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BIOLOGY OF REPRODUCTION 85, 490–497 (2011)
Published online before print 2 June 2011.
DOI 10.1095/biolreprod.110.090431


100 ll corn oil; Sigma) or corn oil by oral gavage, one dose each at 5 and 6 wk...


FIG. 1. Fetal exposure to BPA leads to early vaginal opening in FVB/N
female mice. Pregnant FVB/N fe...


FIG. 3. Fetal exposure to bisphenol A or vehicle control results in


FIG. 7. Tamoxifen treatment abrogates estrogen (solid line) or bisphenol
A (dashed line) induced gro...

mammary gland cancer are supported by previous studies using
rats. We report a ...


cohorts, BPA clearly fueled cell growth and tumor formation
via the estrogen receptor.
In conclusion...











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Bpa increases mammary cancer risk

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EL Bisphenol A incrementa los riesgos de Cáncer de mama

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Bpa increases mammary cancer risk

  1. 1. BIOLOGY OF REPRODUCTION 85, 490–497 (2011) Published online before print 2 June 2011. DOI 10.1095/biolreprod.110.090431 Bisphenol A Increases Mammary Cancer Risk in Two Distinct Mouse Models of Breast Cancer1 Kristen Weber Lozada2,3 and Ruth A. Keri3,4,5 Departments of Pharmacology3 and Genetics4 and Division of General Medical Sciences-Oncology,5 Case Western Reserve University, Cleveland, Ohio Bisphenol A (BPA) is an industrial plasticizer that leaches from food containers during normal usage, leading to human exposure. Early and chronic exposure to endocrine-disrupting environmental contaminants such as BPA elevates the potential for long-term health consequences. We examined the impact of BPA exposure on fetal programming of mammary tumor susceptibility as well as its growth promoting effects on transformed breast cancer cells in vivo. Fetal mice were exposed to 0, 25, or 250 lg/kg BPA by oral gavage of pregnant dams. Offspring were subsequently treated with the known mammary carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). While no significant differences in postnatal mammary development were observed, both low- and high-dose BPA cohorts had a statistically significant increase in susceptibility to DMBAinduced tumors compared to vehicle-treated controls. To determine if BPA also promotes established tumor growth, MCF-7 human breast cancer cells were subcutaneously injected into flanks of ovariectomized NCR nu/nu female mice treated with BPA, 17beta-estradiol, or placebo alone or combined with tamoxifen. Both estradiol- and BPA-treated cohorts formed tumors by 7 wk post-transplantation, while no tumors were detected in the placebo cohort. Tamoxifen reversed the effects of estradiol and BPA. We conclude that BPA may increase mammary tumorigenesis through at least two mechanisms: molecular alteration of fetal glands without associated morphological changes and direct promotion of estrogen-dependent tumor cell growth. Both results indicate that exposure to BPA during various biological states increases the risk of developing mammary cancer in mice. bisphenol A, BPA, breast cancer, development, DMBA, endocrine disruptors, estrogen, mammary cancer, mammary glands, mouse, puberty, rodents (rats, mice, guinea pigs, voles), tamoxifen INTRODUCTION Early and chronic exposure to pervasive environmental contaminants raises the possibility of long-term health consequences. In recent years, the increased exposure to environmental synthetic estrogens, such a bisphenol A (BPA), has been speculated to be involved in the increasing incidence of breast cancer [1, 2]. BPA is an industrial plasticizer that is 1 Supported by grant RO1ES015768 from the National Institutes of Health (NIH). 2 Correspondence: Ruth A. Keri, Department of Pharmacology, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4965. FAX: 216 368 1300; e-mail: keri@case.edu Received: 20 December 2010. First decision: 11 February 2011. Accepted: 11 May 2011. Ó 2011 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363 490 Downloaded from www.biolreprod.org. utilized in the production of many products, including baby bottles, food and water containers, medical supplies, and dental fillings [3]. As a component of polycarbonate plastic, over 6 billion pounds of BPA are produced each year [4]. BPA leaches from plastic containers during normal usage, and detectable amounts can be found in many commercial food products [5, 6]. It is chronically ingested by humans, and multiple surveys have found that ;95% of adults and children tested have detectable concentrations of total urinary BPA in industrialized nations, indicating that exposure is both ubiquitous and continuous [7]. BPA has also been measured in