Comparison of cytochrome P-450 species which catalyze the hydroxylations of the aromatic ring of estradiol and estradiol 17-sulfate

Abstract For identification of microsomal cytochrome P -450 ( P -450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E 2 ) and estradiol 17-sulfate (E 2 -17-S) were selected as the substrates and incubated with various kinds of purified P -450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P -450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P -450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E 2 . The P -450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P -450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E 2 was converted to such metabolites as estrone and those having a hydroxyl group at positions 6β, 15α or 16α, each production of which was estimated to be catalyzed by single or multiple P -450s.