Formation of depurinating N3Adenine and N7Guanine adducts by MCF-10F cells cultured in the presence of 4-hydroxyestradiol

Metabolic conversion of endogenous estrogens, estradiol (E₂) and estrone (E₁), to the catechol estrogens 4-hydroxyE ₁(E₂) [4-OHE₁(E₂)] has been implicated in the initiation of cancer in rodents and humans. Evidence collected in our laboratories has shown that 4-OHE₁(E₂) are enzymatically oxidized to E₁(E₂)-3,4-quinones [E ₁(E₂)-3,4-Q], which have the potential to damage DNA by forming predominantly depurinating adducts, 4-OHE₁(E ₂)-1-N3Ade and 4-OHE₁(E₂)-1-N7Gua, leading to the accumulation of mutations and probably cell transformation. The human breast epithelial cell line MCF-10F has been transformed by treatment with E ₂ or 4-OHE₂. We have used MCF-10F cells to study the presence of adducts and conjugates after treatment with 4-OHE₂. To mimic the intermittent exposure of breast cells to endogenous estrogens, MCF-10F cells were treated with 1 μM 4-OHE₂ for a 24-h period at 72, 120, 192 and 240 h postplating. Culture media were collected at each point, extracted by solid-phase extraction and analyzed by HPLC connected with a multichannel electrochemical detector and/or ultraperformance liquid chromatography/tandem mass spectrometry. Media from successive treatments with 4-OHE₂ showed the formation of methoxy and cysteine conjugates, and the depurinating adducts 4-OHE₁(E₂)-1-N3Ade. The amount of 4-OHE₁(E₂)-1-N3Ade adducts was higher after the third treatment; smaller amounts of the 4-OHE₁(E₂)-1-N7Gua adducts were detected after the second and third treatments. These results demonstrate that MCF-10F cells oxidize 4-OHE₂ to E₁(E ₂)-3,4-Q, which react with DNA to form the depurinating N3Ade and N7Gua adducts. This DNA damage can play an important role in the 4-OHE ₂-induced mutations and transformation of MCF-10F cells to malignant cells.