Sex-dependent metabolism of xenobiotics.

Sex-dependent differences in xenobiotic metabolism have been most extensively studied in the rat. Because sex-dependent differences are most pronounced in rats, this species quickly became the most popular animal model to study sexual dimorphisms in xenobiotic metabolism. Exaggerated sex-dependent variations in metabolism by rats may be the result of extensive inbreeding and/or differential evolution of isoforms of cytochromes P450 in mammals. For example, species-specific gene duplications and gene conversion events in the CYP2 and CYP3 families have produced different isoforms in rats and humans since the species division over 80 million years ago. This observation can help to explain the fact that CYP2C is not found in humans but is a major subfamily in rats (Table 11). Animal studies are used to help determine the metabolism and toxicity of many chemical agents in an attempt to extrapolate the risk of human exposure to these agents. One of the most important concepts in attempting to use rodent studies to identify sensitive individuals in the human population is that human cytochromes P450 differ from rodent cytochromes P450 in both isoform composition and catalytic activities. Xenobiotic metabolism by male rats can reflect human metabolism when the compound of interest is metabolized by CYP1A or CYP2E because there is strong regulatory conservation of these isoforms between rodents and humans. However, problems can arise when rats are used as animal models to predict the potential for sex-dependent differences in xenobiotic handling in humans. Information from countless studies has shown that the identification of sex-dependent differences in metabolism by rats does not translate across other animal species or humans. The major factor contributing to this observation is that CYP2C, a major subfamily in rats, which is expressed in a sex-specific manner, is not found in humans. To date, sex-specific isoforms of cytochromes P450 have not been identified in humans. The lack of expression of sex-dependent isoforms in humans indicates that the male rat is not an accurate model for the prediction of sex-dependent differences in humans. Differences in xenobiotic metabolism among humans are more likely the consequence of intraindividual variations as a result of genetics or environmental exposures rather than from sex-dependent differences in enzyme composition. A major component of the drug discovery and development process is to identify, at as early a stage as possible, the potential for toxicity in humans. Earlier identification of individual differences in xenobiotic metabolism and the potential for toxicity will be facilitated by improving techniques to make better use of human tissue to prepare accurate in vitro systems such as isolated hepatocytes and liver slices to study xenobiotic metabolism and drug-induced toxicities. Accurate systems should possess an array of bioactivation enzymes similar to the in vivo expression of human liver. In addition, the compound concentrations and exposure times used in these in vitro test systems should mimic those achieved in the target tissues of humans. Consideration of such factors will allow the development of compounds with improved efficacy and low toxicity at a more efficient rate. The development of accurate in vitro systems utilizing human tissue will also aid in the investigation of the molecular mechanisms by which the CYP genes are regulated in humans. Such studies will facilitate the study of the basis for differences in expression of isoforms of CYP450 in humans.

Furan-mediated uncoupling of hepatic oxidative phosphorylation in Fischer-344 rats: an early event in cell death.

Furan is a potent rodent hepatotoxicant and carcinogen. The present study was done to examine the effects of furan on hepatic energy metabolism both in vivo and in vitro in male F-344 rats. Furan produced concentration- and incubation time-dependent irreversible reductions in ATP in freshly isolated F-344 rat hepatocytes. Furan-mediated depletion of ATP occurred prior to cell death and was prevented by including 1-phenylimidazole, a cytochrome P450 inhibitor, in the suspensions. Male F-344 rats were treated with furan (0-30 mg/kg, po) and killed 24 hr later to prepare hepatic mitochondria. Furan produced dose-dependent increases in state 4 respiration and ATPase activity. Both of these changes were prevented by 1-phenylimidazole cotreatment. In a separate series of experiments, mitochondria were prepared from isolated rat hepatocytes following incubation with furan (2-100 microM) for 1-4 hr. Furan produced incubation time- and concentration-dependent increases in state 4 respiration and ATPase activity. Furan-mediated mitochondrial changes were prevented by adding 1-phenylimidazole to the hepatocyte suspensions. These results indicate that the ene-dialdehyde metabolite of furan uncouples hepatic oxidative phosphorylation in vivo and in vitro. In vitro studies using an isolated hepatocyte suspension/culture system demonstrated that the concentration response for furan-mediated mitochondrial changes in suspension corresponded with the concentration responses for cell death after 24 hr. Including 1-phenylimidazole or oligomycin plus fructose in hepatocyte suspensions prevented furan-induced cell death after 24 hr in culture. The results of this study indicate that furan-induced uncoupling of oxidative phosphorylation is an early, critical event in cytolethality both in vivo and in vitro.

The contribution of oxidation and deacetylation to acetaminophen nephrotoxicity in female Sprague-Dawley rats.

