Bisphenol A and Hormone-Associated Cancers: Current Progress and Perspectives

Abstract Bisphenol A (BPA), a carbon-based synthetic compound, exhibits hormone-like properties and is present ubiquitously in the environment and in human tissues due to its widespread use and biological accumulation. BPA can mimic estrogen to interact with estrogen receptors α and β, leading to changes in cell proliferation, apoptosis, or migration and thereby, contributing to cancer development and progression. At the genetic level, BPA has been shown to be involved in multiple oncogenic signaling pathways, such as the STAT3, MAPK, and PI3K/AKT pathways. Moreover, BPA may also interact with other steroid receptors (such as androgen receptor) and plays a role in prostate cancer development. This review summarizes the current literature regarding human exposure to BPA, the endocrine-disrupting effects of BPA, and the role of BPA in hormone-associated cancers of the breast, ovary, and prostate.


INTRODUCTION
B isphenol A (BPA) is an industrial synthetic chemical that is used to make certain plastics and epoxy resins and has been commercially available since 1957. Currently, BPA is one of the highest volume chemicals produced worldwide (more than 6 Â 10 9 lb/year). BPA is widely present in many hard plastic bottles and metal-based food and beverage cans. Although BPA is a colorless solid that is soluble in organic solvents and poorly soluble in water, human exposure does occur when BPA leaches from plastic-lined food and beverage cans, water bottles, and some dental sealants. 1,2 BPA leaching occurs when plastic and epoxy resin-containing bottles and cans are heated or repeatedly washed. Thus, BPA is ubiquitously found in the environment throughout the world. 3,4 BPA has been the focus of widespread concern due to the fact that it interferes with endocrine signaling pathways even at extremely low doses. BPA is a diphenyl compound that contains two hydroxyl groups in the ''para'' position, making it remarkably similar to the synthetic estrogen, diethylstilbestrol. Particularly, many studies have shown that BPA can activate estrogen receptors (ERs) a and b. 5,6 To date, multiple lines of evidence have indicated that BPA has estrogen-like activity and exhibits developmental toxicity in the reproductive organs and inhibitory effects on testosterone synthesis. 7-9 Thus, in vitro and in vivo studies have revealed links between BPA exposure and hormonerelated cancers, including breast, prostate, and ovarian cancers and endometrial carcinoma. In this review, we summarize the current literature regarding human exposure to BPA and the role of BPA in the development of hormone-related cancers.

HUMAN EXPOSURE TO BPA AND ITS METABOLISM
BPA can leach into food and beverages through the daily use of tin cans, baby bottles, reusable plastic water bottles, and polycarbonate plastic containers. The rate of BPA leaching increases when polycarbonate is scratched or discolored. 10,11 Heat and non-neutral pH conditions (either acidic or basic) are two factors that influence BPA release, because hydrolysis of the ester bond linking BPA monomers occurs with changes in temperature and pH, 12,13 such as those that take place when BPA-containing plastics are cleaned with harsh detergents or contain acidic or high-temperature liquids. In a study evaluating BPA exposure in male workers who spray epoxy resin, the concentration of BPA in urine was found to be higher in the epoxy resin sprayers (median 1.06 mmol/mol creatinine) compared to the controls (median 0.52 mmol/mol creatinine) and the level of follicle-stimulating hormone (FSH) was also correlated with urinary BPA. 14 Using different measurement techniques, BPA has been found to be present in human serum, urine, amniotic fluid, and breast milk in the populations of industrialized countries worldwide. 15 In a reference population of 394 adults in the United States, BPA was detected in 95% of urine samples with a median concentration of 1.28 mg/L. 16 The BPA concentration in human serum ranges from 0.2-1.6 ng/mL (0.88-7.0 nM). 