Prolonged exposure to estrogens is actively involved in the development of breast cancers. Available data suggest that the mechanism behind the formation of tumors by estrogens is complex and not well understood. An important part in estrogen-induced carcinogenesis is the growing awareness that reactive oxygen species may serve as signaling agents in carcinogenic pathways. The proposed study seeks to establish the molecular mechanism(s) underlying the role of oxidative stress in estrogen-induced carcinogenesis in an animal model relevant to human breast cancers - namely, the female ACI rat model of estrogen-induced breast cancer - and, by using the human breast epithelial cell line MCF-10F, thus establish the importance of oxidative stress in estrogen-induced breast cancer. Therefore, the investigators will characterize: the role of estrogen metabolism-mediated oxidative stress (Specific Aim 1); the role of estrogen receptors (ERs) in the generation of oxidative stress (Specific Aim 2); the role of oxidative stress in modifying ER-dependent gene regulation (Specific Aim 3); and, the role cytochrome P450 1B1 in estrogen-induced oxidative stress and subsequent carcinogenesis (Specific Aim 4). To accomplish Specific Aims 1-4, rats will be treated with estrogens, anti-estrogens, antioxidants, or with inhibitors of metabolic activation. In in vitro approaches, MCF-10F cells will be genetically manipulated with gene insertions and gene knockouts. These cell lines will then be treated with estrogens, anti-estrogens, antioxidants or inhibitors of metabolic activation to study the roles of estrogen receptors and antioxidant defense genes in the regulation of estrogen-dependent oxidant stress. Treatment of animals will be for seven months and information obtained from groups of animals that have been sacrificed at earlier times will be used to study changes during early treatment periods, as well as during both preneoplastic and neoplastic phases of development. Tumor incidence and histopathology will be characterized. Markers of oxidative stress as well as levels of antioxidant defense enzymes will be quantified both in the target organ breast and the nontarget organ liver; also, expression and regulation of estrogen-estrogen receptor-dependent genes will be quantified. Given the federal register's recent inclusion of estrogens as human carcinogens, the proposed studies have a great potential in the development of therapeutic strategies for the treatment of estrogen-induced neoplasia. ? ? ?
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