Induction of mammary gland ductal hyperplasias and carcinoma in situ following fetal bisphenol A exposure

Exposure of the fetus to excess estrogen is believed to increase the risk of developing breast cancer during adult life. Fetal exposure to low doses of the xenoestrogen bisphenol A resulted in long-lasting effects in the mouse mammary gland that were manifested during adult life. It enhanced sensitivity to estradiol, decreased apoptosis, increased the number of progesterone receptor-positive epithelial cells at puberty and increased lateral branching at 4 months of age. We now report that fetal exposure to 2.5, 25, 250 and 1000 microg bisphenol A/kg body weight/day induces the development of ductal hyperplasias and carcinoma in situ at postnatal day 50 and 95 in rats. These highly proliferative lesions have an increased number of estrogen receptor-alpha positive cells. Thus, fetal bisphenol A exposure is sufficient to induce the development of preneoplastic and neoplastic lesions in the mammary gland in the absence of any additional treatment aimed at increasing tumor development.     

Exposure to low doses of oxybenzone during perinatal development alters mammary gland morphology in male and female mice

Oxybenzone (benzophenone-3) is an ultraviolet radiation filter commonly used in personal care products including sunscreens, textiles and inks, and food and beverage containers, among others. Due to its widespread use, human exposures to oxybenzone are widespread. Oxybenzone is considered an endocrine disrupting chemical due to its antiestrogenic and antiandrogenic properties. We evaluated the effects of oral exposures to oxybenzone on the growth and morphology of the mammary gland, body weight and anogenital distance in BALB/c mice exposed to 30, 212 or 3000 μg/kg/day in utero and during lactation. Developmental exposures to oxybenzone reduced the size and growth of mammary gland in males prior to and during puberty. In exposed females, oxybenzone reduced mammary cell proliferation, decreased the number of cells expressing estrogen receptor α, and altered mammary gland morphology in adulthood. These results suggest that even low doses of oxybenzone can disrupt hormone sensitive organs during critical windows of development.     

Mammary gland differentiation by early life exposure to enantiomers of the soy isoflavone metabolite equol

The role of soy in reducing breast cancer risk has been suggested to be associated with early exposure to isoflavones, which alter mammary gland morphology. The objective of the study was to determine the effect of dietary exposure to the enantiomers of a key soy isoflavone metabolite, equol, on mammary gland development and later chemoprotection using the DMBA-induced animal model of breast cancer. Animals were exposed to S-(−)equol or R-(+)equol (250 mg/kg diet) during the neonatal (0–21 days) or prepubertal (21–35 days) periods only. Histological evaluation of the mammary glands showed that both enantiomers fed neonatally via the dam led to significant precocial mammary gland differentiation. By day 50, early S-(−)equol or R-(+)equol exposure resulted in a decrease in immature terminal end structures and an increase in mature lobules, suggesting an early ‘imprinting’ effect. Despite these morphological changes to the mammary gland, neonatal and prepubertal exposure to equol had no long-term chemoprevention against mammary tumors induced by DMBA, although for R-(+)equol there was a trend to delaying tumor formation. In summary, early exposure to equol was not chemopreventive, but neither did it increase tumor formation in response to DMBA, suggesting exposure in early life does not influence breast cancer risk.     

Arsenic-induced cancer cell phenotype in human breast epithelia is estrogen receptor-independent but involves aromatase activation

