Evidence for the involvement of N-methylthiourea, a ring cleavage metabolite, in the hepatotoxicity of methimazole in glutathione-depleted mice: structure-toxicity and metabolic studies

In mice depleted of GSH by treatment with buthionine sulfoximine (BSO), methimazole (2-mercapto-1-methylimidazole, MMI) causes liver injury characterized by centrilobular necrosis of hepatocytes and an increase in serum alanine transaminase (SALT) activity. MMI requires metabolic activation by both P450 monooxygenase and flavin-containing monooxygenase (FMO) before it produces the hepatotoxicity. MMI and its analogues were examined for the ability to increase SALT activity in GSH-depleted mice. Saturation of the C-4,5 double bond in MMI resulted in a complete loss of hepatotoxicity. Similarly, ring fusion of a benzene nucleus to the C-4,5 double bond, forming 2-mercapto-1-methylbenzimidazole, abolished the toxic potency. As for MMI, 2-mercapto-1,4,5-trimethylimidazole, and 2-mercapto-1-methyl-4, 5-di-n-propylimidazole, the toxic potency decreased with the increasing bulk of the 4- and 5-alkyl substituents. Furthermore, methylation of the thiol group of MMI totally reduced its toxicity. These structural requirements and the known toxicity of thiono-sulfur compounds led us to the hypothesis that MMI would undergo epoxidation of the C-4,5 double bond by P450 enzymes and, after being hydrolyzed, the resulting epoxide would be then decomposed to form N-methylthiourea, a proximate toxicant. Before N-methylthiourea would produce toxicity, it would be further biotransformed to its S-oxidized metabolites mainly by FMO. Evidence for this hypothesis was provided by the facts that N-methylthiourea and glyoxal as the accompanying fragment were identified as urinary metabolites in mice treated with MMI and that N-methylthiourea caused a marked increase in SALT activity when administered to mice in combination with BSO.     

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.     

Nephrotoxicity of thiabendazole in mice depleted of glutathione by treatment with DL-buthionine sulphoximine

Thiabendazole [2-(4'-thiazolyl)benzimidazole; TBZ] is widely used as an anthelmintic and a fungicide. TBZ (50-400 mg/kg body weight administered by oral intubation) produced nephrotoxicity in male ICR mice pretreated with an inhibitor of glutathione synthesis, DL-buthionine sulphoximine (BSO; 4 mmol/kg body weight, ip). The toxicity was characterized by increases in kidney: body weight ratio and serum urea nitrogen concentration and by tubular necrosis. The nephrotoxic effects were both dose and time dependent. TBZ in combination with BSO also produced decreases in p-aminohippurate accumulation and acetylation by renal cortical slices. TBZ (up to 1200 mg/kg/body weight) alone resulted in no nephrotoxicity. Administration (ip) of glutathione monomethyl ester, which is readily hydrolysed to glutathione after being transported into cells, completely protected against the toxicity caused by TBZ in combination with BSO; this result suggests that glutathione depletion is a major factor underlying the toxicological interaction between TBZ and BSO. Treatment with three inhibitors of renal microsomal cytochrome P-450-dependent monooxygenases, piperonyl butoxide, methoxsalen and carbon disulphide, all equally prevented the nephrotoxicity of TBZ given in combination with BSO. These results suggest that metabolism of TBZ is a necessary step in TBZ-induced nephrotoxicity in glutathione-depleted mice.     

A convenient method to discriminate between cytochrome P450 enzymes and flavin-containing monooxygenases in human liver microsomes

 Liver microsomes are a frequently used probe to investigate the phase I metabolism of xenobiotics in vitro. Structures containing nucleophilic heteroatoms are possible substrates for cytochrome P450 enzymes (P450) and flavin-containing monooxygenases (FMO). Both enzymes are located in the endoplasmatic reticulum of hepatocytes and both need oxygen and NADPH as cofactors. The common method to distinguish between the two enzyme systems is to use the thermal inactivation of FMO and to inhibit P450 completely with carbon monoxide, N-octylamine or N-benzylimidazole. In the literature no indication could be found that the heat inactivation of FMO does not affect any of the human P450 enzymes or that the overall P450 inhibitors inhibit the different human P450 enzymes sufficiently and do not affect the FMO. The effect of N-benzylimidazole and heat inactivation was tested on specific activities of seven P450 enzymes in human liver microsomes, 1A2, 2A6, 2C9, 2C19, 2D6, 3A4/5, and 2E1, using methoxyresorufin O-demethylation, coumarin 7-hydroxylation, (S)-warfarin 4-hydroxylation, (S)-(+)-mephenytoin 4-hydroxylation, dextrometorphan O-demethylation, oxidation of denitronifedipine, and chlorzoxazone 6-hydroxylation respectively. The sulfoxidation of methimazole (MMI) was used as a specific probe for the determination of FMO activity. Methimazole sulfoxidation was compared with the well known assay for FMO metabolism, the formation of N,N-dimethylaniline (DMA) N-oxide, to be confirmed as an exclusively FMO mediated reaction. The participation of P450 and FMO in the sulfoxidation of four sulfur containing pesticides, ametryne; terbutryne, prometryne and methiocarb was investigated using human liver microsomes. All four reactions were demonstrated to be catalysed predominantly by cytochrome P450. Received: 13 March 1996/Accepted: 20 June 1996     

