Human liver microsomal steroid metabolism: Identification of the major microsomal steroid hormone 6β-hydroxylase cytochrome P-450 enzyme

Abstract

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6β hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (≥75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2β and 15β hydroxylation also occurred, proceeding at ~10% and 3-4% the rate of microsomal 6β hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6β-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6β-hydroxylase catalyst with all three steroid substrates. Steroid 6β-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r < 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6β hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2β and 15β hydroxylation in parallel to the 6β-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6β hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2α, 16α, or 7α hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6β hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6β hydroxylation in human liver microsomes, and furthermore suggest that steroid 6β hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form. © 1988.