The Involvement of Human Placental Microsomal Cytochrome P-450 in Aromatization

Abstract

The aromatization of androstenedione is inhibited in the presence of aminoglutethimide, diethylaminoethyl-2,2-diphenyl valerate HCl (SKF 525-A), and 10-3m KCN, but not carbon monoxide or metyrapone. The role of cytochrome P-450 in this reaction could not, therefore, be unequivocally established by inhibition studies. However, it was determined that steroid binding to placental microsomal cytochrome P-450 is absolutely specific for aromatase substrates, intermediates, and inhibitors. Moreover, the substrate 19-nortestosterone, aromatization of which is inhibited by CO, is a competitive inhibitor of both the aromatization and cytochrome P-450 binding of androstenedione, and vice versa. These results suggest that a single species of cytochrome P-450 is responsible for the aromatization of both steroids. Furthermore, both 19-hydroxyandrostenedione and 19-oxoandrostenedione also compete with androstenedione and 19-nortestosterone for the same species of cytochrome P-450. This suggests that all three hydroxylation reactions of C19 aromatization as well as the aromatization of 19-nortestosterone are carried out at the same enzyme site. In addition, an antibody raised against porcine hepatic NADPH-cytochome c reductase, which specifically inhibits cytochrome P-450 mediated reactions, is a potent inhibitor of the aromatization of both C19 and C18 estrogen precursors. The extent of inhibition of the CO-sensitive aromatization of C18 substrates by the antibody is similar to that observed for CO-sensitive drug hydroxylations occurring in liver microsomes. On the other hand, inhibition of the aromatization of the non-CO-sensitive substrate, androstenedione, by the antibody was atypical in that a much greater inhibitory effect on aromatization than on cytochrome c reduction was found. The distinct patterns of inhibition of aromatization by the reductase antibody are consistent with the hypothesis that the rate of entry of the first electron into the oxidized P-450 substrate complex may be rate limiting in the aromatization of C19 but not C18 steroids. The difference in the rate of entry of the first electron would be reflected in the extent to which the reduced P-450 substrate complex accumulates during the steady state. Since CO inhibition occurs at the level of the reduced P-450 substrate complex, variation in the steady state concentration of this intermediate could explain the difference in CO sensitivity observed between C19 and C18 substrates.