THE LITHOCHOLIC ACID 6-BETA-HYDROXYLASE CYTOCHROME-P-450, CYP 3A10, IS AN ACTIVE CATALYST OF STEROID-HORMONE 6-BETA-HYDROXYLATION

CYP 3A10 is a hamster liver cytochrome P-450 (P450) that encodes lithocholic acid 6beta-hydroxylase, an enzyme that plays an important role in the detoxification of the cholestatic secondary bile acid lithocholate. Western-blot analysis revealed that the expression of CYP 3A10 protein is male-specific in hamster liver microsomes, a finding that is consistent with earlier analysis of CYP 3A10 mRNA. Since it has not been established whether the specificities of bile acid hydroxylase P450s, such as CYP 3A10, are restricted to their anionic bile acid substrates, we investigated the role of CYP 3A10 in the metabolism of a series of neutral steroid hormones using cDNA directed-expression in COS cells. The steroid hormones examined, testosterone, androstenedione and progesterone, were each metabolized by the expressed CYP 3A10, with 6beta-hydroxylation corresponding to a major activity in all three instances. CYP3A10-dependent steroid hydroxylation was increased substantially when the microsomes were prepared from COS cells co-transfected with NADPH:P450 reductase cDNA. In this case, the expressed P450 actively catalysed the 6beta-hydroxylation of testosterone (288 +/- 23 pmol of product formed/min per mg of COS-cell microsomal protein), androstenedione (107 +/- 19 pmol/min per mg) and progesterone (150 +/- 7 pmol/min per mg). Other major CYP 3A10-mediated steroid hydroxylase activities included androstenedione 16alpha-hydroxylation, progesterone 16alpha- and 21-hydroxylation, and the formation of several unidentified products. CYP 3A10 exhibited similar V(max) values for the 6beta-hydroxylation of androstenedione and lithocholic acid (132 and 164 pmol/min per mg respectively), but metabolized the bile acid with a 3-fold lower K(m) (25 muM, as against 75 muM for androstenedione). Together, these studies establish that the substrate specificity of the bile acid hydroxylase CYP 3A10 is not restricted to bile acids, and further suggest that CYP 3A10 can play a physiologically important role in the metabolism of two classes of endogenous P450 substrates: steroid hormones and bile acids.