OXIDATION OF AFLATOXIN B-1 BY BACTERIAL RECOMBINANT HUMAN CYTOCHROME-P450 ENZYMES

Human cytochromes P450 (P450) 1A2 and P450 3A4 were expressed in Escherichia coli, purified, and used in reconstituted oxidation systems. The optimal system for P450 3A4 included a mixture of phospholipids, sodium cholate, cytochrome b(5), GSH, and MgCl2. Relatively high catalytic activities were obtained with such a system for aflatoxin (AF) B-1 3 alpha-hydroxylation and 8,9-epoxidation. P450 3A4 was more active than P450 1A2 in forming genotoxic AFB(1) oxidation products. Analysis of the AFB(1) products indicated that P450 3A4 formed AFQ(1) and the exo-8,9-epoxide; P450 1A2 formed AFM(1), a small amount of AFQ(1), and both the exo- and endo-8,9-epoxides. The endo epoxide is essentially nongenotoxic in the umu test, as found previously in bacterial mutagenicity assays [Iyer, R. S., Coles, B. F., Raney, K. D., Thier, R., Guengerich, F. P., and Harris, T. M. (1994) J. Am. Chem. Sec. 116, 1603-1609]. 7,8-Benzoflavone (alpha-naphthoflavone, alpha NF) stimulated AFB(1) (exo) 8,9-epoxidation and inhibited 3 alpha-hydroxylation in human liver microsomes and a reconstituted P450 3A4 system but was a potent inhibitor of all reactions catalyzed by P450 1A2. Plots of AFB(1) 3 alpha-hydroxylation and 8,9-epoxidation vs AFB(1) concentration were sigmoidal in both human liver microsomes and the reconstituted P450 3A4 system. The results are consistent with the view that P450 3A4 is a major human liver P450 enzyme involved in AFB(1) activation, although the in vivo situation may be more complex due to the presence of the enzyme in the gastrointestinal tract.