, a selective inducer of the cytochrome P450IIIA gene subfamily, enhanced EN-induced hepatotoxicity, whereas pretreatment with the cytochrome P450 inhibitor, cobalt protoporphyrin, reduced the liver injury. Depletion of hepatic non-protein sulfhydryls (NPSHs), an indicator of GSH, by combined treatment with buthionine sulfoximine (BSO) and diethyl maleate (DEM) produced marked elevations in serum transaminases by 6 hr after EN treatment. Administered on its own, EN decreased hepatic NPSH content and when combined with the BSO/DEM pretreatment, the liver was nearly completely devoid of NPSHs. Protection from EN-induced hepatotoxicity was observed in animals administered L-2-oxothiazolidine-4-carboxylic acid, a cysteine precursor. Together, these observations suggest the involvement of cytochrome P450 in EN bioactivation and GSH in detoxification. The results corroborate previous in vitro observations pertaining to the mechanism of EN-induced cytotoxicity towards primary cultures of rat hepatocytes. Although the doses of EN used in this study were far in excess of therapeutic doses, under certain circumstances, this metabolism-mediated toxicologic mechanism could form the basis for idiosyncratic liver injury in patients receiving EN therapy.     

Sex- and Age-Dependent Acetaminophen Hepato- and Nephrotoxicity in Sprague-Dawley Rats: Role of Tissue Accumulation, Nonprotein Sulfhydryl Depletion, and Covalent Binding

Acetaminophen (APAP) produces sex-dependent nephrotoxicity andhepatotoxicity in young adult Sprague-Dawley (SD) rats and age-dependenttoxicity in male rats. There is no information re garding thesusceptibility of aging female SD rats to APAP toxicity. Therefore,the present studies were designed to determine if sex-dependentdifferences in APAP toxicity persist in aging rats and to elucidatefactors contributing to sex- and age-dependent APAP hepatotoxicityand nephrotoxicity. Young adult (3 months old) and aging (18months old) male and female rats were killed from 2 through24 hr after receiving APAP (0–1250 mg/kg, ip) containing[ring-¹⁴C]APAP. Trunk blood was collected for determinationof blood urea nitrogen (BUN) concentration, serum alanine aminotransferase(ALT) activity, and plasma APAP concentration; urine was collectedfor determination of glucose and protein excretion; and liverand kidneys were removed for determination of tissue glutathione(GSH) concentration, APAP concentration, and covalent binding.APAP at 1250 mg/kg induced nephrotoxicity (as indicated by elevationsin BUN concentration) in 3-month-old females but not males,whereas APAP induced hepatotoxicity (as indicated by elevationsin serum ALT activity) in 3-month-old males but not females.Sex differences in APAP toxicity were no longer apparent in18-month-old rats. APAP at 750 mg/kg ip produced liver and kidneydamage in 18-month-old but not 3-month-old male and female rats.No consistent sex- or age-dependent differences in serum, hepatic,and renal APAP concentrations were observed that would accountfor differences in APAI toxicity. No sex- or age-dependent differencesin tissue GSH depletion or covalent binding of radiolabel fromAPAP in livers or kidneys were observed following APAP administration.Utilizing an affinity-purified polyclonal antibody raised againstAPAP, arylated proteins with electrophoretic mobility similarto those observed in mice were prominent in rat livers followingAPAP administration to 3- and 18-month-old rats of both sexes.In contrast, no arylated proteins were detected in any rat kidneysfollowing APAP administration. Absence of immunochemically detectableproteins in rat kidney following APAP administration is in directcontrast to observations in mice and supports the hypothesisthat mechanisms of APAP hepatotoxicity and nephrotoxicity inrats and mice are distinctly different. In conclusion, sex differencesin APAP toxicity are observed only in young adult (3-month-old)rats and sex differences are organ-specific with males moresusceptible to hepatotoxicity and females more susceptible tonephrotoxicity. Aging rats are more susceptible to APAP-induceddamage to both the liver and the kidney than are 3-month-oldrats but sex differences are no longer apparent in 18-month-oldrats. The mechanisms contributing to sex- and age-dependentdifferences in APAP toxicity cannot be attributed to differencesin tissue APAP concentrations, GSH depletion, or covalent binding.     