maternal serum and ovarian follicular fluid, as well as in fetal plasma and amniotic fluid, indicating passage across the placenta [8, 9]. Recent studies have also demonstrated placental transport of BPA-glucuronide and reactivation of this apparently inactive metabolite in rat fetuses [10]. Hence, there is significant risk of BPA exposure during critical developmental periods that are particularly sensitive to changes in the estrogenic environment. This may have lasting effects on hormone responsiveness and homeostatic control of various tissues much later in life [11]. Reports on the long-term reproductive effects of in vivo BPA exposure have been diverse because of the disparity in the use of animal models, strain, dose, timing, and route of exposure [12–14]. The current Environmental Protection Agency (EPA) reference dose for BPA is 50 lg kgÀ1 dayÀ1. This level was derived by identifying the lowest observable adverse effect level (LOAEL; 50 mg kgÀ1 dayÀ1) in Fischer 344 rats and reducing that dose by three orders of magnitude. However, both transient and permanent effects have been observed in various reproductive tissues of animal models with doses that are lower than the EPA reference dose. Doses below this reference are considered ‘‘low dose’’ because they are within the range of human exposure and less than that typically used in toxicology studies [15–17]. Exposure to endocrine disruptors during prenatal development may result in developmental effects and long-term modification of organ systems in adults [18–20], and BPA has been speculated to exert several estrogenic effects on the rodent mammary gland [21]. This may be an indirect response to the modulation of the timing of puberty because early commencement of puberty can affect the development of the mammary gland through premature exposure to ovarian hormones, such as estrogen and progesterone, both of which affect growth and development [22]. Indeed, exposure to BPA during the perinatal period causes precocious puberty in female CD-1 mice [3], and this has been associated with masculinization of the anteroventral periventricular preoptic area in the brain [23]. This may occur through an estrogen-dependent mechanism, as prenatal exposure to estrogen also induces precocious puberty in ICR/Jcl mice [24]. In the mammary gland, fetal exposure of female CD1 mice to BPA causes differences in ductal invasion as well the number of ducts and terminal end and alveolar buds in adults ABSTRACT
  2. 2. BISPHENOL A AND MOUSE MAMMARY CANCER RISK 100 ll corn oil; Sigma) or corn oil by oral gavage, one dose each at 5 and 6 wk of age. At least 10 mice were included in each group. Mice were then palpated weekly for the detection of mammary tumors. Tumor latency was measured using Kaplan-Meier survival analysis. On the detection of tumors, mice were killed and tumors collected. Any mice that were obviously sick or that died during the experiment from causes other than mammary cancer were censored from the study. Xenograft Experiments NCR nu/nu females were obtained from the Case Comprehensive Cancer Center, Athymic Animal Facility. Recipient females were ovariectomized at 8 wk of age and implanted with a placebo (37.5 mg/60-day release), 17bestradiol (1.7 mg/60-day release), or low-dose BPA pellet (37.5 mg pellet/60day release; Innovative Research of America, Sarasota, FL). After recovery from surgery, 1 3 106 MCF-7 cells in 150 ll of Matrigel (R&D Systems, Minneapolis, MN) were subcutaneously injected into the flanks of the mice. Tumor latency was then assessed by weekly palpation; tumor growth was monitored by weekly measurement with calipers. Each treatment group contains a minimum of five mice. After 60 days, mice were implanted with a second 60-day release implant to ensure sustained exposure to 17b-estadiol or BPA. Tumor volume was calculated with the equation (l 3 w2)/2, where w is the smallest diameter measured. Tamoxifen Treatment Following tumor formation in NCR nu/nu mice xenografted with MCF-7 cells and treated with either BPA or estradiol, a small cohort was also treated with the selective estrogen receptor modulator tamoxifen (1 mg mouseÀ1 dayÀ1 [Sigma] or 100 ll vehicle control, 50% PEG400/50% water [Sigma]) by oral gavage for 5 continuous days per week (n ¼ 3 per group). Tumor growth or regression was monitored by weekly measurement with calipers. Mammary Gland Whole Mounts MATERIALS AND METHODS Animals Animal care and use was conducted according to established guidelines approved by the Institutional Animal Care and Use Committee at Case Western Reserve University. All animals were treated humanely and with regard to the alleviation of suffering. Animals were housed in a temperature- and humiditycontrolled animal facility