Accumulating data suggest arsenic may be an endocrine disruptor and tentatively linked to breast cancer by some studies. Therefore, we tested the effects of chronic inorganic arsenic exposure on the normal estrogen receptor (ER)-negative breast epithelial cell line, MCF-10A. Cells were chronically exposed to a low-level arsenite (500 nM) for up to 24 weeks. Markers of cancer cell phenotype and the expression of critical genes relevant to breast cancer or stem cells (SCs) were examined. After 24 weeks, chronic arsenic-exposed breast epithelial (CABE) cells showed increases in secreted MMP activity, colony formation, invasion, and proliferation rate, indicating an acquired cancer cell phenotype. These CABE cells presented with basal-like breast cancer characteristics, including ER-α, HER-2, and progesterone receptor negativity, and overexpression of K5 and p63. Putative CD44⁺/CD24^?/low breast SCs were increased to 80 % over control in CABE cells. CABE cells also formed multilayer cell mounds, indicative of loss of contact inhibition. These mounds showed high levels of K5 and p63, indicating the potential presence of cancer stem cells (CSCs). Epithelial-to-mesenchymal transition occurred during arsenic exposure. Overexpression of aromatase, a key rate-limiting enzyme in estrogen synthesis, occurred with arsenic starting early on in exposure. Levels of 17β-estradiol increased in CABE cells and their conditioned medium. The aromatase inhibitor letrozole abolished arsenic-induced increases in 17β-estradiol production and reversed cancer cell phenotype. Thus, chronic arsenic exposure drives human breast epithelia into a cancer cell phenotype with an apparent overabundance of putative CSCs. Arsenic appears to transform breast epithelia through overexpression of aromatase, thereby activating oncogenic processes independent of ER.     

Estrogens in the wrong place at the wrong time: Fetal BPA exposure and mammary cancer

Iatrogenic gestational exposure to diethylstilbestrol (DES) induced alterations of the genital tract and predisposed individuals to develop clear cell carcinoma of the vagina as well as breast cancer later in life. Gestational exposure of rodents to a related compound, the xenoestrogen bisphenol-A (BPA) increases the propensity to develop mammary cancer during adulthood, long after cessation of exposure. Exposure to BPA during gestation induces morphological alterations in both the stroma and the epithelium of the fetal mammary gland at 18 days of age. We postulate that the primary target of BPA is the fetal stroma, the only mammary tissue expressing estrogen receptors during fetal life. BPA would then alter the reciprocal stroma-epithelial interactions that mediate mammogenesis. In addition to this direct effect on the mammary gland, BPA is postulated to affect the hypothalamus and thus in turn affect the regulation of mammotropic hormones at puberty and beyond.     

Epigenetic influences of low-dose bisphenol A in primary human breast epithelial cells

Substantial evidence indicates that exposure to bisphenol A (BPA) during early development may increase breast cancer risk later in life. The changes may persist into puberty and adulthood, suggesting an epigenetic process being imposed in differentiated breast epithelial cells. The molecular mechanisms by which early memory of BPA exposure is imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood. The aim of this study was to examine epigenetic changes in breast epithelial cells treated with low-dose BPA. We also investigated the effect of BPA on the ERα signaling pathway and global gene expression profiles. Compared to control cells, nuclear internalization of ERα was observed in epithelial cells preexposed to BPA. We identified 170 genes with similar expression changes in response to BPA. Functional analysis confirms that gene suppression was mediated in part through an ERα-dependent pathway. As a result of exposure to BPA or other estrogen-like chemicals, the expression of lysosomal-associated membrane protein 3 (LAMP3) became epigenetically silenced in breast epithelial cells. Furthermore, increased DNA methylation in the LAMP3 CpG island was this repressive mark preferentially occurred in ERα-positive breast tumors. These results suggest that the in vitro system developed in our laboratory is a valuable tool for exposure studies of BPA and other xenoestrogens in human cells. Individual and geographical differences may contribute to altered patterns of gene expression and DNA methylation in susceptible loci. Combination of our exposure model with epigenetic analysis and other biochemical assays can give insight into the heritable effect of low-dose BPA in human cells.     

Segregated responses of mammary gland development and vaginal opening to prepubertal genistein exposure in Bscl2−/− female mice with lipodystrophy