17,18 The U.S. Environmental Protection Agency has defined a reference dose of BPA to be 50 mg kg/day, 19 and the European Union has set a no-observed adverse-effect level (NOAEL) of 5 mg kg/day. The metabolism of BPA has been extensively studied using in vivo and in vitro systems. The first-pass metabolism of ingested BPA occurs in the intestine and/or liver, which greatly limits its systemic bioavailability. 20,21 In rats, BPA is metabolized to DNA-reactive bisphenol-o-quinone through 5-hydroxybisphenol and bisphenol semiquinone. 22 A study comparing the BPA-metabolizing activity of 11 forms of human hepatic cytochromes P450 (CYPs) showed that BPA is mainly metabolized by the CYP2C subfamily in human liver, and it inhibits human steroidogenic CYP17 activities. 23 BPA is also conjugated with glucuronic acid by rat hepatic microsomes. 24 BPA is able to extensively form BPA-monoglucuronide in rat hepatocytes or perfused liver, which is consistent with the in vivo finding that BPA-glucuronide is the major metabolite found in urine. 20,25,26 Indeed, BPA-glucuronide is also the major metabolite in human hepatocytes, although minor amounts of BPA sulfate and the diconjugate are also present in human hepatocytes. 27 Thus, BPA conjugation could be a determinant of its in vivo estrogenic effects, because the monoglucuronide lacks estrogenic activity. 6 In a study to determine the extent of monoglucuronide formation in monolayer cultures of hepatocytes from rats, mice, and humans, the initial rates of metabolism in hepatocytes followed the order of mice > rats > humans. 27 However, when extrapolated to the whole liver, the hepatic capacity for BPA glucuronidation is predicated to be humans > rats > mice, suggesting that firstpass metabolism and rapid elimination of BPA are probable following oral exposure. 27 Fetuses can also be exposed to BPA, because it can freely cross the placenta and then bind to a-fetoprotein, resulting in enhanced bioavailability during neonatal development. 28,29 This exposure puts fetuses and young children at risk for changes in secondary sexual characteristics as well as neural, behavioral, and immune disorders.

ENDOCRINE-DISRUPTING EFFECTS OF BPA
The estrogenic activity of BPA represents the major endocrine-disrupting effect of BPA and was revealed in 1993 by a study showing that BPA can be released from polycarbonate flasks during autoclaving. 30 BPA is a weak agonist to estrogen receptor b, but compared to estradiol, its activity is 1000-fold less. 31 However, this low binding affinity does not mean that the biological activity of BPA in human is negligible, because evaluation based only on in vitro binding assays may lead to underestimation. In humans and experimental animals, BPA was originally thought to disrupt the estrogen-triggered pathways by forming a transcriptional complex that can bind the estrogen responsive element (ERE). 32,33 In this scenario, BPA normally interacts with co-activators and co-repressors. For instance, the interaction of BPA with ER-a and ER-b has been observed to have both agonist and antagonist activities on ER-a in vitro, 34 and even at remarkably low doses, BPA can induce estrogen-like activities in cells that are similar or even stronger than those of estrogen. 35,36 In addition to ER-a and ER-b, BPA also binds to the ER-related receptor g (ERR-g). 37 The binding of BPA to ERR-g preserves its basal constitutive activity and also protects it from deactivation by the selective estrogen receptor modulator 4-hydroxytamoxifen. 37 Interestingly, ERR-g has been found in high concentrations in the placenta, which may explain reports of high BPA accumulation there and associated developmental and transplacental effects. 38 However, additional studies suggested that BPA can also activate non-classical estrogen pathways during BPA-mediated rapid responses, including: 1) binding to the plasma membrane receptor, 39 such as in the example of BPA binding a membrane version of the ER-a on pituitary cells and provoking Ca 2þ influx via L-type channels 40 ; 2) activating the cAMP-responsive element binding protein in a calcium-dependent manner 41 ; and 3) binding to a distinct domain of ERs compared to E2. 42 However, accumulating evidence suggests that the actions of BPA on cell signaling pathways vary among cell types and a combination of a rapid mechanism and longer effects is involved in its effects on estrogen-responsive gene expression.
