) Studies of the role of estrogens (Es) in the etiology of breast cancer have focused on their receptor- mediated actions as mitogens. However, such actions of Es can not fully explain either laboratory or epidemiological data. However, Es can undergo P450-mediated metabolic activation to generate electrophilic species which are known initiators of carcinogenesis. This concept has evolved from studies of the biogenesis and characteristics of 2- and 4-hydroxylated catechol metabolites of Es (2- and 4-OH-CEs) which may damage DNA via oxygen free radicals generated by redox cycling of CEs or by causing depurinating adducts via their quinone metabolites. The mechanisms involved are analogous to those by which xenobiotic procarcinogens undergo P450-mediated metabolic activation to proximal carcinogens. The overall hypothesis states that 2- and 4-OH-CEs generated in the gland, in particular 4-OH-CEs, are important determinants of P450- mediated Es role in carcinogenesis, in particular, in the early initiating stages of the disease. According to this postulate P450s which can generate CEs and their quinone derivatives and support redox cycling of CEs provide an interface between chemical and hormonal carcinogenesis, since these forms of P450 Es are inducible by xenobiotics. The relevance of this postulate to breast cancer is supported by recent cytochemical findings that all of the forms of P450 required for such a mechanism are, in fact, expressed in ductal epithelial cells in the normal human breast, that is, in the cells of origin of most breast cancers. The forms of P450 identified include P45O1A1 and 3A4 which can catalyze formation of 2-OH-CEs and redox cycling of CEs, as well as recently identified P4501B1 which, in its dioxin-inducible form in MCF-7 cells, is an E-4-hydroxylase. This is the first P450 known which generates 4- OH-CEs preferentially, the class of CEs which the investigator proposes may play a special role in carcinogenesis since they are potent long acting Es and may be less readily inactivated than 2-OH-CEs. The specific goal of proposed experiments is to determine if the constitutive form of P4501B1 expressed in human mammary gland, like that induced by dioxins in cultured cells, is an E- 4-hydroxylase and the extent to which its sequence conforms to that induced by dioxin. Probes for screening cDNA libraries from human breast parenchyma will be obtained by polymerase chain reaction applied to reverse transcripts (RT/PCR) to RNA prepared from the same tissue and using asymmetrical primers based on the conserved sequences used to identified P4501B1 in human breast by in situ hybridization. The libraries will be screened with RT/PCR amplified probes to obtain full length sequence for P4501B1 which will then be used to produce P4501B1 protein in yeast expression vector for analysis for E-2- and E-4-hydroxylase activities. The investigator proposes to measure E-2- and 4- hydroxylase activity in samples of normal human breast parenchyma and assess the contribution made to these activities by different forms of P450 using specific antibodies. These studies may provide new information relevant to the etiology of breast cancer and focus attention on factors in normal human breast that may contribute in particular to early, initiating stages of carcinogenesis.
