Many PAHs are potent mammary carcinogens in rodent models, but their role in human breast cancer is poorly understood. We have cloned cytochrome P4501B1 (CYP1B1) and have shown that CYP1B 1-null mice are resistant to several cancers, which are induced by PAHs in normal mice. We will test the hypothesis that CYP1B 1 plays a major role in PAH-induced breast cancer, and that the contribution is greater than for CYP1A1 for one or more of the following reasons: (1) CYP1B1 is constitutively expressed in breast epithelia, whereas CYP1A1 requires induction by the PAH, via the Ah-receptor (AhR); (2) CYP1B1, which is also via the AhR, is expressed in multiple breast cell types, including luminal and basal epithelia and a highly proliferative progenitor cell population that may be very susceptible to transformation. CYP1A1 is primarily expressed in more differentiated luminal epithelia; (3) PAHs are preferentially metabolized to carcinogenic dihydrodiol epoxides (PAHDE) by CYP1B1 relative to CYP1A1. Human primary breast epithelia from reduction mammoplasty tissues will be resolved into the respective cell types. The PAHs to be examined are: dibenzo[a,l]pyrene (diBP), representative of the most potent fjord class; benzo[a]pyrene (BP), a bay-region PAH; and 7,12-dimethylbenz[a]anthracene (DMBA), a pseudo-fjord PAR. Metabolism and PAHIDE-DNA adduct formation will be analyzed in mice (wild type and CYP1B 1-null) in vivo in relation to mammary cancer development following BP, DMBA, and diBP exposure. Similar metabolic analyses will be completed in separated primary human breast epithelia for comparison of CYP1B1 and CYP1A1 expression and AhR activation. The role of CYP1B 1 will be further defined by anti-sense suppression or over-expression in the near normal human breast epithelial cell line, MCF-10F. These metabolically activated PAHs cause growth arrest at low concentrations. We will identify markers of this response, and we will examine the role of CYP1B1 in PAHDE-DNA adduct formation. The transformation of MCF-10F cells (including expression variants) to cells that form anchorage-independent colonies will be used to test the involvement of CYP1B 1 in these changes, which correlate with the early stages of carcinogenesis. This work will examine PAR activation for the first time in well characterized primary human breast cells that are cultured to replicate in vivo ductal morphology. The correlation with CYP1B 1 expression and activity will provide insight into the relationship of the mouse model to human breast cancer.
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