Cytochrome P450-mediated metabolism and cytotoxicity of aflatoxin B(1) in bovine hepatocytes.
Aflatoxin B(1) (AFB(1)) biotransformation comprises cytochrome P450-mediated reactions resulting in hydroxylated and demethylated metabolites as well as AFB(1) 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 AFB(1). Here we show the time- and dose-dependent rate of AFB(1)-metabolism in bovine hepatocytes. Aflatoxin M(1) (AFM(1)) is the most prominent metabolite formed within the first 2-8 hr of incubation, whereas AFB(1)-dhd is detectable in medium mainly after a prolonged incubation period. The delayed formation of AFB(1)-dhd corresponds to the cytotoxicity demonstrated by the MTT assay. alpha-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 AFB(1). Initial experiments confirmed that alpha-naphthoflavone and ketoconazole inhibited ethoxyresorufin O-deethylation and testosterone 6beta-hydroxylation also in bovine hepatocytes. Both inhibitors reduced AFM(1) and AFB(1)-dhd formation concentration dependently, suggesting that both enzyme groups contribute to the formation of these metabolites. However, the formation of AFM(1) was less inhibited by both compounds than the formation of AFB(1)-dhd.
Kuilman
M E
ME
Department of Veterinary Pharmacology, Pharmacy and Toxicology, Utrecht University, PO Box 80152, 3508 TD, The, Utrecht, Netherlands.
Maas
R F
RF
Fink-Gremmels
J
J
D016604:Aflatoxin B1 / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000633:toxicity*; D016607:Aflatoxin M1 / Q000096:biosynthesis; D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D001711:Biotransformation; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D019363:Cytochrome P-450 CYP1A1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D007654:Ketoconazole / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D001571:Benzoflavones; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; C092991:aflatoxin B1-8,9-dihydrodiol-Tris buffer complex; C011512:alpha-naphthoflavone; D016607:Aflatoxin M1; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D013250:Steroid Hydroxylases; D019363:Cytochrome P-450 CYP1A1; C022657:steroid hormone 6-beta-hydroxylase; D007654:Ketoconazole