Estrogens and their metabolites have been implicated in the etiology of breast cancer. Tamoxifen is an anti-estrogen that is used in the treatment of breast cancer patients. However, tamoxifen treatment has been associated with endometrial cancer, and has been shown to induce hepatocarcinoma in rodents. The hypothesis driving the proposed research postulates that estrogens and tamoxifen are weak carcinogens in humans, reflecting their ability to form covalent adducts with DNA. The goal of the proposed research is to establish the mutagenic potential of selected tamoxife and estrogen DNA adducts. Oligodeoxynucleotides containing a single defined DN adduct will be prepared from activated forms of estrogens and tamoxifen, or by chemical synthesis. The miscoding properties of these adducts will be quantified. The particular adducts to be studied include estrogen or tamoxifen adducts at the N2 position of Guanine and the N6 position of Adenine, and thes adducts will be generated using various different activated metabolites of estrogen and tamoxifen. Using mammalian DNA polymerases, steady state kinetics will be used to measure frequencies of nucleotide insertion opposite each lesion and chain extension from the 3' terminus. In this way the in vitro miscoding specificity for each adduct will be established. Miscoding propertie will be analyzed under steady-state conditions, using templates with the adducts in different sequence contexts, and using three different mammalian polymerases. Site specifically adducted oligos will also be used to build plasmids in order to establish the miscoding specificity of selected adducts i vivo, in mammalian cells. The proposed experiments are designed to provide quantitative information on the mutagenicity of selected estrogen and anti-estrogen DNA adducts. The tamoxifen DNA adducts prepared for in vitro experiments will also be employed for as standard markers in studies to detect adducts in the DNA of patients undergoing tamoxifen treatment; preliminary studies have established the presence of such adducts in endometrial tissue. Dr. Shibutani will explore the possibility of using the detection of tamoxifen DNA adducts as a biomarker to investigate the relationship between tamoxifen therapy and the development of endometrial cancer.
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