Environmental exposure is a significant contributor to 85% of all breast cancers. It is difficult to establish that an environmental chemical is carcinogenic in humans. The National Institute of Environmental Health Sciences (NIEHS) in the U.S. is charged with determining potential cancer risk for humans from chemicals. Such a determination requires both experimental data, usually an expensive 2-year induced rodent carcinogenesis assay, and epidemiological data in humans. We have developed a unique method of culturing human breast epithelial cells, both normal and malignant, and propose to use non-tumor adjacent cell explants as the basis for development of an assay that tests environmental chemicals directly for the ability to transform breast cells into malignancy. We will adapt the rodent assay by using naturally mutated non-tumor adjacent cells from outside breast tumors. These human cells share mutations with matched breast tumors but are only a few steps away from tumor formation. To these cells we will provide rounds of exposure to a known carcinogen, resulting in a cell population that is one step away from transformation. We will then test putative carcinogens for their ability to complete this carcinogenic pathway as tumor promoters, resulting in tumor foci in culture. We will use this new transformation assay to test 3 environmental chemicals found in Florida. Chrysene, a product of combustion (eg. tobacco smoking) has been found in Florida soil. Trichloroethylene is present in Florida Superfund sites, and benzene is a contaminant from the Deepwater Horizon spill. This new test will more inexpensively establish that a chemical can transform a human breast epithelial cell to malignancy in culture. To ensure that this transformation is consistent with breast tumorigenesis, we will analyze the gene expression from foci that form after exposure and compare these to stage I tumor cultures we have established.