with a 12-h light:dark cycle. All experimental mice were housed in polypropylene cages with wood bedding and glass water bottles with rubber stoppers. Weaned mice and lactating females were fed a phytoestrogen-free diet, LabDiet 5001 and 5008, respectively (Purina LabDiet, St. Louis, MO). Vaginal Opening and Mammary Development Experiments FVB/N mice (The Jackson Laboratory, Bar Harbor, ME) were bred and observed for a copulatory plug. On evidence of mating, female mice were placed in a separate cage. On Postcoital Day 8, female mice were treated with vehicle (100 ll mineral oil), 25 lg/kg bisphenol A (Sigma, St. Louis, MO) or 250 lg/kg bisphenol A by oral gavage daily until parturition. BPA was dissolved in 50% PBS/50% DMSO and the appropriate concentration added to mineral oil. At parturition, offspring were culled to a maximum of six mice per litter. Female offspring were then randomly divided into experimental groups. Each experimental group contained a minimum of five mice from at least three litters. Female mice were observed daily for vaginal opening beginning on Postnatal Day 16. For analysis of development, mammary glands were collected at 3, 5, and 8 wk of age. Ductal length was measured at 3 and 5 wk of age as the distance from the midpoint of the lymph node to the terminal end bud. The distance to the three longest ducts was measured, and these measurements were averaged for each gland. For forced involution studies, female mice (exposed prenatally to BPA) that were 8 wk of age were mated and nursed a litter for 4 days, after which the pups were removed. Mammary glands were collected 5 days after pup removal. Tumor Susceptibility Experiments We utilized the FVB/N strain, which is the most frequently used mouse model for examining mammary cancer susceptibility because of its intrinsic propensity to develop mammary tumors with various genetic manipulations [34–36]. Female mice exposed prenatally to BPA or vehicle control as described above were given 7,12-dimethyl benz[a]anthracene (DMBA) (1 mg/ When animals were 3, 5, and 8 wk of age or Day 5 postinvolution, mammary gland number 9 was collected, fixed in Kahle’s solution for 2–4 h, and stained by immersion in Carmine Alum stain (2% carmine, 5% aluminum potassium sulfate in water) at 48C. Stained glands were dehydrated in repeated ethanol washes, cleared with xylene, and mounted on glass slides with Permount. At least five mice from two independent litters were measured for each treatment group. MKI67 Immunostaining Tumors were fixed in 4% paraformaldehyde, sectioned, and mounted on slides. Sections were cleared with xylene and dehydrated in ethanol washes. Fifty milliliters of citrate buffer were used for antigen retrieval at 1208C for 10 min using a Biocare Medical pressure cooker. Sections were incubated with a rabbit anti-mouse MKI67 antibody (Abcam, ab66155) at 4 lg/ml, overnight at 48C, followed by incubation with an HRP-conjugated secondary antibody (Vectastain ABC kit; Vector Labs, Burlingame, CA). Positive cells were identified with 3,3 0 -diaminobenzidine (Liquid DAB þ Substrate Chromogen System; DAKO, Carpinteria, CA), and the sections were counterstained with hematoxylin. Tissue sections were dehydrated through decreasing-concentration ethanol washes, cleared with xylene, and mounted under coverslips. Three sections per mouse (n ¼ 3) and three fields per section were counted for positive cells, with a minimum of 500 cells counted per field. Statistical Analysis Results are expressed as a mean 6 SEM. We performed unpaired Student ttests to assess significance of differences and considered a P-value of ,0.05 to be statistically significant. RESULTS Effect of Prenatal Exposure to Bisphenol A To begin evaluating the impact of BPA on mammary tumorigenesis, we first examined whether fetal exposure of FVB/N mice to BPA would induce precocious puberty or alter the development of the mammary gland as described for perinatal BPA exposure in CD1 mice [25–27]. Beginning on Prenatal Day 8, prior to the onset of mammary morphogenesis Downloaded from www.biolreprod.org. compared to vehicle-exposed controls [25, 26]. Fetal exposure also increased ductal area and ductal extension in embryonic Day 18 female mammary glands in the same strain of mice [27]. Terminal end buds are particularly sensitive to carcinogenic events, [28, 29]; hence, their increase following BPA exposure suggests that these mice may be at increased risk for mammary tumorigenesis. Supporting this postulate, intraductal mammary epithelial hyperplasias have been observed in CD1 mice exposed to BPA during fetal life or perinatally [30]. While these lesions are suggestive of a potential procarcinogenic effect of BPA, it is unknown whether such changes result