Hepatotoxicity of butylated hydroxytoluene and its analogs in mice depleted of hepatic glutathione

Butylated hydroxytoluene (2,6-di-tert-butyl-4-methylphenol, BHT) has been reported to be a lung toxicant. Mice treated with BHT (200-800 mg/kg, po) in combination with an inhibitor of glutathione (GSH) synthesis, buthionine sulfoximine (BOS; 1 hr before and 2 hr after BHT, 4 mmol/kg per dose, ip) developed hepatotoxicity characterized by an increase in serum glutamic pyruvic transaminase (GPT) activity and centrilobular necrosis of hepatocytes. The hepatotoxic response was both time- and dose-dependent. BHT (up to 800 mg/kg) alone produced no evidence of liver injury. As judged by the observation of normal serum GPT, drug metabolism inhibitors such as SKF-525A, piperonyl butoxide, and carbon disulfide prevented the hepatotoxic effect of BHT given in combination with BSO. On the other hand, pretreatment with cedar wood oil resulted in increased hepatic injury in mice treated with both BHT and BSO. Pretreatment with phenobarbital also tended to increase hepatic injury as judged by changes in serum GPT. These results suggest that BHT is activated by a cytochrome-P-450-dependent metabolic reaction and that the hepatotoxic effect is caused by inadequate rates of detoxification of the reactive metabolite in mice depleted of hepatic GSH by BSO administration. The hepatotoxic potencies of BHT-related compounds also were examined in BSO-treated animals. For hepatotoxicity, the phenolic ring must have benzylic hydrogen atoms at the 4 position and an ortho-alkyl group(s) that moderately hinders the hydroxyl group. These structural requirements essentially are the same as those for the toxic potency in the lung (T. Mizutani, I. Ishida, K. Yamamoto, and K. Tajima (1982), 62, 273-281) and support the hypothesis that BHT-quinone methide plays a role in producing liver damage in mice with depressed hepatic GSH levels.     

Loss of rat liver microsomal cytochrome P-450 during methimazole metabolism. Role of flavin-containing monooxygenase

The role of flavin-containing monooxygenase (FMO) in the decrease in cytochrome P-450 content during the microsomal metabolism of methimazole (N-methyl-2-mercaptoimidazole) was investigated by heat inactivation of FMO. Incubation of liver microsomes from untreated Fischer 344 rats with NADPH and methimazole resulted in a 25% loss of cytochrome P-450 detectable as its ferrous-carbon monoxide complex. The same extent of cytochrome P-450 loss was observed with 1 and 20 mM methimazole, suggesting saturation of the process. There was no significant loss of cytochrome P-450 when microsomal FMO was heat-inactivated prior to incubation with NADPH and methimazole. Heat pretreatment of the microsomes did not affect cytochrome P-450 concentrations and cytochrome P-420 was not observed. These results indicate that FMO-catalyzed metabolism of methimazole is necessary for the loss of cytochrome P-450 in microsomes from untreated rats. Sulfite and N-methylimidazole, the ultimate products of methimazole metabolism, did not cause a significant loss of cytochrome P-450. There was no loss of cytochrome P-450 when glutathione was included in the incubation with methimazole, suggesting that cytochrome P-450 loss was due to an interaction with oxygenated metabolites of methimazole formed by FMO. Losses of cytochrome P-450 were also observed after incubation of microsomes from phenobarbital- (31%) of beta-naphthoflavone-pretreated rats (44%) with NADPH and methimazole. In contrast to microsomes from untreated rats, heat inactivation of FMO did not prevent the loss of cytochrome P-450 in microsomes from the pretreated rats. These results indicate that both phenobarbital and beta-naphthoflavone induce isozymes of cytochrome P-450 capable of directly activating methimazole.     

