A number of risk factors, including dietary fat intake, family history, and exposure to exogenous and endogenous chemicals, have been implicated in the etiology of breast cancer incidence. Epidemiological studies have identified an association of elevated levels of estrogens with breast cancer development. In particular, the natural hormone, 17B-estradiol (E2) and its major metabolite, 4-hydroxyestradiol (4-E2), which are known animal carcinogens, have been implicated in human breast cancer and it is believed that these substances cause free radical-mediated, direct and/or indirect DNA damage. Novel, polar DNA adducts have been detected in the mammary tissues of both rats and humans by devising new systems to employ 32P-postlabeling/TLC. Adduct spectra between rat and human mammary DNA exhibited strong similarities. Further, the polar adducts were significantly enhanced in estrogen-sensitive tissues, particularly mammary, after treatment of S/D rats with estradiol. In light of the known redox-cycling activity of 4-E2, a major metabolite of estradiol, these results suggest a link between polar DNA adduct formation and metabolic processing of E2. We hypothesize that the polar DNA adducts detected in the mammary tissues originate from free radical mediation. We also hypothesize that significant inter-subject differences occur in the accumulation of pre-mutational DNA lesions, and these differences could in part explain the wide range of susceptibility seen in the induction of some cancers. Specific studies will be as follows: (1) To analyze DNA damage in mammary epithelium of rats treated with the endogenous estrogen and its metabolites, as well as the equine estrogen, equilin and equilenin. Blood lymphocytes will be included as surrogates for comparison. Antioxidant intervention experiments will be performed to support the hypothesis. Also measured will be the tissue and plasma levels of E2 and its metabolites by sensitive HPLC-CoulArray for correlating with oxidative DNA adduct burden. (2) To analyze DNA damage in epithelial cells of breast tissues of cancer-free women and breast cancer patients and surrogate blood lymphocytes, and blood lymphocytes of women receiving hormone replacement therapy. Tissue levels of E2 and its metabolites will be correlated with oxidative DNA adduct burden and breast cancer incidence. (3) To develop and apply LC-MS/MS and CE-MS/MS technology in conjunction with 32P-postlabeling for identifying DNA adducts in human breast tissues. Data resulting from this highly sensitive approach will reveal the etiological nature of exposure and will determine inter-subject differences in mammary DNA damage that may be associated with susceptibility differences. Furthermore, if tissue levels of estradiol metabolite(s) and/or oxidative burden are found to correlate positively with breast cancer incidence, then future clinical studies to reduce breast cancer risks can be planned with antioxidant intervention.