In addition to its estrogen-like activity, BPA also binds to other nuclear receptors, such as androgen receptor (AR). For example, BPA could compete with 5a-dihydrotestosterone (DHT) to bind to the androgen receptor. 43,44 Several studies demonstrated the anti-androgenic activity of BPA in cell systems through the formation of an AR/BPA complex that prevents endogenous androgens from regulating androgendependent gene transcription. 43,45,46 BPA and its chlorinated derivatives also were shown to bind to thyroid hormone receptor (TR). 47,48 Interestingly, BPA is an antagonist to the TR, whereas BPA derivatives are TR agonists that promote proliferation of rat pituitary cells. 49

OVARIAN CANCER
Although the pathophysiology of ovarian cancer remains poorly understood, accumulating evidence suggests that sex steroids (such as estrogens) are involved in the development of ovarian cancer. 50,51 Approximately 50% of human ovarian epithelial cancer cells express higher levels of ER than do cells of benign tumors and normal ovary. 52,53 Both ER-a and ER-b are expressed in normal and transformed ovarian cells, with high abundance of ER-b in granulose cells and of ER-a in theca and interstitial cells. 54 Although an association between hormone replacement therapy and ovarian cancer remains elusive, 55,56 prospective epidemiological studies in postmenopausal women have suggested that estrogen-only replacement therapy increases the incidence and mortality of ovarian cancer. 57,58 Estrogen taken as an oral contraceptive in the postmenopausal years may increase risk of ovarian cancer. 59,60 It is believed that estrogen can provide a hormonal environment that facilitates tumor progression and/or may regulate proliferation and apoptosis of ovarian cells directly. 46 In this context, BPA exposure could mimic the effects of estrogen in ovarian cells.
Indeed, a previous epidemiological study showed that BPA is present in serum, follicular fluid, fetal serum, and full-term amniotic fluid (15-to 18-fold induction compared to other fluids), suggesting accumulation of BPA in fetuses and significant exposure during the prenatal period. 61 BPA interrupts ovarian steroidogenesis by altering the steroidogenic enzymes. 62 However, it remains unknown whether BPA-disrupted steroidogenesis contributes to ovarian carcinogenesis. In humans, BPA exposure increases the incidence or exacerbates the clinical course of polycystic ovary syndrome. 18,63 In rodent models, it is well documented that neonatal exposure to BPA is associated with altered ovarian morphology, an increased number of cystic ovaries, cystic endometrial hyperplasia, and reduction in the pool of primordial follicles in the rat ovary, which is associated with an increased proliferation rate likely mediated by an estrogenic pathway. [64][65][66][67][68] Prenatal exposition to BPA also causes a variety of development abnormities of the ovary (eg, endometriosis, altered number of primordial developing follicles, ovarian lesions, and inhibition of meiotic progression of oocytes). [69][70][71] Nevertheless, it remains unclear whether the effects of BPA on the prenatal or neonatal developing ovary can increase the overall risk for ovarian cancer in adulthood. In adult CD-1 mice, long-term BPA exposure (18 months) also induces a significant increase in cystic ovaries and cystic endometrial hyperplasia, which are premalignant and neoplastic lesions. 68 Furthermore, BPA was shown to regulate the expression of a battery of genes in ovarian tissues, some of which are associated with oncogenic signaling or ovarian cancer development. For instance, BPA treatment is able to up-regulate Cdk4, Ccne1, cyclin D1, ER-a, IGF-1R, and Bcl2 but downregulate p21 and Aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2), resulting in cell proliferation and inhibition of apoptosis. 11,72 -74 BPA has also been shown to regulate the transforming growth factor beta (TGF-b), JAK/STAT3, MAPK/ERK, and PI3K/Akt signaling pathways and to interact with leptin to inhibit caspase-3 expression and activity. 75,76 BPA also stimulates granulosa-lutein cells to express matrix metalloproteinase-9 (MMP-9), an extracellular matrix protein that is associated with progression of ovarian cancer. 77,78 The mechanism of BPA's action in ovarian tissues can be estrogenic pathway-dependent or -independent. For example, BPA exposure elicits aneuploidy of both eggs and embryos, a similar phenotype to that observed in ER-b null female mice, and presumably exerts this action via regulation of ER-b pathway genes. 79 In another study, transplacental exposure to BPA induced changes in the expression of genes associated with estrogenic activity in ovaries of Sprague-Dawley rats. 80 However, BPA-regulated expression of CDK4, Ccne1, Bax, and Bcl2 is independent of the estrogenic pathway, 72 whereas BPAinduced cell proliferation in estrogen-responsive ovarian cancer cells is independent of BPA-induced MAPK activation, indicating that a tissue-specific mechanism is responsible for the regulation of cell growth by BPA. 81 Furthermore, BPA is able to cooperate with leptin to inhibit caspase-3 expression and activity in ovarian cancer cells. 74 According to a recent report, endocrine-disrupting chemicals promote the growth of ovarian cancer cells via the ER-CXCL12-CXCR4 signaling axis. 82 However, there is still a lack of epidemiological data identifying associations between BPA exposure and the incidence of ovarian cancer. Available evidence raises concern regarding cancer susceptibility in ovarian tissues after prenatal or postnatal BPA exposure. Additional studies are required to determine the potential effects of BPA exposure on the development of ovarian cancer in humans.