Inhibitory effect of esculetin on oxidative damage induced by t-butyl hydroperoxide in rat liver

Increasing evidence regarding free radical-generating agents and inflammatory processes suggests that accumulation of reactive oxygen species can cause hepatotoxicity. A short-chain analog of lipid hydroperoxide, t-butyl hydroperoxide (t-BHP), can be metabolized to free radical intermediates by cytochrome P-450 in hepatocytes, which in turn can initiate lipid peroxidation, affect cell integrity and result in cell injury. In this study, we used t-BHP to induce hepatotoxicity in vitro and in vivo and determined the antioxidative bioactivity of esculetin, a coumarin compound. Our investigations showed that pretreatment with esculetin (5-20 microg/ml) significantly decreased the leakage of lactate dehydrogenase (LDH) and alanine transaminase (ALT), and also decreased the formation of malondialdehyde (MDA) in primary cultured rat hepatocytes induced by a 30-min treatment with t-BHP. An in vivo study in rats showed that pretreatment with esculetin (i.p.) at concentrations of 0.5 and 5 mg/kg for 5 days before a single i.p. dose of t-BHP (0.1 mmol/kg) significantly lowered the serum levels of the hepatic enzyme markers (ALT and AST) and reduced oxidative stress in the liver. Histopathological evaluation of the rat livers revealed that esculetin reduced the incidence of liver lesions induced by t-BHP, including hepatocyte swelling, leukocyte infiltration, and necrosis. Based on the results described above, we speculate that esculetin may play a chemopreventive role via reducing oxidative stress in living systems.     

EFFECTS OF MOTORCYCLE EXHAUST ON CYTOCHROME P-450-DEPENDENT MONOOXYGENASES AND GLUTATHIONE S-TRANSFERASE IN RAT TISSUES

The effects of motorcycle exhaust (ME) on cytochrome P-450 (P-450) -dependent monooxygenases were determined using rats exposed to the exhaust by either inhalation, intratracheal, or intraperitoneal administration. A 4-wk ME inhalation significantly increased benzo[a]pyrene hydroxylation, 7-ethoxyresorufin O-deethylation, and NADPH-cytochrome c reductase activities in liver, kidney, and lung microsomes. Intratracheal instillation of organic extracts of ME particulate (MEP) caused a dose- and time-dependent significant increase of monooxygenase activity. Intratracheal treatment with 0.1 g MEP extract/ kg markedly elevated benzo[a]pyrene hydroxylation and 7- ethoxyresorufin O-deethylation activities in the rat tissues 24 h following treatment. Intraperitoneal treatment with 0.5 g MEP extract/ kg/d for 4d resulted in significant increases of P-450 and cytochrome b contents and NADPH-cytochrome c reductase 5 activity in liver microsomes. The intraperitoneal treatment also markedly increased monooxygenases activities toward methoxyresorufin, aniline, benzphetamine, and erythromycin in liver and benzo[a]pyrene and 7-ethoxyresorufin in liver, kidney, and lung. Immunoblotting analyses of microsomal proteins using a mouse monoclonal antibody (Mab) 1-12-3 against rat P-450 1A1 revealed that ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment increased a P-450 1A protein in the hepatic and extrahepatic tissues. Protein blots analyzed using antibodies to P-450 enzymes showed that MEP intraperitoneal treatment caused increases of P-450 2B, 2E, and 3A subfamily proteins in the liver. The ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment resulted in significant increases in glutathione S -transferase activity in liver cytosols. The present study shows that ME and MEP extract contain substances that can induce multiple forms of P-450 and glutathione S-transferase activity in the rat.     

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.     

Induction and inhibition of rat hepatic drug metabolism by N-substituted imidazole drugs

