Carcinogenic Metabolites formed from Antiestrogens

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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 which 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 qfCarcinogenesis andor cytotoxiciO, for certain antiestrogens. For example, tamoxifen can be metabolized to three inoids 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 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. PERFORMANCESITE(s)(organizationc, ity,state) Department of Medicinal Chemistry and Pharmacognosy University of Illinois at Chicago 833 S. Wood St. Chicago, IL 60612-7231 KEYPERSONNELS. eeinstructionosnPage11. Usecontinuationpagesasneededtoprovidetherequiredinformationintheformatshownbelow. Name Organization RoleonProject Judy L. Bolton, Ph.D. University of Illinois at Chicago P.I. John M. Pezzuto, Ph.D. University of Illinois at Chicago Co-Investigator Steven Swanson, Ph.D. University of Illinois at Chicago Co-Investigator Richard B. van Breemen, Ph.D. University of Illinois at Chicago Co-Investigator Emily Pisha, Ph.D. University of Illinois at Chicago Research Associate Linning Yu, B.S. University of Illinois at Chicago Research Assistant Fagen Zhang, Ph.D. University of Illinois at Chicago Research Associate Yanan Yang, B.S. University of Illinois at Chicago Research Assistant PHS398(Rev.4/98) Page2 g g Numberpagesconsecutivealyt thebottomthroughoutheapplication.Donotusesuffixesuchas 3a,3b. Principal Investigator/Program Director (Last, first, middle): Bolton, Judy L. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. Type density and size must conform to limits and specifications provided in the PHS 398 Instructions. RESEARCH GRANT TABLE OF CONTENTS _Numbe_ Face Page ......................................................................................................................................................... 1 2- 2 Description,