Cytochrome P450-mediated metabolism and cytotoxicity of aflatoxin B1 in bovine hepatocytes.

Aflatoxin B1 (AFB1) biotransformation comprises cytochrome P450-mediated reactions resulting in hydroxylated and demethylated metabolites as well as AFB1 epoxides. As the latter are highly nucleophilic, the species-specific rate of epoxidation and the ability for rapid conjugation to glutathione by glutathione S-transferase determines the individual susceptibility to AFB1. Here we show the time- and dose-dependent rate of AFB1-metabolism in bovine hepatocytes. Aflatoxin M1 (AFM1) is the most prominent metabolite formed within the first 2–8 hr of incubation, whereas AFB1-dhd is detectable in medium mainly after a prolonged incubation period. The delayed formation of AFB1-dhd corresponds to the cytotoxicity demonstrated by the MTT assay. α-Naphthoflavone and ketoconazole, inhibitors of CYP1A and CYP3A, respectively in humans, were used to evaluate the contribution of specific P450 isoenzymes in bovine biotransformation of AFB1. Initial experiments confirmed that α-naphthoflavone and ketoconazole inhibited ethoxyresorufin O-deethylation and testosterone 6β-hydroxylation also in bovine hepatocytes. Both inhibitors reduced AFM1 and AFB1-dhd formation concentration dependently, suggesting that both enzyme groups contribute to the formation of these metabolites. However, the formation of AFM1 was less inhibited by both compounds than the formation of AFB1-dhd.