in overt mammary tumors or increased mammary tumor susceptibility [3, 31]. In contrast to the mouse studies, fetal exposure of Sprague-Dawley and Wistar rats to BPA leads to an increase in susceptibility to carcinogen-induced mammary tumors, although no spontaneous tumors were observed [32, 33]. These data suggest that exposure to BPA during the prenatal period could alter mammary susceptibility to additional insults; however, the specific carcinogenesis studies have been limited to rat models. We questioned whether the tumor-promoting effects of BPA could be observed in other species and whether the impact of this compound was restricted to prenatal exposures or whether BPA could promote the growth of established breast tumors. Herein, we report that fetal exposure to BPA also increases mammary tumor susceptibility in mice and that BPA has growth-promoting effects on human breast cancer xenografts. Together, these studies indicate that BPA can act at multiple stages to increase the acquisition of breast cancer. 491
  3. 3. 492 LOZADA AND KERI FIG. 1. Fetal exposure to BPA leads to early vaginal opening in FVB/N female mice. Pregnant FVB/N females were treated with 25 lg kgÀ1 dayÀ1 bisphenol A or vehicle control by oral gavage beginning on Postcoital Day 8. Female offspring were observed for vaginal opening beginning on Postnatal Day 16. Bars are means 6 SD (*P , 6 E-05). Each experimental group contained a minimum of five mice from at least three litters. Susceptibility to 7,12-Dimethylbenz[a]anthracene-Induced Cancer We next investigated whether fetal BPA exposure may exert nonstructural effects on the mammary gland that could alter its susceptibility to carcinogenic insults. We employed a model of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancer [42, 43]. Fetal BPA exposure was repeated with a new cohort of pregnant female mice with the same dosing schedule (vehicle control or BPA [25 lg kgÀ1 dayÀ1 or 250 lg kgÀ1 dayÀ1]) as described above, and female offspring were Downloaded from www.biolreprod.org. that occurs on Embryonic Days 10–11 [37–39], pregnant dams were treated with vehicle control or 25 lg kgÀ1 dayÀ1 BPA by oral gavage [40]. At parturition, exposure to BPA was stopped, and litters were culled to a similar size. Female offspring were observed daily for vaginal opening, an external physical indicator of puberty, beginning on Postnatal Day 16 [41]. Female FVB/N mice exposed prenatally to vehicle control exhibited vaginal opening on Days 22–24, while mice exposed to BPA exhibited accelerated vaginal opening on Days 21–22 (Fig. 1). The difference in age of vaginal opening was statistically significant between vehicle- and BPA-treated groups (P , 6 E-05). These results demonstrate that prenatal exposure to BPA is manifesting an effect similar to the previously reported effects of prenatal exposure to estrogens and can influence changes in the hormonal milieu that establish the onset of puberty. To test the effect of prenatal BPA exposure on mammary gland development, pregnant female mice were treated as described above. The offspring were randomly separated into various experimental end points, and the mammary glands were harvested and whole mounted. Glands were collected at various ages, including prepuberty, puberty, postpuberty, pregnancy, lactation, and involution. At no time point examined were there any notable morphological differences in mammary gland development observed between the BPAand vehicle-treated cohorts of offspring (Fig. 2A; data not shown). There was no difference in average duct length at 3 and 5 wk of age between the BPA and vehicle cohorts (Fig. 2B). By 8 wk of age, the ductal tree had filled the fat pad in both cohorts. These results indicate that prenatal exposure to BPA has no overt morphological effect on the development of the mammary gland in FVB/N mice at the various time points investigated. FIG. 2. Fetal exposure to bisphenol A (25 lg kgÀ1 dayÀ1) by oral gavage has no detectable effect on morphological development of the mammary gland in FVB/N female mice. Pregnant FVB/N females were treated with 25 lg kgÀ1 dayÀ1 bisphenol A or vehicle control by oral gavage beginning on Postcoital Day 8. Mammary glands were collected from female offspring at 3, 5, and 8 wk of age as well as involution on Day 5 following delivery and nursing of one litter. A) Histology of Carmine Alum-stained whole mounts. B) The distance from mid-lymph node to terminal end bud was quantified. At least five mice from two independent litters were measured for each treatment group. Negative values occur in the 3-wk cohorts because the ducts have not yet reached the midpoint of the lymph node. subsequently exposed to one dose of DMBA (1 mg mouseÀ1 dayÀ1) at 5 wk of age and an additional single dose of DMBA at 6 wk of age. Tumor latency was assessed by weekly palpation. Both the low-dose (25 lg kgÀ1 dayÀ1) and the highdose (250 lg kgÀ1 dayÀ1) BPA groups revealed a statistically significant increase in susceptibility to tumor formation compared to vehicle controls (Fig. 3). The high-dose BPA
