The goal of this project is to elucidate the role of five enzymes in the metabolic processes we think are involved in activation/deactivation of estrogens in the pathway leading to tumor initiation. We have hypothesized that estrogens initiate breast, prostrate and other human cancers by reaction of catechol estrogens-3,4-quinones (CE-3,4-Q) with DNA to form adducts that lead to oncogenic mutations. In this process, the estrogens, estrogen and 17beta-estradiol are metabolized to the 4- catechol estrogens (4-CE), which are further oxidized to CE-3,4-Q. Hydroxylation of estrogens to 2-CE is catalyzed by cytochrome P450 (CYP) 1A1. Hydroxylation of estrogens to 4-CE is catalyzed by the activating enzyme cytochrome CYP1B1, but further oxidation of the 4- CE to CE-4,4-Q is usually blocked by catechol-O-methyltransferase (COMT)-catalyzed methylation of the 4-CE, a deactivating step. The CE- Q are reduced to CE by quinone oxidoreductase (QOR), a deactivating event. Another key event may be the conversion of androgens to estrogens by CYP19, aromatase. To complement the information gained in Project 1, we plan to investigate two of the factors that can affect the activity of these enzymes in human tissues: the level of expression of the enzyme and its genetic variants. We propose to accomplish the following specific aims: (1) Assay the expression of aromatase, CYP1A1, CYP1B1, QOR and COMT in the mammary gland of female ACI rats and prostate gland of male Noble rats by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and by their catalytic activity in microsomes. (2) Assay the expression of aromatase, CYP1A1, CYP1B1, QOR and COMT by RT-PCR and their polymorphic variations in human breast and prostate samples obtained from women and men with and without breast or prostate cancer. The results of these studies will provide evidence on the role of the five enzymes in the initiation of breast and prostate tumors, as well as complementing the information gained in Project 1. These findings will help us understand the role of estrogens in initiation cancer and form the basis of developing preventative strategies.
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