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 -450 NF ( 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 P -450 NF or a rat homolog, P -450 PB-2a. Anti- P -450 NF 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 -450 NF gene subfamily ( P -450 IIIA subfamily). These observations establish that P -450 NF 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.