  4. 4. BISPHENOL A AND MOUSE MAMMARY CANCER RISK 493 FIG. 3. Fetal exposure to bisphenol A or vehicle control results in increased susceptibility to mammary carcinogenesis after exposure to 7,12-dimethylbenz[a]anthracene (DMBA). Pregnant FVB/N females were treated with 25 lg kgÀ1 dayÀ1 or 250 lg kgÀ1 dayÀ1 bisphenol A or vehicle control by oral gavage beginning on Postcoital Day 8. Prenatally exposed female offspring were then treated with DMBA (1 mg/mouse) by oral gavage one dose each at 5 wk and again at 6 wk of age. Female mice were palpated weekly to monitor the formation of mammary tumors. KaplanMeier survival analysis is shown. At least 10 mice from each treatment group represent at least three independent litters. MCF-7 Cell Xenografts in NCR nu/nu Mice The studies above indicate that BPA exposure during early development can increase susceptibility to carcinogenic events later in life. We next assessed whether BPA also promotes the growth of established tumor cells to form an overt tumor through an estrogenic mechanism. In this case, we utilized a xenograft mouse model involving MCF-7 human breast cancer cells and immunocompromised NCR nu/ nu female mice. Recipients were ovariectomized at 8 wk of age to remove the effects of endogenous ovarian hormones on the xenografted mammary cells. MCF-7 cells require estrogenic input from either the intact ovary or estrogen pellet implant in ovariectomized mice to form tumors [45]. At the time of ovariectomy, the mice were implanted with a placebo (37.5 mg kgÀ1 60-day releaseÀ1), 17b-estradiol (1.7 mg/60-day release), or BPA pellet (37.5 mg pellet/60-day release). After recovery from surgery, 1 3 106 MCF-7 cells suspended in Matrigel were subcutaneously injected into the flanks of the ovariectomized mice. The mice were palpated weekly and tumors measured by calipers. The 17b-estradioltreated mice formed measurable tumors by 6 wk postinjection. Similarly, BPA-treated females formed tumors by 7 wk postinjection (Fig. 5). In contrast, no tumors formed in the placebo cohort. By the conclusion of the experiment, five of seven mice in the 17b-estradiol-treated cohort, five of six mice in the BPA-treated cohort, and zero of seven mice in the FIG. 4. Histology of 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumors from FVB/N female mice. Tumors were collected from FVB/N females 1 wk after detection of a palpable tumor, fixed in 4% paraformaldehyde, and stained with hematoxylin and eosin. The only tumor that developed in the vehicle treated mice is shown with representative images of tumors from BPA exposed animals. Magnification 320. placebo-treated cohort formed tumors. On average, the tumors formed in the 17b-estradiol-treated cohort were 3.0 times larger in volume than the BPA-treated cohort at 9 wk post tumor cell implantation (P , 0.001). These results indicate that BPA alone can promote the growth of estrogendependent breast cancer cells in vivo. FIG. 5. 17b-estradiol (solid line) or bisphenol A (dashed line) promote growth of MCF-7 tumors in NCR nu/nu mice. Eight-week-old NCR nu/nu females were ovariectomized and implanted with a 17b-estradiol (1.7 mg/ 60-day release), bisphenol A (37.5 mg pellet/60-day release), or placebo pellet (37.5 mg pellet/60-day release). One week later, 1 3 106 MCF-7 cells were injected subcutaneously into the flanks of each female (placebo and 17b-estradiol, n ¼ 7; BPA, n ¼ 6). Mice were palpated for tumor formation, and mammary tumor size was measured weekly. Average tumor volume is statistically different between 17b-estradiol and bisphenol A treated cohorts, as indicated: *P , 0.001. Downloaded from www.biolreprod.org. cohort exhibited a mean tumor latency of 50.8 wk, and the lowdose BPA cohort exhibited a mean tumor latency of 69.3 wk. Only one vehicle-treated mouse developed a DMBA-induced tumor at 111 wk. There was a statistically significant difference in tumor latency in both the low- and the high-dose BPAtreated cohorts compared to controls (P , 0.05). Histologically, all tumors were chemically induced pure squamous cell carcinomas of the mammary gland, including the single tumor that arose in the control group (Fig. 4) [44]. Hence, fetal BPA exposure did not alter the type of mammary tumor that forms in response to DMBA. These results indicate that BPA may increase mammary tumorigenesis but does not exert a dominant effect on the histopathology of resulting tumors.