BREAST CANCER
Estrogen and estrogen signaling pathways play pivotal roles in the development of the mammary gland and breast carcinogenesis. Specifically, both ER-a and ER-b are first expressed at embryo stage E12.5 in the mesenchyme surrounding the bud and drive mammary ductal growth during prenatal and neonatal development. Thus, the effects of BPA exposure on mammary glands and breast carcinogenesis have been studied extensively in vitro and in vivo. Multiple lines of evidence demonstrate that fetal exposure to low doses of BPA alters cell proliferation, apoptosis, and timing in the development of mammary glands, which may further predispose the mammary gland to carcinogenesis. 83 Indeed, BPA increases ductal density and sensitivity to estrogens after BPA exposure, which is generally shown in human breast carcinogenesis. 84 Fetal exposure to BPA results in the in situ development of mammary gland carcinoma in a rat model with significant increases in preneoplastic lesions and intraductal proliferation. 85,86 However, the exact mechanisms by which fetal exposure to BPA is linked to adult breast cancer remain elusive. One study showed that BPA is able to induce expression of WNT-4 and receptor activator of nuclear factor kappa-B ligand (RANKL), the two key molecules of hormone function in the regulation of mammary stem cell proliferation and carcinogenesis. 87 Overall, either disruption of the hypothalamic-pituitary-gonadal axis or direct actions on estrogen-sensitive organs by BPA may be involved in the susceptibility of mammary gland tissue to malignant transformation. The most recent study showed the effect of low-dose BPA on the early differentiation of human embryonic stem cells into mammary epithelial cells. 88 Circulating serum xenoestrogens also affect mammographic breast density. 89 BPA was shown to inactivate the p53 axis and to lead to the deregulation of proliferation kinetics and cell death in non-malignant human breast epithelial cells. 90 Taken together, the results of these studies demonstrate that BPA is likely to have significant effects on mammary epithelium and carcinogenesis.
In addition to the long-lasting effects of fetal exposure to BPA, extensive studies have examined the effects of BPA on adult normal mammary gland and transformed mammary gland cells. For example, BPA treatment promoted cell proliferation and increased cell size in mammary gland sphere cultures, but inhibited apoptosis and induced chemoresistance of different ER-positive breast cancer cell lines. 73,91 -97 Direct exposure of ER-positive breast cancer cells to BPA affected multiple oncogenic signaling pathways, including: 1) vascular endothelial growth factor (VEGF) signaling, which is associated with breast tumor angiogenesis 98 ; 2) the DNA repair pathway 99 ; 3) ERK1/2 activation 100,101 ; and 4) STAT3 signaling. 95 Interestingly, one report showed that BPA stimulated the GPER/EGFR/ERK pathway in the ER-null breast cancer line SKBR3, indicating the contribution of an ER-independent mechanism in BPAmediated cancer progression. 102 A human population-based study demonstrated that increases in serum BPA levels are associated with an increase in mammographic breast density after adjustment for age, body mass index, and other potentially confounding factors. 89 BPA also induced a profile of tumor aggressiveness in high-risk cells from breast cancer patients characterized by high histologic grade and large tumor size, resulting in decreased recurrence-free patient survival. 103

PROSTATE CANCER
It is well documented that steroids play a key role in the initiation and progression of prostate cancer. In addition to androgens, estrogen is involved in the etiology of prostate cancer, and the use of anti-estrogens has been recently recognized to have a therapeutic effect in prostate cancer management. 104,105 Both ER-a and ER-b are expressed in normal prostate and prostate cancer cells. ER-a and ER-b are found primarily in stromal and differentiated epithelium, respectively. 106,107 It has been proposed that the prostate gland is more sensitive to estrogen exposure during the critical developmental period of the embryo stage than during adulthood. 108 Recent evidence suggests that BPA exposure does affect prostate cells and cancer development. 109,110 For example, transient exposure of rats to low, environmentally relevant doses of BPA or estradiol during development enhances prostate gland susceptibility to adult-onset precancerous lesions and hormonal carcinogenesis. 