Although estrogen-induced cancer has been studied for many years, the mechanism is poorly understood. It has been demonstrated that endogenous compounds such as estrogens and dietary or environmental xenobiotics such as heterocyclic amines are metabolized to reactive compounds that form DNA adducts. Heterocyclic amines are formed when meat products such as beef, chicken, pork and fish are cooked at high temperatures. These amines, including 2-amino-l-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), have been shown to cause breast cancer. The current school of thought is that PhIP is metabolized by CYP1A2 in the liver to N-OH-PhIP. This reactive metabolite travels to the breast where it is further metabolized by sulfotransferases (SULT1E) to conjugates that are highly reactive and bind to DNA. It is difficult to conceive that N-OH-PHIP would bypass the many conjugation systems, scavengers, and binding proteins that are found in route from the liver to the breast without reacting with them. Therefore, the investigators hypothesize that breast tissue contains all of the machinery necessary to completely metabolize PhIP to DNA-binding metabolites. They will test this hypothesis by using a normal human breast cell line (MCF-10A). They hope to demonstrate that MCF-10A cells are capable of metabolizing PhIP and N-OH-PhIP to DNA-binding chemicals by using 32P post-labeling to assay for DNA adducts. Various inhibitors of CYP1A2 and SULT1E will be used to confirm that these enzymes are responsible for the metabolic conversion of PhIP to the reactive species. Since these inhibitors are nonspecific, they will be used genetically altered MCF-10A cells that overexpress and/or underexpress the CYP1A2 and SULT1E genes and assay them for their ability cause PhIP-DNA adducts. Finally, they will use the microarray analysis to determine differential gene expression induced by PhIP. These genes may be related to cancer. The results of this study will hopefully provide a better understanding of the mechanism of estrogen-induced cancer which would allow the formulation of better chemotherapeutic and chemopreventive strategies.
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