title: Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme.
abstract: Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-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 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-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 less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta 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 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta 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 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: D J
authors.initials: DJ
authors.lastname: Waxman
authors.forename: C
authors.initials: C
authors.lastname: Attisano
authors.forename: F P
authors.initials: FP
authors.lastname: Guengerich
authors.forename: D P
authors.initials: DP
authors.lastname: Lapenson
mesh_terms: D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D003429:Cross Reactions; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System; D005260:Female; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D051381:Rats; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism; D014217:Troleandomycin / Q000378:metabolism
chemical_list: D013739:Testosterone; D000735:Androstenedione; D011374:Progesterone; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D014217:Troleandomycin; D013250:Steroid Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C022657:steroid hormone 6-beta-hydroxylase