Bioactivation of aflatoxin B 1 by human liver microsomes: Role of cytochrome P450 IIIA enzymes

Abstract

Based on our previous observations (H. S. Ramsdell and D. L. Eaton, 1990, Cancer Res., 50, 615–620) that the proportion of aflatoxin B 1 (AFB 1) converted to the highly reactive AFB 1-8,9-epoxide in microsomal incubations varies with substrate concentration, we have examined the hypothesis of T. Shimada and F. P. Guengerich (1989, Proc. Natl. Acad. Sci. USA, 86, 462–465) that cytochrome P450 IIIA4 is principally responsible for the activation (epoxidation) of AFB 1 by human liver microsomes. The initial rates of formation of AFB 1-8,9-epoxide and hydroxylated AFB 1 metabolites were determined in microsomes prepared from livers of organ donors ( n = 14) at AFB 1 concentrations of 124 and 16 μ m. Microsomal oxidation of nifedipine, catalyzed primarily by P450 IIIA enzymes, was also determined by HPLC. Rates of formation of AFB 1 metabolites and nifedipine oxidation were poorly correlated at either AFB 1 concentration ( r 2 = 0.13–0.41). A somewhat better correlation between AFB 1 epoxidation and nifedipine oxidation was observed at 124 μ m AFB 1 ( r 2 = 0.41) than at 16 μ m AFB 1 ( r 2 = 0.26). Treatment of pooled microsomes with troleandomycin, an apparently specific inhibitor of P450 IIIA enzymes, resulted in 35% inhibition of AFB 1-8,9-epoxide formation at the high AFB 1 level but had little effect at 16 μ m AFB 1. An antibody against rat cytochrome P450 IIIA1 significantly inhibited AFB 1 epoxidation at high, but not low, AFB 1 concentrations, whereas AFQ 1 formation was strongly inhibited at all substrate levels examined. These results are consistent with the hypothesis that cytochrome P450 IIIA enzyme(s) can form AFB 1-8,9-epoxide, but are effective at only relatively high substrate concentrations. Another P450 enzyme(s) appears to be principally responsible for AFB 1-8,9-epoxide formation at the low AFB 1 levels that would be typical for dietary exposures.