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Sequential Action of Phase I and II Enzymes Cytochrome P450 1B1 and Glutathione S-Transferase P1 in Mammary Estrogen Metabolism

¹ Departments of Pharmacology and
² Pathology, Vanderbilt University, Nashville, Tennessee

The Phase I enzyme cytochrome P450 1B1 (CYP1B1) has been postulated to play a key role in estrogen-induced mammary carcinogenesis by catalyzing the oxidative metabolism of 17ß-estradiol (E₂) to catechol estrogens (2-OHE₂ and 4-OHE₂) and highly reactive estrogen quinones (E₂-2,3-Q and E₂-3,4-Q). The potential of the quinones to induce mutagenic DNA lesions is expected to be decreased by their conjugation with glutathione (GSH) either nonenzymatically or catalyzed by glutathione S-transferase P1 (GSTP1), a Phase II enzyme. Because the interaction of the Phase I and Phase II enzymes is not well defined in this setting, we prepared recombinant purified CYP1B1 and GSTP1 to examine their individual and combined roles in the oxidative pathway and used gas and liquid chromatography/mass spectrometry to measure the parent hormone E₂, the catechol estrogens, and the GSH conjugates. 2-OHE₂ and 4-OHE₂ did not form conjugates with GSH alone or in the presence of GSTP1. However, incubation of GSH and CYP1B1 with 2-OHE₂ resulted in nearly linear conjugation through C-4 and C-1 (i.e., 2-OHE₂-4-SG and 2-OHE₂-1-SG), whereas the reaction of 4-OHE₂ yielded only 4-OHE₂-2-SG. When CYP1B1 and GSTP1 were added together, the rate of conjugation was accelerated with a hyperbolic pattern of product formation in the order 4-OHE₂-2-SG > 2-OHE₂-4-SG >> 2-OHE₂-1-SG. Incubation of E₂ with CYP1B1 and GSTP1 resulted in the formation of 4-OHE₂, 2-OHE₂, 4-OHE₂-2-SG, 2-OHE₂-4-SG, and 2-OHE₂-1-SG. The production of GSH-estrogen conjugates was dependent on the concentrations of E₂ and GSTP1 but overall yielded only one-tenth of the catechol estrogen production. The concentration gap between catechol estrogens and GSH-estrogen conjugates may result from nonenzymatic reaction of the labile quinones with other nucleophiles besides GSH or may reflect the lower efficiency of GSTP1 compared with CYP1B1. In summary, both reactions are coordinated qualitatively in terms of product formation and substrate utilization, but the quantitative gap would leave room for the accumulation of estrogen quinones and their potential for DNA damage as part of estrogen-induced mammary carcinogenesis.

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