Here we report that prenatal exposure to BPA increased tumor susceptibility in a dose-dependent manner in a DMBA-induced model of mouse mammary gland carcinogenesis. We also found not only that BPA exposure during early developmental stages increases susceptibility to mammary gland cancer but also that adult exposure directly promotes growth of estrogen dependent tumors in vivo.
In our first model of mammary gland cancer susceptibility, we utilized FVB/N mice. FVB/N, an inbred strain, demonstrates sensitivity to the formation of mammary gland cancer, unlike other models, such as C57BL/6, which are resistant to such tumors [34
]. For this reason, the FVB/N strain has been used extensively for analysis of mammary gland morphogenesis and tumor susceptibility [35
]. Characterization of BPA-induced tumor susceptibility in an inbred strain such as FVB/N should greatly facilitate identifying molecular mechanisms involved because genetic contributors to this process can be readily examined. This contrasts with previous studies utilizing outbred mice because genetic variability across these strains adds a further level of complexity that can hamper precise analyses of molecular mechanisms. Most important, we have found that prenatal exposure of this strain to BPA results in a substantial increase in carcinogen-mediated tumor susceptibility. Determining whether BPA also increases susceptibility to oncogene-induced tumorigenesis will be essential for examining whether the increase in susceptibility is restricted to a DNA damaging agent or whether other tumorigenic events are also facilitated by the BPA-induced molecular changes that occur during early mammary gland development.
Potential routes of human exposure to BPA are being discovered continually, such as the recent study demonstrating the potential for BPA leaching from printed receipts [46
]. As the primary route of human exposure is ingestion, we exposed mice to BPA by oral gavage. When administered in this manner, BPA is subject to first-pass metabolism in the liver, and remaining BPA is deposited throughout the body. The timing, as well as route of exposure, is critical; hence, we chose the prenatal time period during which development of the mammary gland is initiated. In mice, mammary gland development begins at E10, when the anlage of the mammary gland begins to become visible as a small placode, with a small epithelial bud forming around Day 12.5 [37
]. We began treatment at E8, well before this critical time point, and continued daily exposure until birth. BPA crosses the placental barrier because it can be measured in the amniotic fluid and fetal serum [47
], and conjugated forms from the mother can also be reactivated in the fetus [29
]. Exposure of the fetus to BPA could potentially be more detrimental than adult exposure because of critical windows of fetal development that are particularly susceptible to endocrine disruption [50
]. The period of anlagen morphogenesis has been described as one such period of susceptibility for the mammary gland [51
In addition to the potential to cross the maternal/placental barrier and accumulate in the fetus, BPA has been shown to have pathological effects at very low doses. It follows a nonmonotonic dose-response curve. The dose-response curve forms an inverted-U shape, with very low and high doses of BPA eliciting profound responses in vivo [52
]. To this end, we chose a low dose (25 μg kg−1
), which is half of the EPA recommended daily safe dose, and a high dose (250 μg kg−1
), which is 10 times higher than that of our low dose. Both of these doses have previously been utilized in other mouse models of BPA exposure where perturbation of the reproductive track has been demonstrated [13
]. At both the low and the high doses of BPA delivered by oral gavage, we observed early vaginal opening, an indicator of the onset of puberty in mice. These data confirm previous studies indicating that fetal BPA exposure alters the timing of female puberty and confirm that the exposure paradigm was consistent with previous reports. However, the impact of BPA exposure on the puberty did not translate to morphological differences in the mammary gland during the ages we investigated. By 8 wk of age, the mammary tree had filled in the fat pad in both cohorts, and there was no significant difference in ductal length from the lymph node in 3- and 5-wk-old mice. These results differ from similar rodent experiments that have previously been reported. In several studies, prenatal and perinatal exposure to BPA by osmotic pump results in marked differences in mammary gland development, including differences in ductal invasion, number of ducts, and terminal end and alveolar buds in adult female CD1 mice [25
]. Prenatal exposure by osmotic pump also increases ductal area and ductal extension in D18 embryonic mammary glands in CD-1 mice [27
]. Morphological changes have also been reported for female Sprague-Dawley rats that were prenatally exposed to BPA [20
]. Similar to the mammary gland, our study revealed that no overt effects of prenatal low- or high-dose BPA were observed on the morphology or histology of the ovaries. Other groups have previously demonstrated an effect of BPA exposure on the ovary, specifically reporting a disruption of oogenesis and an impact on meiosis, resulting in an increase in aneuploid oocytes and embryos [18
]. While we did not collect information on the number and types follicles, the mice that were exposed in utero to BPA were mated and were able to become pregnant, carry a litter to term, and nurse the litter to weaning age (data not shown). This indicates that the mice were fully fertile at a young age; however, we have not assessed whether these animals may undergo premature ovarian failure due to an exhaustion of normal oocytes.
