Cheryl M. Schaeberle

Perinatal Exposure to Environmentally Relevant Levels of Bisphenol A Decreases Fertility and Fecundity in CD-1 Mice

Background Perinatal exposure to low-doses of bisphenol A (BPA) results in alterations in the ovary, uterus, and mammary glands and in a sexually dimorphic region of the brain known to be important for estrous cyclicity. Objectives We aimed to determine whether perinatal exposure to environmentally relevant doses of BPA alters reproductive capacity. Methods Female CD-1 mice that were exposed to BPA at 0, 25 ng, 250 ng, or 25 μg/kg body weight (BW)/day or diethylstilbestrol (DES) at 10 ng/kg BW/day (positive control) from gestational day 8 through day 16 of lactation were continuously housed with proven breeder males for 32 weeks starting at 2 months of age. At each delivery, pups born to these mating pairs were removed. The cumulative number of pups, number of deliveries, and litter size were recorded. The purity of the BPA used in this and our previous studies was assessed using HPLC, mass spectrometry, and nuclear magnetic resonance. Results The forced breeding experiment revealed a decrease in the cumulative number of pups, observed as a nonmonotonic dose–response effect, and a decline in fertility and fecundity over time in female mice exposed perinatally to BPA. The BPA was 97% pure, with no evidence of contamination by other phenolic compounds. Conclusions Perinatal exposure to BPA leads to a dose-dependent decline in the reproductive capacity of female mice. The effects on the cumulative number of pups are comparable to those previously reported in mice developmentally exposed to DES, a compound well known to impair reproduction in women. This association suggests the possibility that early BPA exposure may also affect reproductive capacity in women.

Perinatal bisphenol A exposure increases estrogen sensitivity of the mammary gland in diverse mouse strains.

Background Studies of low-dose effects of xenoestrogens have yielded conflicting results that may be attributed to differences in estrogen sensitivity between the rodent strains examined. Perinatal exposure of CD-1 mice to low doses of the xenoestrogen bisphenol A (BPA) alters peripubertal mammary gland development. Future studies to assess the role of estrogen receptors as mediators of BPA action require estrogen receptor knock-out mice that were generated on a C57Bl6 background. The sensitivity of the C57Bl6 strain to estradiol and BPA is unknown. Objectives In the present study we examined whether the mammary glands of CD-1 and C57Bl6 mice exhibited similar responses to 17β-estradiol (E2) and whether perinatal exposure to BPA equally enhanced sensitivity of the mammary glands to E2 at puberty. Methods Immature mice were ovariectomized and treated for 10 days with one of eight doses of E2. Morphological mammary gland parameters were examined to identify doses producing half-maximal effects. Mice were exposed perinatally to 0 or 250 ng BPA/kg body weight (bw)/day from gestational day 8 until postnatal day (PND) 2. On PND25, female offspring were ovariectomized and given an estrogen challenge of 0, 0.5, or 1 μg E2/kg bw/day for 10 days. Morphometric parameters of the mammary gland were compared between strains. Results Both strains exhibited similar responses to E2. Perinatal BPA exposure altered responses to E2 at puberty for several parameters in both strains, although the effect in CD-1 was slightly more pronounced. Conclusion Both mouse strains provide adequate models for the study of perinatal exposure to xenoestrogens.

Does Cancer Start in the Womb? Altered Mammary Gland Development and Predisposition to Breast Cancer due to in Utero Exposure to Endocrine Disruptors

We are now witnessing a resurgence of theories of development and carcinogenesis in which the environment is again being accepted as a major player in phenotype determination. Perturbations in the fetal environment predispose an individual to disease that only becomes apparent in adulthood. For example, gestational exposure to diethylstilbestrol resulted in clear cell carcinoma of the vagina and breast cancer. In this review the effects of the endocrine disruptor bisphenol-A (BPA) on mammary development and tumorigenesis in rodents is used as a paradigmatic example of how altered prenatal mammary development may lead to breast cancer in humans who are also widely exposed to it through plastic goods, food and drink packaging, and thermal paper receipts. Changes in the stroma and its extracellular matrix led to altered ductal morphogenesis. Additionally, gestational and lactational exposure to BPA increased the sensitivity of rats and mice to mammotropic hormones during puberty and beyond, thus suggesting a plausible explanation for the increased incidence of breast cancer.

