Tamoxifen remains the endocrine therapy of choice in the treatment of all stages of hormone-dependent breast cancer. In addition, clinical trials are in progress to determine the potential of tamoxifen to act as a chemopreventive agent in women considered at high risk for developing breast cancer. However, several studies have raised concern over the safety of chronic treatment with this drug. Alternative SERMs may not be genotoxic because of different routes of metabolism that could lead to a decrease in amount and/or type of ultimate carcinogen(s). The central hypothesis of this project is that the formation of quinoids is an important mechanism of carcinogenesis and or cytotoxicity, for certain antiestrogens. For example, tamoxifen can be metabolized to three quinoids including two quinone methides and one o-quinone. The following specific aims are proposed: 1. Role of quinoids in the carcinogenic and cytotoxic effects of antiestrogens. We plan to examine the carcinogenic potential of quinoids formed from SERMs in cell lines. The biochemical effects of the antiestrogen quinoids will be investigated in human breast and endometrial cancer cell lines, which are either estrogen receptor positive or negative. 2. Investigate the effect of quinoids structure on electrophilic and/or redox reactivity. The rates of reaction of the SERM quinoids with water and GSH will be measured. Reactions of selected intermediates with either estrogenic or antiestrogenic activity with deoxynucleosides and DNA will also be investigated and adduct structures elucidated. Redox active metabolites will be tested by monitoring changes in the concentrations of reduced cofactors, determining the formation of reactive oxygen species, and examining oxidative damage to DNA. 3. Determine if the antagonist/agonist activity of antiestrogen metabolites correlates with the extent of DNA damage in cell lines. We predict that excessive binding to the estrogen receptor, which then translocates to the nucleus, will be correlated with an increase in DNA damage. The Ishikawa cell system will be used to determine the estrogenic or antiestrogenic effect of the SERM metabolites. The results from the Ishikawa cell experiments will be compared to studies measuring binding of the antiestrogen metabolites to the estrogen receptors. Cellular DNA from estrogen receptor positive and negative cells lines will be isolated after treatment with the test compound. The DNA will be hydrolyzed to deoxynucleosides and examined for DNA damage. Finally, we will determine the extent of DNA damage induced in vivo by the most carcinogenic/cytotoxic antiestrogen metabolites using the rat liver model. These studies will elucidate the relative importance of alkylation and free radical formation for each antiestrogen, thereby enabling correlations of reactivity with structure from which general principles influencing the behavior of antiestrogen reactive metabolites in cells will emerge.
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