Hepatotoxicity of ketoconazole in Sprague-Dawley rats: glutathione depletion,flavin-containing monooxygenases-mediated bioactivation and hepatic covalent binding

1. This study has examined ketoconazole (KT)-induced hepatotoxicity in vivo and in vitro, using male Sprague-Dawley rats with [3 H]KT (1.5 µCi?mg ? 1) at 40 and 90?mg KT kg ? 1 doses. Blood and liver samples were collected from 0 to 24?h for alanine aminotransaminase (ALT), glutathione (GSH) and covalent binding analyses. 2. Covalent binding occurred as early as 0.5?h, peaked at 2?h (0.026 ± 0.01 nmol KT?mg ? 1 protein) and 8?h (0.088 ± 0.04 nmol KT?mg ? 1 protein) for 40 and 90?mg KT kg ? 1 doses, respectively. ALT levels increased at 0.5?h for the 40 and 90?mg KT kg ? 1 doses (44.3 and 56.4 U ml ? 1, respectively) relative to control, 22.7 U ml ? 1. At 24?h, the 90?mg KT kg ? 1 dose reduced hepatic GSH levels from 9.92 ± 1.1 to 4.76 ± 0.3 nmol GSH?mg ? 1 protein. 3. The role of the flavin-containing monooxygenases (FMO) utilized Sprague-Dawley microsomes with 1, 10 and 100 µM [3 H]KT. Maximum covalent binding occurring at 100 µM KT. Heat inactivation of microsomal FMO significantly decreased covalent binding by 75%, whereas 1 mM GSH significantly reduced covalent binding by 65%. 4. Thus, KT-induced hepatotoxicity is dose- and time-dependent and appears to be FMO mediated, in part, to metabolites that may react with protein and, possibly, GSH.     

Carnitine deficiency: a possible risk factor in paracetamol hepatotoxicity

We have addressed in the current study the postulate whether or not carnitine deficiency would represent a risk factor in hepatotoxicity. Carnitine-deficient male Swiss albino rats were obtained following administration of d-carnitine (500 mg/kg, IP) for 10 consecutive days. Serum and liver carnitine levels, both total and free, were assessed to confirm carnitine depletion. Hepatotoxicity was induced by challenging animals with a single dose of paracetamol (1 g/kg, IP). Serum tumor necrosis factor (TNF-α) concentration, and serum activities of aspartate amino transferase (AST), alanine amino transferase (ALT) and alkaline phosphatase (ALP) were undertaken as biomarkers for toxicity. Liver contents of reduced glutathione (GSH), malondialdehyde (MDA), total nitric oxide (NO) and myeloperoxidase (MPO) activities were also investigated. Histopathological examination of liver sections was achieved to confirm the biochemical alterations. d-carnitine altered all biochemical markers and also induced mild tissue inflammation with dilatation and congestion of central and portal veins. Paracetamol produced an obvious hepatotoxicity model that was well characterized biochemically and morphologically. Combined administration of d-carnitine and paracetamol synergistically provoked marked toxicity that was more profound than either agent given alone. The present work was further extended to elucidate any hepatoprotective effect of carnitine supplementation in such toxicity paradigm. It was apparent that l-carnitine notably ameliorated all biochemical markers and also mitigated the gross histologic alterations induced by paracetamol. Data obtained so far would suggest that carnitine deficiency could possibly be a sequela as well as a causative clue for paracetamol hepatotoxicity.     

Oltipraz-induced amelioration of acetaminophen hepatotoxicity in hamsters. I. Lack of dependence on glutathione

These studies were designed to test the hypothesis that oltipraz (OTP) provided protection against AAP intoxication in a sensitive species, the hamster; and further, to show that the sparing effect was related to the marked increase in hepatic reduced glutathione (GSH) levels. Dose-response and time-course experiments demonstrated that maximal increases in liver GSH occurred at 48 hr after an oltipraz dose of approximately 2.0 mmol/kg (po). Accompanying greater GSH levels were increased glutathione disulfide (GSSG) levels. Decreased indices of the oxidation state of glutathione and of hepatic pyridine nucleotides indicated a greater share of glutathione existed as GSH and that increased reducing equivalents were present, respectively. Additionally, glutathione disulfide reductase activity was greater in OTP-treated groups. Glutathione S-transferase activities were only marginally increased. OTP treatment did not elicit observable hepatotoxicity, whereas AAP (2.6 mmol/kg, ip) resulted in a reproducible model of liver damage. OTP-treated groups were protected from AAP-induced toxicity, as shown by decreased plasma appearance of liver enzymes and unremarkable histopathology. However, the degree of liver GSH depletion by AAP was fourfold greater in non-OTP treated groups compared to those which had received the dithiolthione. To test the importance of increased hepatic GSH, the biosynthesis of glutathione was interrupted. Buthionine sulfoximine (BSO) treatment decreased hepatic GSH, the biosynthesis of glutathione was interrupted. Buthionine sulfoximine (BSO) treatment decreased hepatic GSH content to 50% of control in hamsters which either had or had not received OTP. The groups receiving BSO and AAP incurred 83% lethality, while no lethality, unremarkable liver histopathology, and plasma enzyme levels consistent with control were found in the group receiving OTP, BSO, and AAP. Treatment with BSO only had no influence on hepatotoxicity parameters. These results indicate that the increased GSH levels in the OTP-treated hamster are coincidental to the sparing effect of OTP and are not central to the protection scheme in AAP-induced hepatotoxicity.     