Perinatally Administered Bisphenol A as a Potential Mammary Gland Carcinogen in Rats

Background: Environmental exposure to bisphenol A (BPA) affects mammary gland development in rodents and primates. Prenatal exposure to environmentally relevant doses of BPA increased the number of intraductal hyperplasias and ductal carcinomas in situ by 50 days of age in Wistar-Furth rats. Objective: We aimed to determine whether BPA exposure of dams during gestation only or throughout lactation affects the incidence of mammary gland neoplasia in female offspring. Methods: We treated pregnant Sprague-Dawley rats with BPA at 0, 0.25, 2.5, 25, or 250 μg BPA/kg BW/day from gestational day (GD) 9 to birth and from GD9 to postnatal day (PND) 21. Mammary glands from BPA-exposed offspring were examined at four time points for preneoplastic and neoplastic lesions. To assess circulating BPA levels, we exposed pregnant rats to vehicle or 250 μg BPA/kg BW/day during gestation only or during gestation/lactation and analyzed sera from dams, fetuses, and nursing pups for total and unconjugated BPA. Results: Total and unconjugated BPA were detected in sera from 100% of dams and fetuses and 33% of pups exposed to 250 μg BPA/kg BW/day. Unconjugated BPA levels in exposed dams and fetuses (gestational) and in exposed dams and pups (gestational/lactational) were within levels found in humans. Preneoplastic lesions developed in BPA-exposed female offspring across all doses as early as PND50. Unexpectedly, mammary gland adenocarcinomas developed in BPA-exposed offspring by PND90. Conclusions: Our findings suggest that developmental exposure to environmentally relevant levels of BPA during gestation and lactation induces mammary gland neoplasms in the absence of any additional carcinogenic treatment. Thus, BPA may act as a complete mammary gland carcinogen. Citation: Acevedo N, Davis B, Schaeberle CM, Sonnenschein C, Soto AM. 2013. Perinatally administered bisphenol A acts as a mammary gland carcinogen in rats. Environ Health Perspect 121:1040–1046; http://dx.doi.org/10.1289/ehp.1306734

The mammary gland response to estradiol: monotonic at the cellular level, non-monotonic at the tissue-level of organization?

The role of hormones in mammary gland development has been studied in detail using surgical and genetic models. These studies have indicated roles for estrogen in ductal elongation and terminal end bud formation. However, no comprehensive study has quantified how different doses of estrogen affect morphological parameters of mammary gland development. Additionally, comparisons between the estrogen-responsiveness of the mammary gland and uterus, the model organ for estrogen action are incomplete. In this study, immature mice were ovariectomized and implanted with osmotic pumps releasing one of eight doses of 17beta-estradiol for 10 days. As expected from the classical uterotrophic assay, the uterus showed a monotonic dose-response curve for all measured endpoints. In contrast, the mammary gland showed a non-monotonic, inverted-U shaped response to estrogen with regard to morphometric parameters, and a monotonic response with regard to gene expression parameters. These results indicate that estrogen has opposing effects in mammary gland morphogenesis depending on estrogen dose, i.e. low to moderate doses induce terminal end bud formation and ductal elongation while higher doses inhibit these processes. This non-monotonic dose-response in the mammary gland may reflect complex interactions, where estrogen can act on multiple targets either as an agonist or antagonist.

The male mammary gland: A target for the xenoestrogen bisphenol A

Males of some strains of mice retain their mammary epithelium even in the absence of nipples. Here, we have characterized the mammary gland in male CD-1 mice both in whole mounts and histological sections. We also examined the effects of bisphenol A (BPA), an estrogen mimic that alters development of the female mouse mammary gland. BPA was administered at a range of environmentally relevant doses (0.25-250μg/kg/day) to pregnant and lactating mice and then the mammary glands of male offspring were examined at several periods in adulthood. We observed age- and dose-specific effects on mammary gland morphology, indicating that perinatal BPA exposures alter the male mammary gland in adulthood. These results may provide insight into gynecomastia, the most common male breast disease in humans, where proliferation of the mammary epithelium leads to breast enlargement.

Dynamic Metabolic Disruption in Rats Perinatally Exposed to Low Doses of Bisphenol-A

