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.