Characterization of the NADPH-Dependent Metabolism of 17b-Estradiol to Multiple Metabolites by Human Liver Microsomes and Selectively Expressed Human Cytochrome P450 3A4 and 3A5

We characterized the NADPH-dependent metabolism of 17bestradiol (E2) by liver microsomes from 21 male and 12 female human subjects. A large number of radioactive estrogen metabolite peaks were detected following incubations of [H]E2 with male or female human liver microsomes in the presence of NADPH. The structures of 18 hydroxylated or keto estrogen metabolites formed by these microsomes were identified by gas chromatography/mass spectrometry analysis. 2-Hydroxylation (the formation of 2-OH-E2 and 2-OH-E1) was the dominant metabolic pathway with all human liver microsomes tested. The average ratio of 4-OH-E2 to 2-OH-E2 formation was ;1:6. A new monohydroxylated E2 metabolite (chemical structure unidentified) was found to be one of the major metabolites formed by human liver microsomes of both genders. 6b-OH-E2 and 16b-OH-E2 were also formed in significant quantities, but products of estrogen 16a-hydroxylation (16a-OH-E2 1 16aOH-E1) were quantitatively minor metabolites. In addition, many other estrogen metabolites such as 6-keto-E2, 6a-OH-E2, 7aOH-E2, 12b-OH-E2, 15a-OH-E2, 15b-OH-E2, 16b-OH-E1, and 16-keto-E2 were also formed in relatively small quantities. The overall profiles for the E2 metabolites formed by male and female human liver microsomes were similar, and their average rates were not significantly different. The activity of testosterone 6b-hydroxylation (a selective probe for CYP3A4/5 activity) strongly correlated with the rate of formation of 2-OH-E2, 4-OHE2, and several other hydroxyestrogen metabolites by both male and female liver microsomes. The dominant role of hepatic CYP3A4 and CYP3A5 in the formation of these hydroxyestrogen metabolites was further confirmed by incubations of selectively expressed human CYP3A4 or CYP3A5 with [H]E2 and NADPH. Endogenous estrogens [such as 17b-estradiol (E2) and estrone (E1)] can be hydroxylated at multiple positions (as illustrated in Fig. 1) by drug-metabolizing enzymes present in liver as well as in extrahepatic organs (reviewed by Martucci and Fishman, 1993; Zhu and Conney, 1998a). Cytochrome P450 (CYP) family enzymes are the major enzymes that catalyze the NADPH-dependent oxidative metabolism of estrogens to various hydroxylated or keto metabolites (Martucci and Fishman, 1993; Zhu and Conney, 1998a). In most animals as well as in humans, the hepatic tissues contain the highest levels of total CYP-dependent drug-metabolizing enzymes and possibly also the largest numbers of different CYP isoforms. By using a versatile HPLC separation method coupled with radioactivity detection, investigators recently showed that incubations of radiolabeled E2 with rat or mouse liver microsomes (a crude preparation containing many different CYP isoforms) resulted in the formation of at least 15 estrogen metabolites (Suchar et al., 1995, 1996; Zhu et al.,