Hydroxylation of pregnenolone at the 7α- and 7β-positions by mouse liver microsomes.: Effects of cytochrome p450 inhibitors and structure-specific inhibition by steroid hormones

Hydroxylations of pregnenolone (PREG) at the 7 alpha- and 7 beta- positions have been reported in numerous murine tissues and organs, including liver, and the responsible cytochrome P450 (P450) species await identification. Using thin-layer chromatography and gas chromatography-mass spectrometry and crystaliization to constant specific activity, we report identification of 7 alpha-hydroxy-PREG and 7 beta-hydroxy-PREG metabolites produced in mouse liver microsomes and kinetic studies of their production with apparent K-M values of 2.45 +/- 0.124 mu M and 3.41 +/- 0.236 mu M for 7 alpha- and 7 beta-hydroxylation, respectively. Investigation of P450 inhibitors and of steroid hormone effects on both 7 alpha- and 7 beta-hydroxylation of PREG showed that 1) different P450 were involved because metyrapone and antipyrine inhibited solely 7 alpha- and 7 beta-hydroxylation, respectively; 2) P450 2A2, 2D6, 2B1, and 2B11 were nor responsible for 7 alpha and 7 beta-hydroxylation of PREG because respective specific inhibitors furafylline, quinidine, and chloramphenicol triggered no inhibition; 3) P450 1A1 was responsible for only part of the 7 beta-hydroxylation of PREG because alpha-naphthoflavone, which inhibits specifically P450 1A1, did not suppress entirely 7 beta-hydroxylation while ketoconazole, antipyrine, and metyrapone extensively decreased the 7 beta-hydroxylation; 4) comparison of these findings with those obtained with brain microsomes suggests that tissue-specific P450 species are responsible for the 7 alpha- and 7 beta-hydroxylation of PREG; and 5) 7 alpha-hydroxylation of PREG may be shared with other 3 beta-hydroxysteroids such as isoandrosterone, 5-androstene-3 beta, 17 beta-diol, and dehydroepiandrosterone, which acted in a competitive manner. Taken together, these findings will be of use for identification of the P450 species responsible for 7 alpha- and 7 beta-hydroxylation of PREG and for studies of their activities ill liver and other organs. (C) 1998 by Elsevier Science Inc.