Abstract It has been speculated that the increasing incidence of breast cancer might be linked to the increased exposure to environmental synthetic estrogens, such as bisphenol A (BPA), which is a most pervasive chemical in modern life as a component of polycarbonate plastics and epoxy resins used widely for food and beverage containers and dental sealants. Perinatal exposure to low, environmentally relevant doses of BPA in rodents resulted in the induction of pre-neoplastic ductal hyperplasia, carcinoma in situ, and increased susceptibility to tumorigenesis. However, the underlying mechanism for these observations is unclear. The murine mammary stem cells (MaSCs) are present in fetal mammary rudiments and could be the putative targets for BPA-induced tumorigenesis. More recently, MaSCs of different lineages have been matched with different subtypes of breast cancer by their specific gene-expression signatures. We thus hypothesize that mammary gland exposed to BPA at a susceptible window may lead to its susceptibility to tumorigenesis through a MaSC and/or stem cell niche mediated mechanism. To test this hypothesis, we exposed 21-day old Balb/C mice to BPA by gavage at 25 µg/kg/day during puberty for 3 weeks, and then isolated primary mammary cells at different time points (6-week, 2 and 4-month) for MaSC quantification using an in vitro mammosphere formation and 3D-ECM sphere differentiation assay as well as the in vivo cleared mammary fat pad regeneration assay. Our findings indicate that low dose BPA exposure at puberty can accelerate puberty onset, increase lateral branches and hyperplasia in adult mammary glands. Further, a single oral dose of DMBA at 30 mg/kg administrated at 2-month old resulted in a 2.4-fold increase in hyperplasia of mammary glands harvested at 4-month old when compared with BPA treated group without DMBA. Most significantly, puberty BPA exposure increased luminal MaSCs during gland development and this resulted in an increase of luminal cells in adult glands. In addition, puberty BPA exposure also altered basal MaSCs in such a way that regenerated glands from these basal MaSCs yielded higher preneoplastic lesion than control basal MaSCs, indicating puberty BPA exposure render basal MaSCs more susceptible to transformation. We also found significant increase of gene expression in steroid hormonal receptors (e.g. ERs and PR) in the stromal compartment (or stem cell niche), which suggest that the changes we observed above for the luminal and basal MaSCs may be caused indirectly through altered stromal environment upon BPA exposure during early life stage. Future studies are necessary to differentiate the effect of BPA exposure on MaSCs or the MaSC niche. In conclusion, our study showed that BPA-induced morphogenesis changes in mammary gland had a stem cell origin. More importantly, our findings suggest that BPA-induced susceptibility of mammary glands to tumorigenesis may also have a stem cell origin. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-06.