Young adult female rats are more susceptible to acetaminophen (APAP) induced nephrotoxicity than are male rats. The purpose of the present study was to assess the contribution of oxidation and deacetylation to the expression of APAP nephrotoxicity. Male and female rats received APAP (1100 mg kg(-1) i.p.) alone or following pretreatment with 1-aminobenzotriazole (ABT), a suicide inhibitor of cytochromes P450, or tri-o-tolylphosphate (TOTP), an irreversible carboxyesterase inhibitor. Rats were sacrificed 6 or 24 h following administration of 1100 mg APAP kg(-1) containing [ring-14C]APAP. Blood urea nitrogen (BUN) concentration was used as an index of nephrotoxicity. Renal and hepatic non-protein sulfhydryl (NPSH) contents and covalent binding of radiolabel derived from APAP were determined 6 h following APAP administration. Pretreating female rats with ABT, TOTP, or both compounds prevented the APAP-induced elevation in BUN concentration at 24 h. Pretreatment with ABT or ABT plus TOTP prevented APAP-induced depletion of both hepatic and renal NPSH content at 6 h in female rats. In male rats, APAP treatment did not significantly affect hepatic NPSH content. However, renal NPSH content in males was significantly decreased following APAP treatment and the decrease was prevented when rats were pretreated with ABT or ABT plus TOTP. Covalent binding of radiolabel derived from APAP was significantly greater in female kidney as compared to male kidney. Further, covalent binding in female kidney was significantly decreased when rats were pretreated with ABT, TOTP or both. These data suggest that both oxidative metabolism and deacetylation may contribute to APAP-induced nephrotoxicity in rats.

Contribution of oxidation and deacetylation to the bioactivation of acetaminophen in vitro in liver and kidney from male and female Sprague-Dawley rats.

Young adult female Sprague-Dawley (SD) rats are more susceptible to acetaminophen (APAP)-induced nephrotoxicity than are age-matched male SD rats. Mechanisms contributing to sex-dependent APAP nephrotoxicity may involve differences in APAP bioactivation via cytochrome P450-dependent metabolism to N-acetyl-p-benzoquinoneimine and/or deacetylation to para-amino-phenol. APAP bioactivation by oxidation and deacetylation was assessed by examining the effects of 1-aminobenzotriazole (ABT), a suicide substrate inhibitor of cytochrome P450, and bis-(para-nitrophenyl) phosphate (BNPP), a reversible carboxyesterase inhibitor, on covalent binding of APAP-derived radiolabel. Hepatic and renal S9 fractions prepared from naive male and female rats were incubated with [14C-ring]-APAP in the presence and absence of NADPH. There were no sex-related differences in covalent binding of APAP-derived radiolabel in hepatic or renal S9 fractions from male and female rats. In both sexes, incubation of hepatic or renal S9 fractions with 10 mM ABT significantly reduced covalent binding of APAP-derived radiolabel as compared with covalent binding in the absence of ABT. In contrast, incubation of renal and hepatic S9 fractions with 10 mM BNPP did not alter covalent binding of radiolabel derived from APAP in either males or females. Thus, at least in vitro, differences in bioactivation of APAP in liver and kidney from male and female SD rats do not seem to contribute to sex-dependent APAP toxicity. Carboxyesterases inhibited by BNPP do not seem to contribute to covalent binding of APAP-derived radiolabel in vitro in either liver or kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Aminobenzotriazole-induced destruction of hepatic and renal cytochromes P450 in male Sprague-Dawley rats.

1-Aminobenzotriazole (ABT) is a suicide substrate of both hepatic and pulmonary cytochromes P450. The present studies were designed to compare the effects of ABT on hepatic and renal metabolism. Hepatic and renal microsomes and cytosol were prepared from male Sprague-Dawley rats following ABT pretreatment (0-100 mg/kg ip) for various times. Administration of 100 mg ABT/kg produced profound reductions in P450 content in both liver and kidney within 2 hr; loss of P450 in both tissues persisted for at least 48 hours. ABT-induced destruction of P450 was dose-dependent. Maximal destruction of about 80% of total hepatic P450 occurred at dosages of ABT equal to or greater than 10 mg/kg. Maximal destruction of about 80% of total renal P450 occurred at dosages of ABT equal to or greater than 50 mg/kg. In vitro, ABT rapidly and efficiently destroyed P450 in both hepatic and renal microsomes prepared from naive male Sprague-Dawley rats. Incubation of hepatic or renal microsomes in vitro with ABT produced detectable destruction of P450 within 5 min. Maximal destruction of P450 occurred within 10 min in both hepatic and renal microsomes during in vitro incubation with ABT. ABT-induced destruction of P450 in vitro was concentration-dependent. For hepatic microsomes, maximal destruction of about 70% of P450 required concentrations of ABT equal to or greater than 10 mM. For renal microsomes, maximal destruction of about 80% of P450 required concentrations of ABT equal to or greater than 10 mM. In both liver and kidney, only P450 content and P450-dependent activities were significantly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)