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 litho cholic acid 6beta-hydroxylase, an enzyme that plays an important role in the detoxification of the cholestatic secondary bile acid lithochol ate. Western-blot analysis revealed that the expression of CYP 3A10 pr otein is male-specific in hamster liver microsomes, a finding that is consistent with earlier analysis of CYP 3A10 mRNA. Since it has not be en established whether the specificities of bile acid hydroxylase P450 s, such as CYP 3A10, are restricted to their anionic bile acid substra tes, we investigated the role of CYP 3A10 in the metabolism of a serie s of neutral steroid hormones using cDNA directed-expression in COS ce lls. The steroid hormones examined, testosterone, androstenedione and progesterone, were each metabolized by the expressed CYP 3A10, with 6b eta-hydroxylation corresponding to a major activity in all three insta nces. CYP3A10-dependent steroid hydroxylation was increased substantia lly when the microsomes were prepared from COS cells co-transfected wi th NADPH:P450 reductase cDNA. In this case, the expressed P450 activel y catalysed the 6beta-hydroxylation of testosterone (288 +/- 23 pmol o f product formed/min per mg of COS-cell microsomal protein), androsten edione (107 +/- 19 pmol/min per mg) and progesterone (150 +/- 7 pmol/m in per mg). Other major CYP 3A10-mediated steroid hydroxylase activiti es included androstenedione 16alpha-hydroxylation, progesterone 16alph a- and 21-hydroxylation, and the formation of several unidentified pro ducts. CYP 3A10 exhibited similar V(max) values for the 6beta-hydroxyl ation of androstenedione and lithocholic acid (132 and 164 pmol/min pe r 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 hydr oxylase CYP 3A10 is not restricted to bile acids, and further suggest that CYP 3A10 can play a physiologically important role in the metabol ism of two classes of endogenous P450 substrates: steroid hormones and bile acids.