In vitro model of mammary estrogen metabolism: Structural and kinetic differences between catechol estrogens 2-and 4-hydroxyestradiol

Estrogens and their oxidative metabolites, the catechol estrogens, have been implicated in the development of breast cancer; yet, relatively little is known about estrogen metabolism in the breast. To determine how the parent hormone, 17beta-estradiol (E-2) is metabolized, we used recombinant, purified phase I enzymes, cytochrome P450 (CYP) 1A1 and 1B1, with the phase II enzymes catechol-O-methyltransferase (COMT) and glutathione S-transferase P1 (GSTP1), all of which are expressed in breast tissue. We employed both gas and liquid chromatography with mass spectrometry to measure E-2, the catechol estrogens 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2), as well as methoxyestrogens and estrogen-GSH conjugates. The oxidation of E-2 to 2-OHE2 and 4-OHE2 was exclusively regulated by CYP1A1 and 1B1, regardless of the presence or concentration of COMT and GSTP1. COMT generated two products, 2-methoxyestradiol and 2-hydroxy-3-methoxyestradiol, from 2-OHE2 but only one product, 4-methoxyestradiol, from 4-OHE2. Similarly, GSTP1 yielded two conjugates, 2-OHE2-1-SG and 2-OHE2-4-SG, from the corresponding quinone 2-hydroxyestradiol-quinone and one conjugate, 4-OHE2-2-SG, from 4-hydroxyestradiol-quinone. Using the experimental data, we developed a multicompartment kinetic model for the oxidative metabolism of the parent hormone E2, which revealed significant differences in rate constants for its C-2 and C-4 metabolites. The results demonstrated a tightly regulated interaction of phase I and phase II enzymes, in which the latter decreased the concentration of catechol estrogens and estrogen quinones, thereby reducing the potential of these oxidative estrogen metabolites to induce DNA damage.