In laboratory animals, synthetic and natural estrogens are known to induce tumors. Previously, it was found that estrogens induce target-organ specific DNA damage preceding malignancy in hamster kidney. In this study, it will be attempted to prove that this DNA damage was caused by estrogens metabolically activated to reactive metabolites which then induced unknown endogenous substances to bind covalently to DNA. In particular, it will be attempted to a) clarify the structures of the estrogen-induced DNA adducts, b) determine the pathway of adduct formation, c) establish estrogen induced DNA adducts in human tissue and in animal models other than the Syrian hamster kidney as a cause of estrogen-induced cancer. Specifically the following experiments will be carried out: 1. Characterization of the structure and formation of DNA adducts. DNA adducts will be tested for structural features by chemical and enzymatic reactions. Differing DNA adduct patterns after (bio)chemical alterations will indicate the presence of unsaturated sites, carbonyles, lipid, carbohydrate, carboxyl groups, or other features of the estrogen-induced DNA adducts. Adducts will be synthesized in vitro. Reaction conditions will be varied to a) find in vitro conditions for DNA adduct formation, b) optimize adduct yield, c) obtain sufficient amounts of adducts for structure determination, d) define the conditions and pathway of adduct formation. 2. Increased adduct yields will be searched for in hamster kidney cell subpopulations and also in mammalian and yeast cell lines known to be transformed by estrogens. 3. The occurrence of estrogen-induced DNA adducts will be searched for in human tissues (target organ of estrogen-associated cancer) and in various rodent target organs. 4. Estrogen-induced DNA adduct patterns will be compared with adducts induced by redox-cycling agents (daunomycin) or radiation to investigate the role of redox cycling or radicals. 5. To understand the mechanism of adduct formation in vivo, hamster renal carcinogenesis and adduct formation will be modulated using enzyme inhibitors or antiestrogens. 6. The mechanism of activation of estrogens in hamster kidney by cytochrome P450, peroxidase, and/or prostaglandin endoperoxide synthase will be studied. Especially, the role of redox cycling and radical formation will be investigated.
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