Berardinelli-Seip congenital lipodystrophy 2-deficient (Bscl2^−/−) mice recapitulate human BSCL2 disease with lipodystrophy. Bscl2-encoded seipin is detected in adipocytes and epithelium of mammary gland. Postnatal mammary gland growth spurt and vaginal opening signify pubertal onset in female mice. Bscl2^−/− females have longer and dilated mammary gland ducts at 5-week old and delayed vaginal opening. Prepubertal exposure to 500 ppm genistein diet increases mammary gland area and accelerates vaginal opening in both control and Bscl2^−/− females. However, genistein treatment increases ductal length in control but not Bscl2^−/− females. Neither prepubertal genistein treatment nor Bscl2-deficiency affects phospho-estrogen receptor α or progesterone receptor expression patterns in 5-week old mammary gland. Interestingly, Bscl2-deficiency specifically reduces estrogen receptor β expression in mammary gland ductal epithelium. In summary, Bscl2^−/− females have accelerated postnatal mammary ductal development but delayed vaginal opening; they display segregated responses in mammary gland development and vaginal opening to prepubertal genistein treatment.     

Does Breast Cancer Start in the Womb?

Perturbations in the foetal environment predispose individuals to diseases that become apparent in adulthood. These findings prompted researchers to hypothesize that foetal exposure to environmental oestrogens may play a role in the increased incidence of breast cancer observed in European and US populations over the last 50 years. There is widespread human exposure to bisphenol A, an oestrogenic compound that leaches from dental materials and consumer products. In CD-1 mice, perinatal exposure to environmentally relevant bisphenol A levels induced alterations of the mammary gland architecture. Bisphenol A increased the number of terminal end buds at puberty and terminal ends at 6 months of age and increased ductal lateral branching at 4 months of age. Exposed mice also showed an enhanced sensitivity to oestradiol when ovariectomized prior to puberty. All these parameters are associated in human beings with an increased risk for developing breast cancer. To assess whether bisphenol A induces mammary gland neoplasia, we chose a rat model because it more closely mimics the human disease than mouse models. Examination of the mammary glands of Wistar/Furth rats during early adulthood revealed that gestational exposure to bisphenol A induced the development of pre-neoplastic lesions and carcinoma in situ in the absence of any additional treatment aimed at increasing tumour development. Emerging epidemiological data reveal an increased incidence of breast cancer in women exposed to diethylstilboestrol during gestation. Hence, both animal experiments and epidemiological data strengthen the hypothesis that foetal exposure to xenooestrogens may be an underlying cause of the increased incidence of breast cancer observed over the last 50 years.     

Prepubertal exposure to arsenic(III) suppresses circulating insulin-like growth factor-1 (IGF-1) delaying sexual maturation in female rats

Arsenic (As) is a prevalent environmental toxin readily accessible for human consumption and has been identified as an endocrine disruptor. However, it is not known what impact As has on female sexual maturation. Therefore, in the present study, we investigated the effects of prepubertal exposure on mammary gland development and pubertal onset in female rats. Results showed that prepubertal exposure to 10 mg/kg of arsenite (As(III)) delayed vaginal opening (VO) and prepubertal mammary gland maturation. We determined that As accumulates in the liver, disrupts hepatocyte function and suppresses serum levels of the puberty related hormone insulin-like growth factor 1 (IGF-1) in prepubertal animals. Overall, this is the first study to show that prepubertal exposure to As(III) acts peripherally to suppress circulating levels of IGF-1 resulting in delayed sexual maturation. Furthermore, this study identifies a critical window of increased susceptibility to As(III) that may have a lasting impact on female reproductive function.     

Selective estrogen receptor-beta (SERM-beta) compounds modulate raphe nuclei tryptophan hydroxylase-1 (TPH-1) mRNA expression and cause antidepressant-like effects in the forced swim test

