Cyp7b1 catalyses the 7α-hydroxylation of dehydroepiandrosterone and 25-hydroxycholesterol in rat prostate

Dehydroepiandrosterone (DHEA) is the most prominent circulating steroid in humans, and it is a precursor for sex-steroid synthesis in peripheral tissues. including the prostate, Recently, enzyme-mediated pre-receptor metabolism has been recognized as a key step in determining steroid action in vivo. Hydroxylation of 3 beta -steroids at the 7 alpha -position has been reported in rat and human prostate to be a major inhibitory pathway to sex-steroid synthesis/action. However, the molecular identity of the enzyme responsible is so far unknown. We recently described a novel cytochrome P450 enzyme, cyp7b1, strongly expressed in the hippocampus of rodent brain, which catalyses the metabolism of DHEA, pregnenolone and 25-hydroxycholesterol to 7 alpha -hydroxy products. In the light of this new enzyme, we have examined its possible role in 7 alpha -hydroxylation conversion in rat prostate. NADPH-dependent 7 alpha -hydroxylation was confirmed for 3 beta -hydroxysteroids including DHEA and androstenediol, as well as 25-hydroxycholesterol. Kinetic analysis yielded an apparent K, of 14 +/-1 muM for rin-hydroxylation of DHEA in the prostate gland, a value similar to that recorded for recombinant cyp7b1 enzyme [13.6 muM; Rose, Stapleton, Dott, Kieny, Best, Schwarz, Russell, Bjoorkheim, Seck1 and Lathe (1997) Proc. Natl, Acad, Sci. U.S.A. 94, 4925-4930]. The V-max value of the prostate was 46 +/-2 pmol/min per mg, and this activity was inhibited by clotrimazole, a P450-enzyme blocker. Moreover, RNA analysis (reverse-transcription PCR, Northern blotting and in situ hybridization) revealed a high expression of cyp7b1 mRNA in the rat prostate, restricted to the epithelium. suggesting that cyp7b1 catalyses oxysterol 7a-hydroxylation in the prostate gland.