Cytochrome P450 1B1-mediated estrogen metabolism results in estrogen-deoxyribonucleoside adduct formation

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 17beta-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-OHE(2)-1(alpha,beta)-N7-guanine (4-OHE2-N7-Gua) and 4-OHE(2)-1(alpha,beta)-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 (Km, 4.6+/-0.7 micromol/L; kcat, 45+/-1.6/h) was produced 1.5 times more efficiently than the 4-OHE2-N3-Ade adduct (Km, 4.6+/-1.0 micromol/L; kcat, 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.