Estrogen acts through two molecularly distinct receptors termed estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) which bind estradiol with similar affinities and mediate the effects of estrogen throughout the body. ERα plays a major role in reproductive physiology and behavior, and mediates classic estrogen signaling in such tissues as the uterus, mammary gland, and skeleton. ERβ, however, modulates estrogen signaling in the ovary, the immune system, prostate, gastrointestinal tract, and hypothalamus, and there is some evidence that ERβ can regulate ERα activity. Moreover, ERβ knockout studies and receptor distribution analyses in the CNS suggest that this receptor may play a role in the modulation of mood and cognition. In recent years several ERβ-specific compounds (selective estrogen receptor beta modulators; SERM-beta) have become available, and research suggests potential utility of these compounds in menopausal symptom relief, breast cancer prevention, diseases that have an inflammatory component, osteoporosis, cardiovascular disease, and inflammatory bowel disease, as well as modulation of mood, and anxiety. Here we demonstrate an antidepressant-like effect obtained using two SERM-beta compounds, SERM-beta1 and SERM-beta2. These compounds exhibit full agonist activity at ERβ in a cell based estrogen response element (ERE) transactivation assay. SERM-beta1 and 2 are non-proliferative with respect to breast as determined using the MCF-7 breast cancer cell-based assay and non-proliferative in the uterus as determined by assessing the effects of SERM-beta compounds on immature rat uterine weight and murine uterine weight. In vivo SERM-beta1 and 2 are brain penetrant and display dose dependent efficacy in the murine dorsal raphe assays for induction of tryptophan hydroxylase mRNA and progesterone receptor protein. These compounds show activity in the murine forced swim test and promote hippocampal neurogenesis acutely in rats. Taken together these data suggest that ERβ may play an important role in modulating mood and the ERβ specific compounds described herein will be useful tools for probing the utility of an ERβ agonist for treating neuroendocrine-related mood disturbance and menopausal symptoms.     

Proliferative and estrogenic sensitivity of the mammary gland are modulated by isoflavones during distinct periods of adolescence

Isoflavone (ISO) exposure during adolescence has been demonstrated to modulate the estrogenic and proliferative sensitivity of the adult breast tissue. In this study, we investigated whether ISO exposure restricted to the period of puberty is sufficient to result in similar effects. Female rats were divided into three groups receiving either lifelong an ISO-free diet (IDD) or an ISO-rich diet (ISD), or an ISD from postnatal day (PND) 30 to PND 60 covering the time period of puberty (pISD). Serum concentrations of ISO and metabolites were determined at PND 50 and 80. At PND 50, ISD animals had significant higher equol serum levels than pISD animals. Onset of puberty occurred significantly earlier in the pISD and ISD group compared to the animals fed IDD. Cycle length was shortest in pISD group. To determine estrogen sensitivity of the adult breast tissue, adult rats were ovariectomized and subcutaneously treated either with estradiol (E₂) or with genistein (GEN) for 3 days (PND 77–80). Analysis of Ki-67 and proliferating cell nuclear antigen (PCNA) expression showed a reduced proliferative response of the breast to E₂ in pISD and ISD animals compared to IDD group, while the induction of progesterone receptor (PR) was higher in both IDD and pISD compared to ISD fed rats. Our results demonstrate that ISO exposure during puberty is sufficient to reduce the proliferative response of the adult mammary gland but not to reduce the response of classical E₂ sensitive genes like the PR. In summary, our results demonstrate that animals exposed during different periods of their adolescence to ISO differ in several physiological aspects. In addition, the detected differences in the serum equol levels between pISD and ISD rats and the detected differences in the estrogen sensitivity of the breast clearly underline this assumption.     

Hormonally active doses of isoflavone aglycones promote mammary and endometrial carcinogenesis and alter the molecular tumor environment in Donryu rats

Our research is focused on modifying effects of an isoflavone aglycones (IAs)-rich extract at a hormonally active dose of 150 mg/kg body weight/day on mammary and endometrial carcinogenesis in female Donryu rats. IA administered for 2 weeks in a phytoestrogen-low diet exerted estrogenic activity and induced cell proliferation in the uterus of ovariectomized rats. Furthermore, administration for 4 weeks resulted in elevation of cell proliferation in the mammary glands of 7,12-dimethylbenz[a]anthracene (DMBA)-treated animals. Forty weeks of postpubertal administration of IA to 5-week-old rats after initiation of mammary and endometrial carcinogenesis with DMBA and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) caused significant increase of incidence and multiplicity of mammary adenocarcinoma, multiplicities of endometrial atypical hyperplasia, adenomatous polyps, and an increased trend of uterine adenocarcinomas. Liquid chromatography with tandem mass spectrometry and immunohistochemical analyses revealed significant elevation of tumorigenesis-related proteins such as S100 calcium-binding protein A8, kininogen 1, and annexins 1 and 2 in mammary adenocarcinomas and cadherin EGF LAG seven-pass G-type receptor 2, DEAD box polypeptide 1, and cysteine- and glycine-rich protein 1 in uterine proliferative lesions of IA-treated animals. Those changes are likely to be related to modulation of estrogen receptor (ER), AP1, nuclear factor-kappa B, and actin signaling pathways. Our results indicate that the postpubertal exposure of Donryu rats to IA at an estrogenic dose results in promotion of mammary and uterine carcinogenesis induced by DMBA and ENNG, which might be related to the activation of ER-dependent signaling and alteration of the molecular tumor environment in the mammary gland and endometrium.     

