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) × 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) × 10−5; A·T to G·C 70-73.3%). The estrogen-induced A·T to G·C mutations were detected as G·T heteroduplexes, as would be expected if N3Ade depurinations caused Gua misincorporations by erroneous repair. These heteroduplexes were identified by the T·G-DNA glycosylase (TDG) assay. TDG converts G·T heteroduplexes to G.abasic sites, rendering DNA templates refractory to PCR amplification. Consequently, A·T to G·C mutations present as G·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·T to G·C mutations down to pre-existing frequencies. Our results demonstrate that treatment with estradiol-3,4-quinone, an important metabolite of estrogens, produced A·T to G·C mutations in the DNA of the mammary gland of ACI rats.