Three daily administrations of N-substituted imidazole antimycotics, clotrimazole (CloTZ, 75 mg/kg/day), miconazole (MCZ, 150 mg/kg/day), or tioconazole (TCZ, 150 mg/kg/day), but not the 4,5-disubstituted imidazole cimetidine (350 mg/kg/day) or imidazole (200 mg/kg/day for 4 days), induced rat hepatic cytochrome P-450 and other drug-metabolizing enzymes. These findings paralleled in vitro observations where CloTZ, MCZ, and TCZ were several orders of magnitude more potent as inhibitors of p-nitroanisole O-demethylase activity in control male rat liver microsomes than cimetidine or imidazole. Although no marked difference in inhibitory potency was evident among the N-substituted imidazoles, there were qualitative and quantitative differences in the profiles and extents of induction of various cytochrome P-450-dependent monooxygenases and Phase II conjugation enzymes. Cytochrome P-450 was elevated dramatically by CloTZ (3-4 times the control) and to a lesser extent by MCZ and TCZ (congruent to 1.5 times the control). For all agents, there was an increase in metyrapone binding approximately equivalent to the additional (i.e. above control) cytochrome P-450. Despite the large difference in cytochrome P-450 induction by CloTZ, MCZ, and TCZ, these agents elevated p-nitroanisole demethylase and aniline hydroxylase to similar extents (3-5 X and 1-2 X control, respectively). All agents induced erythromycin and ethylmorphine demethylation in proportion to cytochrome P-450. Ethoxyresorufin O-de-ethylation was not substantially affected by any agent. Large differences in the extent and specificity of induction of microsomal glucuronide conjugations were also evident.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Sulphoxidation of albendazole by the FAD-containing and cytochrome P-450 dependent mono-oxygenases from pig liver microsomes

1. Two distinct microsomal pathways involved in the metabolism of albendazole (ABZ) to albendazole-sulphoxide (SO.ABZ) by pig liver microsomes have been identified and quantified. 2. The binding of ABZ to microsomal cytochrome P-450 (Type I spectrum, Ks = 25.5 microM), the decrease of the rate of sulphoxidation by antibody against NADPH cytochrome c reductase, and the use of purified cytochrome P-450 A demonstrated the contribution of a cytochrome P-450-dependent mono-oxygenase to the metabolism of ABZ. 3. The involvement of FAD-containing mono-oxygenase (FMO) was shown by thermal pretreatment of microsomes, n-octylamine activation of the reaction, and by using purified pig liver FMO. 4. From Km and Vmax values, it would appear that the relative contributions of the two systems depend on the concentration of ABZ.     

Effects of benzothiazole on the xenobiotic metabolizing enzymes and metabolism of acetaminophen

Benzothiazole (BT) is present in tobacco smoke and widely used for industrial and pharmaceutical purposes. In this study we have investigated the influence of BT on the activities of hepatic cytochrome P450 monooxygenases (P450s) and UDP-glucuronyltransferase (UDP-GT), sulphotransferase and glutathione-S-transferase (GST) in male Sprague-Dawley rats. We also examined if BT would change the metabolism and toxification of acetaminophen (AA) through modulation of metabolizing enzymes. Benzothiazole (1 mmol kg(-1), p.o., 5 days) markedly increased the enzyme activities of P4501A1, 1A2, 2B1, 3A4, 2E1, UDP-GT and GST in liver. Pretreatment with BT significantly decreased the amount of total AA recovered in bile to 68.5% of controls, mainly as a consequence of reduced AA-glucuronide conjugate (35.3% of controls), whereas the AA-glutathione conjugate (AA-GS) was augmented to 1.6-fold. After pretreatment with BT, potentiation of the hepatotoxicity by AA (400 mg kg(-1), i.p., 24 h) was observed by measuring serum alanine aminotransferase activities in ICR mice. These results indicate that: BT is a potent inducer of P450s and phase II metabolizing enzymes; and the increase of AA-GS conjugate and aggravation of AA hepatotoxicity by BT may be related to induction of P450s.     

Monooxygenase-mediated metabolism and binding of ethylene thiourea to mouse liver microsomal protein

Ethylenethiourea (ETU), a metabolite and degradation product of the ethylenebisdithiocarbamate fungicides, is a substrate for the flavin-dependent monooxygenase (FMO), a microsomal NADPH requiring enzyme that can oxidize ETU, as well as for the cytochrome P-450 enzyme system. The present study shows that the mouse metabolises ETU preferentially via the FMO system. FMO activity decreases as male, but not female mice, increase in age to 30 weeks. This difference in activity is reflected in decreased overall metabolism of ETU and in a decreased FMO-mediated binding of radiolabelled ETU to mouse liver microsomal protein. The rapid metabolism of ETU by the FMO system may contribute to the lack of acute toxicity and teratogenicity exhibited by the mouse relative to the rat. However, the FMO-mediated binding of ETU metabolites to mouse liver protein is consistent with the chronic hepatotoxicity exhibited by ETU in this species.     