In utero exposure to arsenite contributes to metabolic and reproductive dysfunction in male offspring of CD-1 mice

In utero exposure to arsenite (iAs) is known to increase disease risks later in life. We investigated the effect of in utero exposure to iAs in the drinking water on metabolic and reproductive parameters in male mouse offspring at postnatal and adult stages. Pregnant CD-1 mice were exposed to iAs (as sodium arsenite) in the drinking water at 0 (control), 10 ppb (EPA standard for drinking water), and 42.5 ppm (tumor-inducing dose in mice) from embryonic day (E) 10–18. At birth, pups were fostered to unexposed females. Male offspring exposed to 10 ppb in utero exhibited increase in body weight at birth when compared to controls. Male offspring exposed to 42.5 ppm in utero showed a tendency for increased body weight and a smaller anogenital distance. The body weight in iAs-exposed pups continued to increase significantly compared to control at 3 weeks and 11 weeks of age. At 5 months of age, iAs-exposed males exhibited greater body fat content and glucose intolerance. Male offspring exposed to 10 ppb in utero had higher circulating levels of leptin compared to control. In addition, males exposed to 42.5 ppm in utero exhibited decreased total number of pups born compared to controls and lower average number of litters sired over a six-month period. These results indicate that in utero exposure to iAs at either human relevant concentration or tumor-inducing concentration is a potential cause of developmental origin of metabolic and reproductive dysfunction in adult male mice.     

Postweaning dietary genistein exposure advances puberty without significantly affecting early pregnancy in C57BL/6J female mice

An epidemiological study indicates higher plasma level of genistein in girls with earlier puberty. This study tests the hypothesis in C57BL/6J mice that postweaning (peripubertal) dietary genistein exposure could result in earlier puberty in females assessed by vaginal opening, estrous cyclicity, corpus luteum and mammary gland development. Newly weaned female mice were fed with 0, 5, 100, or 500 ppm genistein diets. Decreased age at vaginal opening, increased length on estrus stage, and accelerated mammary gland development were detected in 100 and 500 ppm genistein-treated groups. Increased presence of corpus luteum was found in 5 ppm genistein-treated group at 6 weeks old only. Increased expression of epithelial-specific genes but not that of ERα or ERβ was detected in 500 ppm genistein-treated mammary glands at 5 weeks old. No significant adverse effect on embryo implantation was observed. These data demonstrate causal effect of dietary genistein on earlier puberty in female mice.     

In utero DDT exposure and breast density before age 50

Prior studies in the Child Health and Development Studies (CHDS) found in utero exposure to the pesticide, dichlorodiphenyltrichloroethane (DDT), increased breast cancer risk by age 52. Mammographic density is considered a primary risk factor for breast cancer. We conducted a study of 309 daughters from the CHDS to examine in utero DDT exposure and mammographic density in midlife. Among daughters with high (>75th percentile) exposure to p,p’-Dichlorodiphenyldichloroethylene (DDE), p,p’-DDT was significantly correlated with increased dense area and percent density regardless of her body mass in midlife. In the subset of women with lower (<75th percentile) p,p-DDE, p,p’-DDT was associated with increased non-dense breast area. This was explained by adjustment for midlife BMI suggesting that p,p’-DDT may be obesogenic. In aggregate our findings indicate that early life p,p’-DDT exposure impacts breast density in a complex way that depends on the hosts biological ability to sequester and process DDT and levels of exposure.     

