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Cytochrome P450 1B1–Mediated Estrogen Metabolism Results in Estrogen-Deoxyribonucleoside Adduct Formation

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

Requests for reprints: Fritz F. Parl, Department of Pathology, Vanderbilt University Medical Center, TVC 4918, Nashville, TN 37232. Phone: 615-343-9117; Fax: 615-343-9563; E-mail: fritz.parl{at}mcmail.vanderbilt.edu.

The oxidative metabolism of estrogens has been implicated in the development of breast cancer; yet, relatively little is known about the mechanism by which estrogens cause DNA damage and thereby initiate mammary carcinogenesis. To determine how the metabolism of the parent hormone 17ß-estradiol (E₂) leads to the formation of DNA adducts, we used the recombinant, purified phase I enzyme, cytochrome P450 1B1 (CYP1B1), which is expressed in breast tissue, to oxidize E₂ in the presence of 2'-deoxyguanosine or 2'-deoxyadenosine. We used both gas and liquid chromatography with tandem mass spectrometry to measure E₂, the 2- and 4-catechol estrogens (2-OHE₂, 4-OHE₂), and the depurinating adducts 4-OHE₂-1(,ß)-N7-guanine (4-OHE₂-N7-Gua) and 4-OHE₂-1(,ß)-N3-adenine (4-OHE₂-N3-Ade). CYP1B1 oxidized E₂ to the catechol 4-OHE₂ and the labile quinone 4-hydroxyestradiol-quinone to produce 4-OHE₂-N7-Gua and 4-OHE₂-N3-Ade in a time- and concentration-dependent manner. Because the reactive quinones were produced as part of the CYP1B1-mediated oxidation reaction, the adduct formation followed Michaelis-Menten kinetics. Under the conditions of the assay, the 4-OHE₂-N7-Gua adduct (K_m, 4.6 ± 0.7 µmol/L; k_cat, 45 ± 1.6/h) was produced 1.5 times more efficiently than the 4-OHE₂-N3-Ade adduct (K_m, 4.6 ± 1.0 µmol/L; k_cat, 30 ± 1.5/h). The production of adducts was two to three orders of magnitude lower than the 4-OHE₂ production. The results present direct proof of CYP1B1-mediated, E₂-induced adduct formation and provide the experimental basis for future studies of estrogen carcinogenesis. [Cancer Res 2007;67(2):812–7]

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