The effect of methimazole on thioamide bioactivation and toxicity.

The hepatotoxicity induced by administration of ethionamide and thionicotinamide (TNA) was shown to be decreased by pre-administration of methimazole (MMI). Pre-administration of MMI was also shown to decrease the levels of excretion of TNA S-oxide. This indicates that thioamide S-oxidation, mediated by the flavin-containing mono-oxygenase, may be linked to the initiation of hepatotoxicity induced by these thioamides. SK&F-525-A, the cytochrome P-450 inhibitor, did not affect either thioamide-induced toxicity or levels of excretion of TNA S-oxide; however, the role of the P-450 isoenzymes cannot be totally ruled out.     

Involvement of cytochrome P450-mediated metabolism in tienilic acid hepatotoxicity in rats

Tienilic acid is reported to be converted into electrophilic metabolites by cytochrome P450 (CYP) in vitro. In vivo, however, the metabolites have not been detected and their effect on liver function is unknown. We previously demonstrated that tienilic acid decreased the GSH level and upregulated genes responsive to oxidative/electrophilic stresses, such as heme oxygenase-1 (Ho-1), glutamate-cysteine ligase modifier subunit (Gclm) and NAD(P)H dehydrogenase quinone 1 (Nqo1), in rat liver, as well as inducing hepatotoxicity by co-treatment with the glutathione biosynthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO). In this study, for the first time, we identified a glutathione-tienilic acid adduct, a stable conjugate of putative electrophilic metabolites with glutathione (GSH), in the bile of rats given a single oral dose of tienilic acid (300mg/kg). Furthermore, a tienilic acid-induced decrease in the GSH level and upregulation of Ho-1, Gclm and Nqo1 were completely blocked by pretreatment with the CYP inhibitor 1-aminobenzotriazole (ABT, 66mg/kg, i.p.). The increase in the serum ALT level and hepatocyte necrosis resulting from the combined dosing of BSO and tienilic acid was prevented by ABT, despite a low hepatic GSH level. These findings suggest that the electrophilic metabolites of tienilic acid produced by CYP induce electrophilic/oxidative stresses in the rat liver and this contributes to the hepatotoxicity of tienilic acid under impaired GSH biosynthesis.     

Hepatotoxicity of pulegone in rats: its effects on microsomal enzymes, in vivo

Oral administration of pulegone (400 mg/kg) to rats once daily for five days caused significant decreases in the levels of liver microsomal cytochrome P-450 and heme. Cytochrome b5 and NAD(P)H-cytochrome c-reductase activities were not affected. Massive hepatotoxicity accompanied by an increase in serum glutamate pyruvate transaminase (SGPT) and a decrease in glucose-6-phosphatase were observed upon treatment with pulegone. A significant decrease in aminopyrine N-demethylase was also noticed after pulegone administration. Menthone or carvone (600 mg/kg), compounds related to pulegone, when administered orally did not cause any decrease in cytochrome P-450 levels. The hepatotoxic effects of pulegone were both dose and time dependent. Pretreatment of rats with phenobarbital (PB) or diethylmaleate (DEM) potentiated the hepatotoxicity caused by pulegone, whereas, pretreatment with 3-methylcholanthrene (3-MC) or piperonyl butoxide protected from it. It appears that a PB induced cytochrome P-450 catalysed reactive metabolite(s) may be responsible for the hepatotoxicity caused by pulegone.     

Induction of rat UDP-glucuronosyltransferase and glutathione S-transferase activities by L-buthionine-S,R-sulfoximine without induction of cytochrome P-450

