Cell turnover is a key process involved in mammary gland development and the pathogenesis of tumor formation; hence we investigated if early prepubertal exposure to BPA and genistein treatment, alone and in combination, exerted effects on cell proliferation and apoptosis. At PND21, rats prepubertally exposed to genistein only exhibited a 50% increase in cell proliferation in mammary TEBs (p < 0.01), a slight but not significant decrease in apoptosis, and a significant increase in cell turnover (2.6-fold). These effects are consistent with previous reports of prepubertal genistein treatment inducing rapid mammary gland growth that enhanced mammary gland maturation and cell differentiation (at PND50) and decreased susceptibility for chemically-induced mammary cancer in adult rats [8–10]. On the other hand, there was no significant change in cell proliferation or apoptosis following prepubertal BPA only exposure at PND21. However, the combined BPA + Gen treatments significantly increased cell proliferation and decreased apoptosis, resulting in a 3.6-fold increase in cell turnover at PND21. Since the results of combinational BPA + Gen treatment are most similar to the outcomes observed for genistein only treatment, we hypothesize that genistein is able to exert its effects on mammary gland maturation and differentiation even in the presence of BPA. While cell proliferation and apoptosis were measured in PND21 rats, it is important to point out that the observed effects at this age may be a consequence of the accumulative effects that occurred from the direct presence of BPA and/or genistein on days 2–21.
Subsequently, we investigated cell proliferation and apoptosis in PND50 rats exposed prepubertally to BPA and genistein, alone and in combination. This is 30 days after the last treatment, and a time point that, due to metabolism, animals are free of circulating genistein and BPA. Nevertheless, mammary TEBs of rats that were exposed prepubertally to BPA alone had increased cell proliferation and cell turnover by 40% and 99%, respectively. However, prepubertal genistein only treatment significantly increased cell apoptosis and decreased cell turnover (55%). While BPA did not alter apoptosis and genistein did not affect cell proliferation, both single treatments changed the ratio of cell proliferation-to-apoptosis. Furthermore, the combination of BPA + Gen exposure on the ratio of cell proliferation to apoptosis was similar to genistein only treatment (decreases), suggesting that prepubertal genistein action could overcome the action of prepubertal BPA treatment towards susceptibility for carcinogenesis.
Because of the differential effects of BPA and genistein on cell proliferation and apoptosis observed on PND50, we utilized western blot analysis to investigate proteins and the pathways targeted by these chemicals. In the mammary glands of rats exposed prepubertally to BPA only, cleaved caspase-3, cleaved PARP and p21 were significantly down-regulated while p-Bad was significantly increased compared to control treated animals, intimating decreased apoptosis. A failure to undergo apoptosis whereby DNA damaged cells are allowed to replicate is widely believed to be a key event in cancer formation and progression [15, 16]. We believe the increase in mammary cancer that occurs in rats exposed prepubertally to BPA only and later to the DNA damaging carcinogen DMBA at PND50 falls in this category [9, 11, 12].
On the other hand, prepubertal genistein only treatment increased cleaved caspase-3, cleaved caspase-9 and cleaved PARP compared to the mammary glands of control treated rats. Therefore, differential regulation of cleaved caspase-3 and cleaved PARP was observed in comparing BPA and genistein treatments. The results of increased cleaved caspase-9 and cleaved PARP in genistein alone and BPA + Gen groups were consistent with our cell apoptosis data. PARP is a well-established substrate for caspase-3 , and cleaved PARP is considered to be a hallmark of apoptosis . Western blot detection of PARP cleavage has been used extensively as an indicator of apoptosis .
The PI3K pathway is an intracellular signalling pathway important in the regulation of apoptosis and cancer development, being overactive when the tumor suppressor PTEN is faulty or deficient . Also, playing a role in apoptosis are the Akt proteins, which promote growth factor-mediated cell survival both directly and indirectly. BAD is a pro-apoptotic protein of the Bcl-2 family. Akt could phosphorylate BAD on Ser136, which makes Bad dissociate from the Bcl-2/Bcl-X complex and lose the pro-apoptotic function . In regard to the action of these proteins, we found that BPA only, but not genistein only, treatment significantly increased the expression of the Akt-1, 2, and 3 isoforms, p-Bad and p-Akt, but decreased on PTEN compared with control treatment, suggesting that BPA could inhibit cell apoptosis by regulation of PTEN, Akt pathway, and p-Bad in rat mammary glands. On the other hand, Akt-1 and p-Akt were significantly decreased in the mammary glands of rats exposed to combinational BPA + Gen treatment compared to BPA only treatment, pointing to the protective effect of genistein over BPA for apoptosis over cell proliferation.
