Involvement of cytochrome P-450c in alpha-naphthoflavone metabolism by rat liver microsomes

Metabolism of alpha-naphthoflavone (ANF) is increased markedly in rat liver microsomes by 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), two inducers of cytochromes P-450c and P-450d (P-450c and P-450d). Although several indirect lines of evidence in the literature suggest that ANF is metabolized by P-450c, Vyas et al. J. Biol. Chem. 258:5649-5659 (1983)] reported that ANF metabolism by 3-MC-induced rat liver microsomes was only partially inhibited by antibodies against P-450c. Our laboratory has previously reported clastogenic effects of metabolites of ANF, and in the present study we reexamined the role of P-450c in ANF metabolism by both uninduced and TCDD-induced rat liver microsomes, using monospecific polyclonal antibodies to P-450c and P-450d. ANF metabolism was inhibited to different extents in TCDD-induced microsomes by different preparations of anti-P-450c. One lot of anti-P-450c produced only 50% inhibition of ANF metabolism in TCDD-induced microsomes, whereas another lot of anti-P-450c inhibited ANF metabolism by 80%. Anti-P-450d had no effect on ANF metabolism. Neither anti-P-450c nor anti-P-450d inhibited ANF metabolism in uninduced rat liver microsomes. In a reconstituted enzyme system, purified P-450c metabolized ANF 47 and 510 times more rapidly than P-450d and P-450b, respectively. Metabolites resulting from oxidation at 7,8- or 5,6-positions (7,8-dihydro-7,8-dihydroxy-ANF, 5,6-dihydro-5,6-dihydroxy-ANF, 5,6-oxide-ANF, and 6-hydroxy-ANF) were formed by all preparations of microsomes. An unknown toxic ANF metabolite was formed only with a reconstituted P-450c system and with 3-MC- or TCDD-induced microsomes. Our results indicate that P-450c is responsible for the majority of the metabolism of ANF in TCDD-induced microsomes, whereas other constitutive isozymes are responsible for the metabolism seen in uninduced liver microsomes. The variable inhibition of ANF metabolism with different lots of anti-P-450c probably reflects the differences in the proportion of antibodies to different epitopes important in the binding or metabolism of this substrate.