The effect of prolonged exposure to buthionine sulfoximine (BSO) on rat hepatic Phase I and Phase II drug-metabolizing enzymes has been examined. Exposure to 30 mM BSO in drinking water for 7 days induced hepatic microsomal UDP-glucuronosyltransferase activity (detergent-activated) toward p-nitrophenol (250%), 1-naphthol (210%), morphine (130%) and testosterone (140%), but not estrone. Glucuronosyltransferase activities were also induced after exposure for as short as 3 and as long as 13 days. When rats were returned to unsupplemented drinking water for 1 day prior to sacrifice following 6 days on 30 mM BSO, comparable induction to that seen after 7 consecutive days on the BSO solution was observed despite liver glutathione concentration having rebounded to 127% of control. Daily ingestion of BSO was similar (1 mmol/rat/day) for all periods of 30 mM BSO-drinking water exposure, with a body weight-adjusted dose range of 3.2-6.3 mmol/kg/day. An analogous inductive response caused by drinking 30 mM BSO for 3 days was elicited for p-nitrophenol and morphine glucuronidation by 6 mmol/kg doses of BSO given as single daily intraperitoneal or intragastric injections for 3 days. Intraperitoneal, intragastric and all BSO-drinking water exposures also significantly induced (130-195%) cytosolic glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene. Significant increases in UDP-glucuronosyltransferase and glutathione S-transferase activities were also observed following 3 days of exposure to BSO in the drinking water at a concentration as low as 5 mM. Cytosolic p-nitrophenol sulfotransferase activity, with one minor exception, was not enhanced by any BSO treatment regimen. Alterations in transferase activities were not accompanied by any major changes in either overall cytochrome P-450 concentration or oxidative reactions selective for two isozymes. Thus, in addition to its well-documented glutathione-depleting property, BSO also selectively induces several Phase II drug-metabolizing enzymes, an effect to be considered in studies employing extended BSO treatment.     

Methoxsalen decreases the metabolic activation and prevents the hepatotoxicity and nephrotoxicity of chloroform in mice

The effects of methoxsalen, a potent inhibitor of cytochrome P-450, on the hepatotoxicity and nephrotoxicity of chloroform have been determined in mice. Hepatic and renal monooxygenase activities and the in vitro covalent binding of chloroform metabolites to hepatic and renal microsomal proteins were decreased by 20-70% in microsomes from mice killed 2 hr after the administration of methoxsalen (250 mumol.kg-1ip) alone. Administration of methoxsalen (250 mumol.kg-1ip), 30 min before [14C]chloroform (1 ml.kg-1ip), did not modify blood levels of [14C]chloroform (and metabolites) but decreased the in vivo covalent binding of [14C]chloroform metabolites to hepatic and renal proteins 4 hr after the administration of [14C]chloroform. This pretreatment markedly decreased serum glutamic pyruvic transaminase activity, blood urea nitrogen, glucosuria, liver and kidney lesions, and mortality 24 hr after the administration of chloroform (0.125-1.5 ml.kg-1ip). Other cytochrome P-450 inhibitors (SKF 525-A or piperonyl butoxide), given at the same molar dose (250 mumol.kg-1ip), exerted no protective effect. Pretreatment with methoxsalen appears to decrease the metabolic activation of chloroform and essentially prevents its hepatotoxicity and nephrotoxicity in mice. Methoxsalen may have use as a tool to determine the role of metabolic activation by cytochrome P-450 in the hepatotoxicity and nephrotoxicity of drugs and chemicals.     

Cyclic conversion of the novel Src kinase inhibitor [7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (TG100435) and Its N-oxide metabolite by flavin-containing monoxygenases and cytochrome P450 reductase.

[7-(2,6-Dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (TG100435) is a novel multi-targeted Src family kinase inhibitor with demonstrated anticancer activity in preclinical species. Potent kinase inhibition is associated with TG100435 and its major N-oxide metabolite [7-(2,6-dichlorophenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-{4-[2-(1-oxy-pyrrolidin-1-yl)-ethoxy]-phenyl}-amine (TG100855). The objectives of the current study were to identify the hepatic enzyme(s) responsible for 1) the total metabolic flux of TG100435, 2) the formation of TG100855, and 3) the subsequent metabolism of TG100855. Flavin-containing monooxygenases (FMO) and cytochrome P450 monooxygenases (P450s) contribute to TG100435 total metabolic flux. TG100435 metabolic flux was completely inhibited by methimazole and ketoconazole, suggesting only FMO- and CYP3A4-mediated metabolism. TG100855 formation was markedly inhibited (~90%) by methimazole or heat inactivation (>99%). FMO3 was the primary enzyme responsible for TG100855 formation. In addition, an enzyme mediated retroreduction of TG100855 back to TG100435 was observed. The N-oxidation reaction was approximately 15 times faster than the retroreduction reaction. Interestingly, the retroreduction of TG100855 to TG100435 in recombinant P450 or liver microsomes lacked inhibition by the P450 inhibitors. TG100435 formation in the human liver microsomes or recombinant P450 increased as a function of cytochrome P450 reductase activity, suggesting potential involvement of cytochrome P450 reductase. The results of this in vitro study demonstrate the potential of TG100435 and TG100855 to be interconverted metabolically. FMO seem to be the major N-oxidizing enzymes, whereas cytochrome P450 reductase seems to be responsible for the retroreduction reaction.