  5. 5. 494 LOZADA AND KERI FIG. 7. Tamoxifen treatment abrogates estrogen (solid line) or bisphenol A (dashed line) induced growth of MCF-7 tumors. Eight-week-old NCR nu/nu females were ovariectomized and implanted with either 17bestradiol (1.7 mg/60-day release), bisphenol A (37.5 mg pellet/60-day release), or placebo pellets (37.5 mg pellet/60-day release). One week later, 1 3 106 MCF-7 cells were injected subcutaneously into the flanks of each female. Seven weeks after tumor formation was first detected, females were treated daily with tamoxifen (1 mg mouseÀ1 dayÀ1) by oral gavage. At 60 days post tumor cell injection, the mice were given a second 17b-estradiol, BPA, or placebo pellet to maintain levels throughout the remainder of the experiment. Mice were palpated and mammary tumors measured weekly (n ¼ 3 for estradiol and BPA). DISCUSSION FIG. 6. A) MCF-7 tumors from mice treated with 17b-estradiol (1.7 mg/ 60-day release) or BPA (37.5 mg/60-day release) are histologically identical. Xenograft tumor sections from each cohort were stained with hematoxylin and eosin. Representative sections are shown. B) MCF-7 cell xenograft tumors from the 17b-estradiol cohort have a higher percentage of proliferating cells as revealed with MKI67 immunostaining. Xenograft tumor sections were immunostained with an anti-MKI67 antibody. Representative sections are shown. Magnification 320. C) The percentage of MKI67 positively stained cells was quantified in three mice from each treatment group. Bars are means 6 SD (*P , 1.2 E-6). Examination of hematoxylin and eosin-stained tumor sections by light microscopy revealed similar histopathology and tumor cell size between BPA and 17b-estradiol-treated groups. Analysis of MKI67 immunohistochemistry, which identifies proliferating cells, revealed that tumors from the 17bestradiol cohort had an average of 2-fold more proliferation than the tumors of the BPA cohort (Fig. 6; P , 1.2 E-6). The difference in the percentage of proliferating cells likely underlies the difference in growth rate and tumor size between the 17b-estradiol- and BPA-treated cohorts. To determine if the effects of BPA were mediated by the estrogen receptor, another cohort of mice was treated with the selective estrogen receptor modulator tamoxifen (1 mg mouseÀ1 dayÀ1). Following formation of measurable MCF-7 cell tumors in NCR nu/nu mice, the animals were treated with tamoxifen for 3 wk while maintaining exposure to BPA or 17bestradiol. Tumors were measured weekly by calipers. Tumor regression was observed in all mice treated with tamoxifen regardless of whether the mice were treated with 17b-estradiol or BPA (Fig. 7). Hence, although there is a difference in the percent of proliferating cells between the 17b-estradiol- and the BPA-treated cohorts, the estrogenic action of BPA is the Here we report that prenatal exposure to BPA increased tumor susceptibility in a dose-dependent manner in a DMBAinduced 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, 35]. 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 Downloaded from www.biolreprod.org. primary mechanism underlying its promotion of MCF-7 tumor cell growth in vivo.
  6. 6. BISPHENOL A AND MOUSE MAMMARY CANCER RISK 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-nitroso-Nmethylurea 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, 55]. 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 estrogendependent 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 17b-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 a in the mammary gland, in the sustained presence of BPA or 17bestradiol [62]. As evidenced by the regression of tumors in both Downloaded from www.biolreprod.org. 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, 38]. 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 [47–49], 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 [52–54]. To this end, we chose a low dose (25 lg kgÀ1 dayÀ1), which is half of the EPA recommended daily safe dose, and a high dose (250 lg kgÀ1 dayÀ1), 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, 21, 31, 33, 55]. 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, 26]. 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, 56–59]. 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 495
  7. 7. 496 LOZADA AND KERI 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. 17. 18. 19. 20. 21. 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