Acetaminophen-induced cytotoxicity on human normal liver L-02 cells and the protection of antioxidants

In vitro cell models, which can partially mimic in vivo responses, offer potentially sensitive tools for toxicological assessment. The objective of this study was to explore the possible mechanisms of acetaminophen (AP)-induced toxicity in human normal liver L-02 cells. The expression of the CYP2E1 enzyme, which is reported to transform AP to its toxic metabolites, was higher in L-02 than in Hep3B cells. Further cell viability and reduced glutathione (GSH) depletion after AP treatment were examined. After exposure to AP for 24?h, cell viability decreased in a concentration-dependent manner. Concentration-dependent GSH depletion was also observed after AP treatment for 48?h, indicating oxidative stress had occurred in L-02 cells. The effects of D, L-buthionine-(S, R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, and N-acetylcysteine (NAC), a precursor of GSH synthesis, on the cytotoxicity induced by AP were also investigated. BSO aggravated the cytotoxicity induced by AP while NAC ameliorated such cell death. Further results showed that 10?mM AP caused cell apoptosis after 48?h treatment based on the DNA fragmentation assay and western blot of caspase-3 activation, respectively. In addition, the protective effects of various well-known antioxidants against AP-induced hepatotoxicity were observed. Taken together, these results indicate that oxidative stress and cellular apoptosis are involved in AP-induced toxicity in human normal liver L-02 cells, and this cell line is a suitable in vitro cell model for AP hepatotoxicity study.     

Mechanism of action of paracetamol protective agents in mice in vivo

The mechanism of action of cysteine, methionine, N-acetylcysteine (NAC) and cysteamine in protecting against paracetamol (APAP) induced hepatotoxicity in male C3H mice in vivo has been investigated by, characterising the effect of the individual protective agents on the metabolism of an hepatotoxic dose of APAP, and determining the efficacy of the protective agents in animals treated with buthionine sulphoximine (BSO), a specific inhibitor of glutathione (GSH) synthesis. Co-administration of cysteine, methionine or NAC increased, while co-administration of cysteamine decreased, the proportion of GSH-derived conjugates of APAP excreted in the urine of mice administered APAP, 300 mg/kg. Pretreatment of animals with BSO abolished the protective effect of cysteine, methionine and NAC, whereas cysteamine still afforded protection against APAP after BSO treatment. In conjunction with other data, these results suggest the most likely mechanism for the protective effect of cysteine, methionine and NAC is by facilitating GSH synthesis, while the most likely mechanism for the protective effect of cysteamine is inhibition of cytochrome P-450 mediated formation of the reactive metabolite of APAP.     

Hepatotoxicity of ketoconazole in Sprague-Dawley rats: glutathione depletion,flavin-containing monooxygenases-mediated bioactivation and hepatic covalent binding

1. This study has examined ketoconazole (KT)-induced hepatotoxicity in vivo and in vitro, using male Sprague-Dawley rats with [(3)H]KT (1.5 micro Ci mg(-1)) at 40 and 90 mg KT kg(-1) doses. Blood and liver samples were collected from 0 to 24 h for alanine aminotransaminase (ALT), glutathione (GSH) and covalent binding analyses. 2. Covalent binding occurred as early as 0.5 h, peaked at 2 h (0.026 +/- 0.01 nmol KT mg(-1) protein) and 8 h (0.088 +/- 0.04 nmol KT mg(-1) protein) for 40 and 90 mg KT kg(-1) doses, respectively. ALT levels increased at 0.5 h for the 40 and 90 mg KT kg(-1) doses (44.3 and 56.4 U ml(-1), respectively) relative to control, 22.7 U ml(-1). At 24 h, the 90 mg KT kg(-1) dose reduced hepatic GSH levels from 9.92 +/- 1.1 to 4.76 +/- 0.3 nmol GSH mg(-1) protein. 3. The role of the flavin-containing monooxygenases (FMO) utilized Sprague-Dawley microsomes with 1, 10 and 100 micro M [(3)H]KT. Maximum covalent binding occurring at 100 micro M KT. Heat inactivation of microsomal FMO significantly decreased covalent binding by 75%, whereas 1 mM GSH significantly reduced covalent binding by 65%. 4. Thus, KT-induced hepatotoxicity is dose- and time-dependent and appears to be FMO mediated, in part, to metabolites that may react with protein and, possibly, GSH.