Effects of cytochrome P450 inhibitors and of steroid hormones on the formation of 7-hydroxylated metabolites of pregnenolone in mouse brain microsomes.
Hydroxylations of pregnenolone (PREG) at the 7 alpha- and 7 beta-positions have been reported in numerous murine tissues and organs and responsible cytochrome P450 (CYP) species await identification. Using thin layer chromatography and gas chromatography-mass spectrometry, we report identification of 7 alpha-hydroxy-PREG and 7 beta-hydroxy-PREG metabolites produced in mouse brain microsome digests and kinetic studies of their production with apparent KM values of 0.5 +/- 0.1 microM and 5.1 +/- 0.6 microM for 7 alpha- and 7 beta-hydroxylation respectively. Investigation of CYP inhibitors and of steroid hormone effects on both 7 alpha- and 7 beta-hydroxylations of PREG showed that: (i) different CYP were involved in 7 alpha- and 7 beta-hydroxylation of PREG because solely 7 alpha-hydroxylation was extensively inhibited by metyrapone, alpha-naphthoflavone, ketoconazole and 3 beta-hydroxysteroids, (ii) CYP 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, (iii) CYP 1A1 was responsible for only part of the 7 beta-hydroxylation of PREG because use of alpha-naphthoflavone, which inhibits specifically CYP 1A1, did not suppress entirely 7 beta-hydroxylation, while ketoconazole, metyrapone and antipyrine, which do not inhibit CYP 1A1, decreased part of the 7 beta-hydroxylation, (iv) 7 alpha-hydroxylation of PREG may be shared with other 3 beta-hydroxysteroids such as isoandrosterone and 5-androstene-3 beta,17 beta-diol which were strong inhibitors, but not with dehydroepiandrosterone which was a non-competitive inhibitor as weak as 3-oxosteroids, and (v) 7 beta-hydroxylation of PREG was not markedly changed by other steroids. Taken together, these findings will be of use for identification of the CYP species responsible for 7 alpha- and 7 beta-hydroxylation of PREG and for studies of their activities in brain.
Doostzadeh
J
J
Laboratoire de Biotechnologie, Conservatoire National des Arts et Métiers, Paris, France.
Morfin
R
R
D000738:Androsterone / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000983:Antipyrine / Q000494:pharmacology*; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D065607:Cytochrome P-450 Enzyme Inhibitors; D003687:Dehydroepiandrosterone / Q000494:pharmacology; D008401:Gas Chromatography-Mass Spectrometry; D006900:Hydroxylation; D008297:Male; D008797:Metyrapone / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism*; D011284:Pregnenolone / Q000378:metabolism*
D065607:Cytochrome P-450 Enzyme Inhibitors; D003687:Dehydroepiandrosterone; D011284:Pregnenolone; D000738:Androsterone; D000983:Antipyrine; D008797:Metyrapone