Although we observed differing results of prenatal exposure to BPA on mammary gland development in FVB/N mice compared to previous reports, our findings on susceptibility to mammary gland cancer are supported by previous studies using rats. We report a dose-dependent increase in susceptibility to mammary gland cancer in DMBA-induced model of mammary gland cancer in mice that were prenatally exposed to BPA. Prenatal exposure to BPA of Wistar rats, coupled with the subsequent exposure to subcarcinogenic doses of N
-methylurea in adulthood, results in increased susceptibility to cancer, with the development of hyperplastic lesions and low penetrance of focal tumors [33
]. Increased susceptibility to mammary cancer also occurs following neonatal and prepubertal exposure to BPA and DMBA in Sprague-Dawley rats [32
]. In mice, it has been reported that prenatal exposure to BPA caused the formation of hyperplastic lesions and ductal carcinoma in situ, without any additional exposure to carcinogens as an adult [31
]. However, it is unclear whether these lesions ultimately progress to cancers. Hence, to our knowledge, our studies are the first to demonstrate that mammary cancer susceptibility is indeed increased in mice following prenatal BPA exposure, indicating that prenatal exposure to BPA increases mammary tumor susceptibility in multiple rodent models.
The terminal end bud of the mammary gland develops with the onset of puberty, contains a large number of stem cells, and is highly susceptible to carcinogenic events [60
]. Thus, an increase in terminal end bud number is frequently associated with increased cancer susceptibility. Fetal exposure to BPA has previously been reported to increase terminal end bud number in CD1 mice, leading to speculation that this may increase mammary tumor susceptibility. However, our studies demonstrated that prenatal exposure to BPA can increase tumor susceptibility in the absence of morphological changes in FVB/N animals. These data suggest that while BPA can alter mammary morphology in some models, unassociated molecular changes must also contribute to the establishment of breast cancer risk. Such changes may involve epigenetic alterations of gene expression as has been observed following in vitro BPA treatment of mammary-derived epithelial cells from adult women [61
]. Identifying the specific mechanism(s) by which BPA establishes cancer risk during this period will require extensive studies that examine BPA alteration of the specific genetic programs that are established during anlagen morphogenesis that are coupled to tumor susceptibility later in life.
In addition to examining the impact of BPA on establishing tumor susceptibility during development, we assessed whether BPA could affect another stage of oncogenesis—specifically, promoting the growth of transformed mammary epithelial cells into overt tumors. We used a standard model of estrogen-dependent breast cancer involving MCF-7 cell xenografts. Surprisingly, while BPA has been shown to promote growth of these cells in vitro, no in vivo analyses have assessed whether BPA can potentiate the growth of hormone-dependent breast cancers. We found that, like 17β-estradiol, BPA can induce the formation of tumors from these cells, albeit with reduced growth rates. These results suggest that high circulating levels of BPA may contribute to estrogen independent growth of breast cancers in postmenopausal women; however, this will require further study to determine if there is a correlation between serum levels of BPA and apparent hormone independence.
BPA has been reported to exert both estrogenic and nonestrogenic effects. To determine if BPA was acting primarily through its estrogenic properties to induce MCF-7 tumor growth, we treated mice with overt tumors with tamoxifen, an established inhibitor of estrogen receptor α in the mammary gland, in the sustained presence of BPA or 17β-estradiol [62
]. As evidenced by the regression of tumors in both cohorts, BPA clearly fueled cell growth and tumor formation via the estrogen receptor.
In conclusion, the results reported here indicate that BPA may increase mammary tumorigenesis through at least two mechanisms. One involves alterations of the developing fetal mammary gland in the absence of morphological changes that increases susceptibility to carcinogenic insults. The other demonstrates promotion of tumor cell growth through estrogenic signaling. Both results indicate that exposure to BPA at various time points throughout the life span increases the risk of developing mammary cancer in mice. If these mechanisms extend to humans, BPA has the potential to increase susceptibility to breast cancer at low doses if exposure occurs at various important developmental time points.