Hydroxylation of pregnenolone at the 7 alpha- and 7 beta- 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 crystallization 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 KM values of 2.45 +/- 0.124 microM and 3.41 +/- 0.236 microM 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 1A2, 2D6, 2B1, and 2B11 were not 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 in liver and other organs.
Doostzadeh
J
J
Laboratoire de Biotechnologie, Conservatoire National des Arts et Métiers, Paris, France.
Cotillon
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Morfin
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D000818:Animals; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D012739:Gonadal Steroid Hormones / Q000737:chemistry / Q000494:pharmacology; D006900:Hydroxylation / Q000187:drug effects; D006914:Hydroxysteroids / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D011284:Pregnenolone / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D012739:Gonadal Steroid Hormones; D006914:Hydroxysteroids; D011284:Pregnenolone