Along with the well-established effects on fertility and fecundity, perinatal exposure to endocrine disrupting chemicals, and notably to xeno-estrogens, is strongly suspected of modulating general metabolism. The metabolism of a perinatally exposed individual may be durably altered leading to a higher susceptibility of developing metabolic disorders such as obesity and diabetes; however, experimental designs involving the long term study of these dynamic changes in the metabolome raise novel challenges. 1H-NMR-based metabolomics was applied to study the effects of bisphenol-A (BPA, 0; 0.25; 2.5, 25 and 250 μg/kg BW/day) in rats exposed perinatally. Serum and liver samples of exposed animals were analyzed on days 21, 50, 90, 140 and 200 in order to explore whether maternal exposure to BPA alters metabolism. Partial Least Squares-Discriminant Analysis (PLS-DA) was independently applied to each time point, demonstrating a significant pair-wise discrimination for liver as well as serum samples at all time-points, and highlighting unequivocal metabolic shifts in rats perinatally exposed to BPA, including those exposed to lower doses. In BPA exposed animals, metabolism of glucose, lactate and fatty acids was modified over time. To further explore dynamic variation, ANOVA-Simultaneous Component Analysis (A-SCA) was used to separate data into blocks corresponding to the different sources of variation (Time, Dose and Time*Dose interaction). A-SCA enabled the demonstration of a dynamic, time/age dependent shift of serum metabolome throughout the rats’ lifetimes. Variables responsible for the discrimination between groups clearly indicate that BPA modulates energy metabolism, and suggest alterations of neurotransmitter signaling, the latter finding being compatible with the neurodevelopmental effect of this xenoestrogen. In conclusion, long lasting metabolic effects of BPA could be characterized over 200 days, despite physiological (and thus metabolic) changes connected with sexual maturation and aging.

Perinatal BPA exposure alters body weight and composition in a dose specific and sex specific manner: The addition of peripubertal exposure exacerbates adverse effects in female mice.

Body weight (BW) and body composition were examined in CD-1 mice exposed perinatally or perinatally and peripubertally to 0, 0.25, 2.5, 25, or 250μg BPA/kg BW/day. Our goal was to identify the BPA dose (s) and the exposure window(s) that increased BW and adiposity, and to assess potential sex differences in this response. Both perinatal exposure alone and perinatal plus peripubertal exposure to environmentally relevant levels of BPA resulted in lasting effects on body weight and body composition. The effects were dose specific and sex specific and were influenced by the precise window of BPA exposure. The addition of peripubertal BPA exposure following the initial perinatal exposure exacerbated adverse effects in the females but appeared to reduce differences in body weight and body composition between control and BPA exposed males. Some effects of BPA on body weight and body composition showed a non-linear dose response.

New insights into fetal mammary gland morphogenesis: differential effects of natural and environmental estrogens

An increased breast cancer risk during adulthood has been linked to estrogen exposure during fetal life. However, the impossibility of removing estrogens from the feto-maternal unit has hindered the testing of estrogen’s direct effect on mammary gland organogenesis. To overcome this limitation, we developed an ex vivo culture method of the mammary gland where the direct action of estrogens can be tested during embryonic days (E)14 to 19. Mouse mammary buds dissected at E14 and cultured for 5 days showed that estrogens directly altered fetal mammary gland development. Exposure to 0.1 pM, 10 pM, and 1 nM 17 β-estradiol (E2) resulted in monotonic inhibition of mammary buds ductal growth. In contrast, Bisphenol-A (BPA) elicited a non-monotonic response. At environmentally relevant doses (1 nM), BPA significantly increased ductal growth, as previously observed in vivo, while 1 μM BPA significantly inhibited ductal growth. Ductal branching followed the same pattern. This effect of BPA was blocked by Fulvestrant, a full estrogen antagonist, while the effect of estradiol was not. This method may be used to study the hormonal regulation of mammary gland development, and to test newly synthesized chemicals that are released into the environment without proper assessment of their hormonal action on critical targets like the mammary gland.

Evidence of Absence: Estrogenicity Assessment of a New Food-Contact Coating and the Bisphenol Used in Its Synthesis

Consumer concerns about exposure to substances found in food contact materials with estrogenic activity (EA) have created substantial demand for alternatives. We assessed the potential EA of both a new bisphenol monomer used to synthesize polymeric coatings for metal food-contact applications and the nonintentionally added substances (NIAS) that may migrate into food. We evaluated tetramethyl bisphenol F (TMBPF) using in vitro and in vivo assays. We extracted the polymeric coating using food simulants ethanol (50% v/v) and acetic acid (3% w/v) and measured migration using tandem liquid chromatography (LC)/mass spectrometry (MS) and LC time-of-flight MS for TMBPF and NIAS, respectively. We also tested migrants for EA using the E-SCREEN assay. TMBPF did not show estrogenic activity in the uterotrophic assay and did not alter puberty in male and female rats or mammary gland development in female rats. Neither TMBPF nor the migrants from the final polymeric coating increased proliferation of estrogen-sensitive MCF7 cells. TMBPF did not show estrogen-agonist or antagonist activity in the estrogen receptor-transactivation assay. TMBPF migration was below the 0.2 parts per billion detection limit. Our findings provide compelling evidence for the absence of EA by TMBPF and the polymeric coating derived from it and that human exposure to TMBPF would be negligible.