111 The role of BPA in prostate tissue is relatively complicated compared to that in the mammary gland or ovary. Both steroid receptors (ERs and AR) play crucial roles in the development and progression of prostate cancer. A number of studies have shown that BPA can regulate the proliferation and migration of prostate cancer cells and induce DNA adducts in prostate cancer cells. 109,112,113 However, the underlying molecular mechanism remains unknown (eg, through BPA-ER or BPA-AR interaction). In a xenograft model of prostate cancer cells that expresses the endogenous BPA-responsive AR-T877A mutant protein, BPA enhances tumor cell proliferation after androgen deprivation and increases the prostate-specific antigen level, indicating that the AR, at least partially, mediates the effects of BPA. 114 In another study, BPA induced a distinct gene expression signature in prostate cancer cells expressing an AR mutation. 115 Particularly, a major action of BPA was to down-regulate ER-b. Considering that ER-b is a molecular antagonist of AR, this study linked the interaction of BPA with ER and AR to shed light on the mechanisms of the cell proliferation-promoting effects of BPA in prostate cancer cells. However, BPA also interacts with AR, inducing nuclear translocation of the tumor-derived receptor (AR-T877A) and subsequent binding to the response elements. 116 As AR-negative prostate cancer cells fail to show growth inhibition after exposure to high doses of BPA, 117 both ER-b and AR could mediate the actions of BPA in advanced prostate adenocarcinomas, but only the role of AR may be essential. In addition to hormone receptor-dependent mechanisms, BPA also induces alterations in the DNA methylation patterns of multiple cell signaling genes in prostate cancer cells, indicating involvement of an epigenetic mechanism. 118 Specifically, BPA enhances the expression of the enzyme phosphodiesterase type 4 variant 4 (PDE4D4), the gene for which is associated with DNA methylation and aging. 118 Further studies are needed to elucidate the effects of BPA exposure on the development of prostate cancer.

CONCLUSION AND FUTURE DIRECTIONS
This review summarized the current available data regarding the role of BPA in hormone-associated cancers. Overall, several conclusions can be made: 1) BPA is a typical xenoestrogen and its estrogenic activity and estrogen-independent activity are likely responsible for its roles in promoting carcinogenesis of multiple cancers ( Figure 1); 2) BPA interacts with other steroid receptor such as AR to promote proliferation of prostate cancer cells; and 3) fetal exposure to BPA could lead to ''long-lasting'' effects on the carcinogenesis of certain organs. Recently, one study showed increased expression of histone trimethylated H3 at lysine 27 of EZH2 after BPA treatment in human breast cancer cells, indicating epigenetic regulation by BPA of cells in carcinogenesis and progression of breast cancer. 119 In another study, BPA treatment induced expression of microRNA-146a, which plays a role in the immune response. 120 Thus, regulation of an epigenetic program and microRNA may represent future directions for the study of BPA's effects on hormone-related cancers, and novel results are expected to expand our understanding of the functional network of BPA in endocrine-associated cancers. A recent report demonstrated that prostate stem-progenitor cells are direct BPA targets and that exposure to BPA at low doses during development increases the hormone-dependent cancer risk in the human prostate epithelium, 121 indicating a novel target in BPAmediated carcinogenesis of prostate cancer. Interestingly, another study also reported that BPA exposure during puberty alters functions and gene expression in mammary stem cells, leading to early neoplasia. 122 These two reports elucidated roles for BPA in the functions of normal or cancerous progenitor and stem cells, which represent key mechanisms of carcinogenesis. Continued characterization of the molecular and signaling mechanisms underlying the hormone-dependent and -independent effects of BPA in the growth, differentiation, and progression of cells in endocrine organs should bring a better understanding of the risks of BPA exposure in humans. Another major gap in this field is the lack of studies showing the associations of long-term BPA exposure and cancer burdens in humans, which would provide definitive evidence for evaluating the risks of BPA exposure for hormone-related cancers.