中药复方联合三苯氧胺对实验大鼠乳腺增生组织及ER、PR表达的影响

目的探讨中药联合三苯氧胺(TAM)周期治疗大鼠乳腺增生的疗效及作用机理。方法将大鼠随机分为空白对照组、疾病模型组、三苯氧胺组、中药复方组、联合用药组5组,经雌二醇及孕酮刺激建立乳腺增生模型。光镜下观察乳腺组织病理学变化,免疫组化法测定雌激素受体(ER)、孕激素受体(PR)阳性表达强度。结果中药联合TAM治疗大鼠乳腺增生,可使乳腺小叶腺泡数明显减少、腺泡腔缩小及腺导管扩张程度减轻,ER、PR的阳性表达强度明显抑制,其疗效优于单纯中药和TAM组(P<0.05或0.01)。结论中药联合TAM治疗可降低实验大鼠乳腺组织中ER、PR含量,使其对雌激素的敏感性下降,对大鼠乳腺增生有明显的抑制作用。     

Hypothesis: Activation of rapid signaling by environmental estrogens and epigenetic reprogramming in breast cancer

Environmental and lifestyle factors are considered significant components of the increasing breast cancer risk in the last 50 years. Specifically, exposure to environmental endocrine disrupting compounds is correlated with cancer susceptibility in a variety of tissues. In both human and rodent models, the exposure to ubiquitous environmental estrogens during early life has been shown to disrupt normal mammary development and cause permanent adverse effects. Recent studies indicate that environmental estrogens not only have the ability to disrupt estrogen receptor (ER) signaling, but can also reprogram the epigenome by altering DNA and histone methylation through rapid, nongenomic ER actions. We have observed xenoestrogen-mediated activation of several nongenomic signaling pathways and have identified a target for epigenetic reprogramming in MCF-7 breast cancer cells. These observations, in addition to data from the literature, support the hypothesis that activation of rapid signaling by environmental estrogens can lead to epigenetic reprogramming and contribute to the progression of breast cancer.     

Dietary quercetin exacerbates the development of estrogen-induced breast tumors in female ACI rats

Phytoestrogens are plant compounds that structurally mimic the endogenous estrogen 17β-estradiol (E₂). Despite intense investigation, the net effect of phytoestrogen exposure on the breast remains unclear. The objective of the current study was to examine the effects of quercetin on E₂-induced breast cancer in vivo. Female ACI rats were given quercetin (2.5 g/kg food) for 8 months. Animals were monitored weekly for palpable tumors, and at the end of the experiment, rats were euthanized, breast tumor and different tissues excised so that they could be examined for histopathologic changes, estrogen metabolic activity and oxidant stress. Quercetin alone did not induce mammary tumors in female ACI rats. However, in rats implanted with E₂ pellets, co-exposure to quercetin did not protect rats from E₂-induced breast tumor development with 100% of the animals developing breast tumors within 8 months of treatment. No changes in serum quercetin levels were observed in quercetin and quercetin + E₂-treated groups at the end of the experiment. Tumor latency was significantly decreased among rats from the quercetin + E₂ group relative to those in the E₂ group. Catechol-O-methyltransferase (COMT) activity was significantly downregulated in quercetin-exposed mammary tissue. Analysis of 8-isoprostane F_2α (8-iso-PGF_2α) levels as a marker of oxidant stress showed that quercetin did not decrease E₂-induced oxidant stress. These results indicate that quercetin (2.5 g/kg food) does not confer protection against breast cancer, does not inhibit E₂-induced oxidant stress and may exacerbate breast carcinogenesis in E₂-treated ACI rats. Inhibition of COMT activity by quercetin may expose breast cells chronically to E₂ and catechol estrogens. This would permit longer exposure times to the carcinogenic metabolites of E₂ and chronic exposure to oxidant stress as a result of metabolic redox cycling to estrogen metabolites, and thus quercetin may exacerbate E₂-induced breast tumors in female ACI rats.