Increased breast cancer incidence has prompted scientists to consider the possible role of hormonally active environmental chemicals. However, epidemiological studies searching for correlations between adult environmental exposures and breast cancer incidence have been mainly inconclusive. Yet, epidemiological studies do suggest that fetal estrogen level fluctuations have long-term consequences on the risk of developing breast cancer as an adult. Studies in mice reveal that prenatal exposure to low doses of the xenoestrogen bisphenol A (BPA) alters the development of these mammary glands. These effects manifested long after exposure ceased. The increased number of terminal end buds and terminal ducts in these mammary glands is particularly relevant since carcinomas originate in these structures. We hypothesize that prenatal exposure to low doses of BPA may increase the risk of mammary cancer and that the mechanism responsible for the neoplastic outcome may include the extemporaneous expression of genes that mediate mammary gland development. Xenoestrogens administered prenatally may alter the expression of estrogen-responsive genes that would then affect downstream genes in the mammary development program and predispose these animals to cancer. To test this hypothesis we will use a rat model whereby exposure to the carcinogen nitroso-methyl-urea (NMU) at puberty induces mammary cancer. A pilot study performed using Wistar rats showed that this model could be modified to assess the effect of fetal exposure to environmentally relevant xenoestrogen levels. It produced data consistent with the proposed hypothesis. Aim 1: To determine the levels at which prenatal BPA exposure results in an increased incidence of mammary cancer. Tumor incidence and latency will be measured in Wistar rats exposed to BPA from gestational day 9 to birth. At 50 days of age, animals will be injected with a sub-carcinogenic dose of NMU. Aim 2: To test the hypothesis that prenatal BPA exposure alters gene expression in the mammary gland during both the period of BPA exposure and throughout life. Total RNA will be isolated from mammary glands and analyzed by DNA microarray technology. Where appropriate, laser-capture microdissection and/or RNA linear amplification techniques will also be used to obtain samples. Up- and down-regulated genes will be confirmed by QRTPCR. Cellular distribution will be assessed by in situ hybridization. Developmental points to be analyzed are: 1) during gestational BPA exposure; 2) 5 days postnatal; 3) peripubertal; and 4) after ductal development is completed. Aim 3: To test the hypothesis that in utero exposure to BPA alters the histoarchitecture of the rat mammary gland and results in the expression of pre-neoplastic phenotypes. This Aim will assess whether exposure to low doses of BPA results in specific morphological alterations in the rat mammary gland that may confer a propensity to carcinogenesis, such as an increase in the number of terminal end buds (TEBs) and terminal ducts (TDs) at the time of NMU administration), the persistence of TEBs beyond puberty, and the appearance of pre-neoplastic lesions at several age points. In addition, the histoarchitecture of the mammary gland at the time-points studied in Aim 2 will be examined. Aims 1, 2 and 3 are intimately linked, because they seek to study the same phenomenon at different levels of biological organization. Gene expression alterations may suggest histoarchitectural consequences and vice-versa. This exploratory research is central to the generation of testable hypotheses about cause-effect relationships linking prenatal hormonal exposure and mammary gland carcinogenesis and may finally provide the direct link between environmental exposures and incidence of breast cancer researchers have been looking for over the past 20 years. Moreover, if, as suspected, low doses of BPA increase the incidence of mammary cancer, this work may have a great impact on the way we study risk factors and conduct epidemiological studies. It may even influence public policy about prevention.