Induction of A•T to G•C mutations by erroneous repair of depurinated DNA following estrogen treatment of the mammary gland of ACI rats

Evidence suggests that the genotoxic mechanism of estrogens (estrone/estradiol) in breast cancer involves their oxidation to 3,4-quinones and reaction with DNA to form depurinating N3Ade and N7Gua adducts. We examined whether estrogen genotoxicity is mutagenic in the mammary gland of the female ACI rat, a model for estrogen-dependent breast cancer. Mutagenesis was studied by PCR amplification of the H-ras1 gene (exons 1-2), cloning in pUC18, transforming Escherichia coli, and sequencing the inserts in plasmids from individual colonies. Mammary glands of both estrogen-responsive (ACI and DA) and resistant (Sprague-Dawley) rats contained pre-existing mutations at frequencies of (39.8-58.8) x 10(-5), the majority (62.5-100%) of which were A-T to G-C transitions. Estradiol-3,4-quinone (200 nmol) treatment of ACI rats caused rapid (6 h to 1 day) mutagenesis (frequency (83.3-156.1) x 10(-5); AT to G-C 70-73.3%). The estrogen-induced A center dot T to G center dot C mutations were detected as G center dot T heteroduplexes, as would be expected if N3Ade depurinations caused Gua misincorporations by erroneous repair. These heteroduplexes were identified by the T center dot G-DNA glycosylase (TDG) assay. TDG converts G center dot T heteroduplexes to G.abasic sites, rendering DNA templates refractory to PCR amplification. Consequently, A center dot T to G center dot C mutations present as G center dot T heteroduplexes in the DNA are eliminated from the spectra. TDG treatment of mammary DNA from estradiol-3,4-quinone-treated ACI rats brought A center dot T to G center dot C mutations down to pre-existing frequencies. Our results demonstrate that treatment with estradiol-3,4-quinone, an important metabolite of estrogens, produced A center dot T to G center dot C mutations in the DNA of the mammary gland of ACI rats. (c) 2006 Elsevier Ltd. All rights reserved.