Equine cytochrome P450 aromatase exhibits an estrogen 2-hydroxylase activity in vitro

Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water release from labelled estradiol and testosterone. Kinetic analysis of estradiol 2-hydroxylation showed an apparent K-m of 23 mu M and a V-max of 18 nmol/min/mg, whereas the values for testosterone aromatization were a K-m of 15.7 nM and a V-max of 34.6 pmol/min/mg. A specific antiserum raised against purified testicular equine P450arom and known to inhibit aromatase activity [1] was also found to inhibit the estrogen hydroxylase activity of equine placental microsomes in a dose-dependent manner with an IC50 value of 15 mu l serum: 0.5 ml incubate. The estrogen hydroxylase activity was inhibited in a dose-dependent manner by two classes of aromatase inhibitors, i.e. steroidal-(4-hydroxyandrostenedione and 7 alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione)-and non-steroidal-(fadrozole and miconazole). The IC50 values were approximately 300 and 890nM for 4-hydroxyandrostenedione and 7 alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione, and 92 and 285 nM, for fadrozole and miconazole, respectively. Furthermore, 4-hydroxyandrostenedione caused a time-dependent inactivation of estrogen hydroxylase activity. We conclude that equine aromatase is able to use estradiol as a substrate, and converts it to catechol estradiol in vitro, possibly using the active site of aromatization. This is the first demonstration that equine aromatase functions as an estrogen 2-hydroxylase, in addition to transforming androgens into estrogen. Copyright (C) 1996 Elsevier Science Ltd.