The Effect Of Genistein In Utero Exposure On MMTV-ErbB-2 Transgenic Mice

Objective: In this study we examined the effect of in utero exposure to genistein on mammary gland development and tumorigenesis in MMTV-ErbB-2 transgenic mice, and investigated the underlying mechanisms.Methods: We treated erbB-2 transgenic mice (on AIN-93G diet) with different doses of genistein by subcutaneous injection of genistein containing peanut oil from day 13 to day 18 of gestation. The groups include: control (50μl of peanut oil alone), 20μg/day/mouse and 100μg/day/mouse (genistein in peanut oil). The effect of in utero treatment on the offspring was examined by sex ratio, body weight, female offspring's vaginal opening time and estrous cycle. Mammary gland morphology at 5 and 10 weeks of age was analyzed by whole mount and HE staining. To investigate the underlying mechanisms, signal transduction in ER and erbB-2 pathways was examined using Western blot, RT-PCR, Immunohistochemistry (IHC).Results: We found that in utero exposure to genistein induced earlier vaginal opening and disturbed estrous cycle patterns, genistein treated mice had a prolonged estrous stage as compared to the control. Morphology analysis of the whole mounts from the offspring at 5 weeks of age showed that in utero exposure to genistein induced enhanced proliferation and outgrowth of mammary glands, as indicated by longer duct extension and increased terminal end bud (TEB) numbers. At 10 weeks of age, the mammary glands in mice with in utero exposure to genistein, especially in the 20μg group, showed a dramatic expansion of the ductal network. More importantly, mice with in utero exposure to 20μg/mouse/day genistein developed tumors earlier than the mice in the control group (P<0.01), although tumor development in the 100μg group was not significantly altered(P>0.05). Mechanistic studies indicated that in utero exposure to genistein induced the expression of estrogen receptorα(ERα) and phosphorylation/activation of ERα, ERK and erbB-2 in mammary tissues. The proliferation in both terminal end buds and ducts is associated with ERα-positive cells. Conclusion: Our data demonstrated that in utero exposure to genistein induce pro-estrogenic effects in the female offspring, and exerted lifelong effects on the morphology of mammary gland. More importantly, in utero exposure to genistein may promote mammary tumor development in erbB-2 transgenic mice in a dose dependent manner. The underlying mechanisms may involve enhanced crosstalk between ER and erbB-2 pathways. It is also suggested that interactions between in utero exposure to certain phytoestrogens and genetic predisposition may play a role in breast cancer initiation, which can be a target for breast cancer prevention and management.