title: Effects of CYP inhibitors on precocene I metabolism and toxicity in rat liver slices.
abstract: We present a comprehensive in vitro approach to assessing metabolism-mediated hepatotoxicity using male Sprague-Dawley rat liver slices incubated with the well characterized hepatotoxicant, precocene I, and inhibitors of cytochrome P450 (CYP) enzymes. This approach combines liquid chromatography mass spectrometry (LC MS) detection methods with multiple toxicity endpoints to enable identification of critical metabolic pathways for hepatotoxicity. The incubations were performed in the absence and presence of the non-specific CYP inhibitor, 1-aminobenzotriazole (ABT) and isoform-specific inhibitors. The metabolite profile of precocene I in rat liver slices shares some features of the in vivo profile, but also had a major difference in that epoxide dihydrodiol hydrolysis products were not observed to a measurable extent. As examples of our liver slice metabolite identification procedure, a minor glutathione adduct and previously unreported 7-O-desmethyl and glucuronidated metabolites of precocene I are reported. Precocene I induced hepatocellular necrosis in a dose- and time-dependent manner. ABT decreased the toxicity of precocene I, increased exposure to parent compound, and decreased metabolite levels in a dose-dependent manner. Of the isoform-specific CYP inhibitors tested for an effect on the precocene I metabolite profile, only tranylcypromine was noticeably effective, indicating a role of CYPs 2A6, 2C9, 2Cl9, and 2E1. With respect to toxicity, the order of CYP inhibitor effectiveness was ABT>diethyldithiocarbamate∼tranylcypromine>ketoconazole. Furafylline and sulfaphenazole had no effect, while quinidine appeared to augment precocene I toxicity. These results suggest that rat liver slices do not reproduce the reported in vivo biotransformation of precocene I and therefore may not be an appropriate model for precocene I metabolism. However, these results provide an example of how small molecule manipulation of CYP activity in an in vitro model can be used to confirm metabolism-mediated toxicity.
authors: Bristol-Myers Squibb, Pharmaceutical Candidate Optimization, Department of Biotransformation, Princeton, NJ 08543, USA.
authors: Van T
authors: VT
authors: Ly
authors: Barry
authors: B
authors: Brock
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism; D001578:Benzopyrans / Q000378:metabolism* / Q000494:pharmacology / Q000633:toxicity*; D002851:Chromatography, High Pressure Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004050:Ditiocarb / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D020719:Glucuronides / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006900:Hydroxylation; D007654:Ketoconazole / Q000378:metabolism / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D015394:Molecular Structure; D009336:Necrosis / Q000139:chemically induced / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013426:Sulfaphenazole / Q000378:metabolism / Q000494:pharmacology; D014191:Tranylcypromine / Q000494:pharmacology; D014230:Triazoles / Q000378:metabolism / Q000494:pharmacology
chemical_list: D001578:Benzopyrans; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D020719:Glucuronides; D014230:Triazoles; D013426:Sulfaphenazole; C033020:1-aminobenzotriazole; C019539:precocene I; D014191:Tranylcypromine; D000255:Adenosine Triphosphate; D003577:Cytochrome P-450 Enzyme System; D004050:Ditiocarb; D007770:L-Lactate Dehydrogenase; D001219:Aspartate Aminotransferases; D005978:Glutathione; D007654:Ketoconazole