Steroid hormones can exert their actions by signalling through intracellular steroid hormone receptors. Regulation of steroid receptor action, including ER-α and ER-β, is complex due to a number of co-regulators, including steroid receptor co-activators, that can determine signalling specificity and intensity, resulting in pleiotropic biological effects [22, 23]. But, it is not only steroid molecules that act via these mechanisms. We have previously reported that prepubertal BPA exposure resulted in up-regulated SRCs1-3, without altering ER-α in the mammary glands of 50 day old rats . Here, we confirm those effects on the SRCs, plus demonstrate that prepubertal BPA exposure results in reduced expression of ER-β. ERβ has been shown to inhibit breast cancer cell growth by reducing cell proliferation and has been termed a tumor suppressor [24–26]. Decreased ER-β has been associated with proliferative preinvasive mammary tumors . So, the decreased expression of ER-β by BPA suggests increased susceptibility for chemically-induced mammary carcinogenesis in adult rats. Furthermore, increased expression of the SRCs could have a dual effect, one to enhance ERβ action as a suppressor of mammary cancer and the other to stimulate “normal” levels of ER-α to enhance tumor-promoting effects. In contrast, prepubertal genistein exposure resulted in down-regulated expression of the SRCs, leading to the opposite effect of BPA for carcinogenesis via SRC regulation, i.e. mammary cancer prevention. Consistent with the actions of genistein only, the SRCs were down-regulated in mammary glands of rats exposed to BPA + Gen, suggesting that genistein may reduce the potential of BPA’s carcinogenic effect towards mammary cancer development.
In summary, prepubertal BPA exposure had little direct effect on cell proliferation and apoptosis and altered protein expression of only one out of 15 proteins investigated at PND21. Nevertheless, in mature rats (30 days later) cell proliferation and the ratio of cell proliferation to apoptosis were significantly increased. Associated with this at PND50 are differential expressions of 15 proteins whose alterations are consistent with increased cell proliferation and reduced apoptosis. These data are supportive of prior reports of rats exposed in a similar manner to BPA prepubertally and at PND50 to DMBA developing more mammary tumors [11, 12]. We hypothesize that DNA damaged cells from DMBA exposure at PND50 that did not undergo apoptosis were allowed to undergo long-term rapid cell proliferation to promote mammary tumor development. Our collaborative epigenetic studies have shown that mammary epithelial cells from PND50 rats exposed prepubertally to BPA revealed concurrent repression in 10 genes homologous to the 16p11.2 loci . These findings suggest that this conserved cluster is susceptible to BPA-mediated repression in rat mammary epithelial cells and may lead to permanent silencing of the 16p11.2 loci, which in turn can effect cell cycle regulation. Also, there were another 75 rat genes found to be down-regulated.
Interestingly enough, the mechanism for genistein chemoprevention is linked to increased cell proliferation that takes place prepubertally. As evident here, prepubertal genistein exposure results in increased cell proliferation in epithelial cells of mammary TEBs in prepubertal rats. This is correlated with increased mammary gland maturation and differentiation of TEBs to lobules [8, 10]. While TEBs are the terminal ductal structures most susceptible to carcinogenesis in the rat mammary gland, lobules are the terminal ductal structures least susceptible to carcinogenesis . Furthermore, prepubertal genistein exposure results in increased apoptosis at PND50, which could reduce the number of DNA damaged cells.
Combinational exposures are considered by many to be rather complex to be studied, especially in vivo. For this reason, we selected two chemicals whose properties we have previously studied and have shown to have opposing outcomes as detailed above. While genistein has been shown to elicit estrogen-like properties, including stimulating cell proliferation at PND21 and promoting mammary gland maturation and cell differentiation, its toxicological properties are questionable or minimal, especially when administered orally. Several laboratory and epidemiological studies have demonstrated genistein and its natural and most abundant source, soy, to suppress mammary cancer [8–10, 29]. In this study we found that combinational BPA + Gen exposure, via the mothers’ milk, from PND2-21 resulted in increased cell proliferation and decreased apoptosis when investigated at PND21. Furthermore, three proteins presumed to be associated with these actions were down-regulated at PND21. At PND50, we found that combinational BPA + Gen exposure resulted in significantly decreased cell proliferation and increased apoptosis. These results were very similar to the actions of prepubertal genistein only exposure. Also, nine proteins associated with cell proliferation and apoptosis were differentially regulated in a manner consistent with suppressing cell proliferation and increased potential for apoptosis at PND50. We interpreted this to mean that genistein could protect against BPA predisposition for mammary carcinogenesis. Our data strongly suggest that the mechanism behind these cellular responses are centred on differential protein expressions of caspases, PARP, Bad, p21, Akts, PTEN, ER-β and SRCs 1–3 in the rat mammary gland. Studies are under way to determine if these cellular actions supported by appropriate changes in long-term protein expressions can be explained by epigenetic mechanisms and translated into genistein suppressing BPA predisposition for mammary cancer.