Prior data obtained from siblings of the animals used in the present study revealed that prenatal exposure to environmentally relevant levels of BPA resulted in alterations of the reproductive tract and mammary gland that were manifested long after exposure ended. These alterations encompassed functional changes such as alteration of estrous cyclicity observed at 3 months of age (27
); morphological changes in the ovary, uterus, and vagina, also observed at 3 months of age (27
); and cellular changes, such as an increase in the ER- and PR-positive cells observed in the lining of the endometrium at 3 months of age (45
). In the mammary gland, an increase in the total area of the ductal tree and increases in the number of terminal ducts, terminal ends, and alveolar buds were observed at 6 months of age (34
The objective of the present study was to assess whether perinatal BPA exposure also affected the development of the mammary gland at puberty, a phenomenon initiated by the rise in estrogen plasma levels. During this period, the ductal tree undergoes growth by invasion of the stroma, accrual of new cells, and lumen formation; these processes are mediated by a highly dynamic structure, the TEB. We observed a positive correlation between ductal length from the center of the lymph node to the leading edge and the age at first proestrus; the slope of this curve was significantly reduced as the BPA dose increased. At 30 d of age, the number of TEBs relative to the ductal area increased significantly in animals exposed perinatally to BPA. Whereas the distal aspect of the TEB is the invasive organ, the solid primordium of body cells near the neck is involved in the formation of the subtending duct and its lumen, a process that is manifested by a high apoptotic rate (38
); the ductal lumen is formed as a consequence of this apoptotic activity. A large and significant decrease in the number of apoptotic cells was observed within the TEBs in animals of both BPA-treated groups. Hence, decreased cell death may be the factor underlying the impaired ductal elongation and the increased number and area of TEBs per ductal area in the BPA-treated animals. Cell proliferation, instead, seemed to be unaffected because no significant changes in the BrdU labeling index of epithelial cells were observed. However, a significant decrease of the labeling index of stromal cells was found in animals exposed to 250 ng BPA/kg bw·d. Because mammary gland morphogenesis is mediated by complex stromal-epithelial interactions, this result suggests that BPA also affects the stromal compartment and may disrupt important stromal-epithelial interactions.
The morphological changes found in 30-d-old animals exposed perinatally to BPA could be attributed, at least in part, to an increased sensitivity to estrogens. Indeed, the magnitude of the response to E2
was significantly enhanced in their siblings that were ovariectomized and exposed to E2
for 10 d. In particular, a significant increase in the number and area of TEBs relative to the ductal area was observed in these ovariectomized E2
-treated animals as well as in their intact siblings that were exposed perinatally to BPA. Although it seems counterintuitive that E2
would decrease ductal growth, we observed that the response of the normal mammary gland to E2
is biphasic, with low doses producing a larger effect than higher doses (Vandenberg L., personal communication). This nonmonotonic behavior was also observed regarding the percent area of the mammary gland occupied by the ductal tree (46
). Alternatively, BPA may affect ductal growth by acting on other yet-unknown end points in addition to increasing the sensitivity to estrogens. Indeed, recent evidence indicates a complex interaction among E2
, IGF-I, and progesterone on ductal morphogenesis (47
Progesterone is the main mediator of lateral branching and alveolar growth, a fact clearly illustrated by the PR null mutant mice (48
). Perinatal BPA exposure resulted in a significant increase in the number of epithelial cells expressing PR. This finding may result from the observed increased estrogen sensitivity of the mammary gland in BPA-exposed animals. In these animals, the ductal PR-positive cells often formed clusters. Lateral branching points and alveoli originate in these clusters (49
). In the normal mammary gland, the transition from uniform PR expression to PR clustering takes place between 8 and 12 wk of age (49
). Wnt 4 expression is a crucial event downstream from PR signaling in the lateral branching process (44
). Perinatal BPA exposure resulted in increased wnt4 mRNA levels at puberty, the only time point assayed; however, this increase did not reach significance. Nevertheless, morphometric analysis of the mammary glands at 4 months of age clearly revealed an increase in the number of lateral branches in the animals exposed to BPA.
These observations suggest that perinatal exposure to BPA significantly enhances the response to estrogens and increases the expression of PR. Furthermore, it suggests that the increased expression of PR may be the mechanism underlying the enhanced side branching observed at 4 months of age and the significant increase in the percentage of alveolar buds observed at 6 months of age (34
The mechanisms by which BPA affects the morphology of the mammary gland long after the period of exposure are largely unknown. One pathway may involve the direct action of BPA on the fetal mammary gland by altering the expression of genes that regulate mesenchymal-epithelial interactions and ductal morphogenesis (50
). Misexpression of these genes has been associated with mammary gland dysgenesis and carcinogenesis (51
regulates certain homeobox genes (51
); thus, it is plausible that BPA may also have this effect.
Prenatal exposure to DES down-regulates the expression of Wnt7a in the uterus (54
) and AbdB Hoxa in the müllerian duct (55
) and induces a posterior shift in Hox gene expression and homeotic anterior transformations of the reproductive tract (56
), and this correlates with structural abnormalities of these organs. The observation that the response to E2
was enhanced in ovariectomized animals exposed perinatally to BPA is compatible with this scenario. An additional pathway by which BPA may indirectly affect mammary gland development is by disrupting the hypothalamic-pituitary-ovarian axis. This would alter the secretory patterns of pituitary and ovarian hormones, which are important in postnatal mammary gland morphogenesis. The observations that perinatal exposure of rats to low-dose BPA reduces serum LH levels after ovariectomy in adulthood (57
), disrupts estrous cyclicity, and results in morphological changes in the ovary (27
) support the potential for alterations in the hypothalamus-pituitary-ovarian axis of BPA-exposed offspring.
What are the implications of these findings regarding human health? Surrogate animal models provide an understanding of human diseases. Although the relationship between the two is not always direct, surrogate models are most useful when used to develop hypotheses linking exposures and health outcomes. They also increase our understanding of the mechanisms underlying these pathologies. For instance, the mouse model has proven to be a generally outstanding model of human DES exposure, thus providing a means to understand the mechanisms underlying the DES syndrome. This excellent performance strengthens the human relevance of the current findings in mice. Within this context, it is useful to speculate how the findings described here may also apply to humans. On the one hand, exposure to estrogens is a main risk factor for the development of breast cancer in humans and also increases the development of mammary cancer induced by chemical carcinogens in rodent models (58
). Thus, the increased sensitivity to E2
suggests that prenatal exposure to BPA may increase the likelihood of neoplastic development. On the other hand, TEBs are the structures in which mammary cancer originates in both rodents and humans (59
). The increase in the number of TEBs/ductal area is also consistent with an increased risk of breast cancer. Another well-established risk factor for breast cancer is increased mammographic density. Mammographic density is attributed to an increased epithelial compartment and increased nonadipose stroma. The mammary glands of BPA-exposed animals contained significantly more ducts due to lateral branching at 4 months of age. Previous data obtained from sisters of animals in this study revealed that at 6 months of age, the number of all the epithelial structures were significantly increased (34
), including terminal ducts, i.e.
the structures in which neoplasia originates in adult animals (59
). These correlations suggest that perinatal exposure to BPA in particular, and to estrogens in general, may increase susceptibility to breast cancer. This hypothesis is being further tested in our laboratories.