**Abstract**

The massive use of bisphenols, actually bisphenol A, in consumer products and food packaging has been associated with certain hazardous conditions for human health, which include their interactions with a family of specific membrane receptors and their effects as endocrine disruptors related to breast cancer. For this reason, bisphenol A was removed from many products, but it has been replaced by structural analogs whose pathways of action and metabolic effects are so far partially unknown. This chapter emphasizes the discovery of bisphenols, their uses in human life, and their impact on health population by focusing on breast cancer. Regarding their mechanisms of action, we have focused on the signaling routes activated by bisphenols following their binding to G protein–coupled receptors.

**Keywords:** estrogen, bisphenols, GPCRs, breast cancer, endocrine disruptors, G protein-coupled estrogen receptor 1 (GPER-1), angiotensin receptors (AT), adrenergic receptors (AR), chemokine receptors

### **1. Introduction**

Significant evidence suggests that endocrine disruption is attributable not only to pharmaceutical products or rare contaminants, but also to exogenous chemical compounds ubiquitously found in everyday life of the modern world. Endocrinedisrupting chemicals (EDCs) enter the human body where they act similarly to endogenous hormones, altering endocrine homeostasis and causing adverse effects on human health [1–7]. Interestingly, the US Food and Drug Administration identified more than 1800 chemical disruptors of endocrine pathways involving estrogen, androgen, and thyroid hormones [8]. EDCs have been related to the development of disorders such as adulthood diabetes, poor semen quality, polycystic ovary syndrome, neurodegenerative disorders, and cancer [1, 8]. Changes in the physiological levels of hormones circulating in the human body may be involved in the high incidence of tumors of the reproductive system in both men and women [8]. Indeed, breast cancer is the most common cancer diagnosed in women worldwide that has been associated in a small percentage with genetic predisposition (*BRCA1* and *BRCA2* mutations) whereas the majority of breast tumors have been categorized as sporadic breast cancer [9]. In fact, lifestyle factors such as smoking, alcohol consumption, sedentary lifestyle, and obesity have been related to the development of the disease. However, an increasing body of evidence suggests that etiology for

breast cancer may be related at least in part to exposure to chemicals of some kind. Indeed, recent studies show that environmental pollutants could also play a role in the pathogenesis and progression of breast cancer. Evidence from epidemiological studies and basic science using *in vitro* and *in vivo* models suggests that exposure to EDCs may be positively correlated with breast cancer development, particularly when the exposure occurred during critical stages of human life. The list of suspected environmental pollutants having a role in breast cancer is extensive and includes polychlorides, biphenyl ethers, phthalates, triclosan, octylphenol, dichlorodiphenyltrichloroethane, and bisphenols (BPs). Bisphenol A (BPA), or 4,40-dihydroxy-2,2-diphenylpropane, is one of the main compounds of this class; BPA is an organic synthetic plastic monomer that was first synthesized in the 1890s as a synthetic estrogen and a key element in the manufacture of cans, reusable water bottles, and medical equipment. BPA regulates several processes, such as cell proliferation, migration, and apoptosis, leading to neoplastic changes due to its ability to mimic the actions of estrogen at multiple levels by activating both α and β estrogen receptors (ERα and ERβ). The effects of BPA on the reproductive system of rats were reported in the 1930s. Now, 91 years later, several studies performed in mice have demonstrated DNA damage, induction of oxidative stress, and epigenetic changes in oocytes [8]. BPA can induce various types of modifications in the reproductive system of men and women, supporting multiple oncogenic signaling routes such as STAT3, PI3K/Akt, and MAPK pathways [8]. Benign lesions that can progress to breast or ovarian cancer due to BPA depend on several molecular and epigenetic mechanisms that will determine whether the endocrine or the reproductive system is affected and will be reviewed in this chapter. Moreover, the effects of BPs on GPCRs associated with breast cancer development or progression are addressed.

### **2. Xenoestrogens derived from anthropogenic activity**

### **2.1 Some historical aspects of bisphenol A and related compounds**

In 1891 Aleksandr Dianin, a Russian chemist from Saint Petersburg, combined phenol with acetone in the presence of an acid catalyst, synthesizing for the first time the chemical substance called 4,40-dihydroxy-2,2-diphenylpropane [10], a molecule that was later recognized by the name of bisphenol A [11]. In 1936, the English scientists Dodds and Lawson reported that BPA exhibited important estrogenic properties inducing complete cornification in vaginal smears of ovariectomized rats treated with this compound [12]. In the 1940s, BPA was basically considered a synthetic estrogen and its potential carcinogenic properties in humans started to be studied [13, 14]. Therefore, BPA is one of the first compounds of anthropogenic origin in which an endocrine-disrupting activity has been verified.

Later in the 1950s, it was found that the reaction of BPA with phosgene generated a polycarbonate, unalterable over time, easy to mold, versatile, and transparent. Due to these multiple qualities, together with its chemical stability, the industry began to use it rapidly and massively to manufacture all types of plastic containers [2]. Currently, BPA has been used to produce various electronic and construction products, automotive parts, medical and clinical articles, toys for children, hygiene and personal care items, and storage products. In addition, it is used for the inner lining of metal cans for preservation of food and beverages [2]. For this reason, BPA is today one of the most used chemical products worldwide. Several studies have suggested that the greatest human exposure to BPA (>90%) is likely to occur through food contamination and, to a lesser extent, by dust ingestion and absorption through the skin or dental surgeries [8].

*Interaction of Bisphenol A with G Protein: Coupled Receptors - New Paradigms in Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.101204*

The proestrogenic activity of BPA resurfaced in the early 1990s when a team led by David Feldman identified through mass spectrometry the presence of this molecule in a growth medium of yeast (*Saccharomyces cerevisiae*) and even in the pure water contained in the autoclaved polycarbonate flasks [15]. In turn, one of the first effects of BPA was evaluated in breast cancer cells. Indeed, in estrogen-sensitive MCF-7 human breast cancer cells (ERα-positive cells), BPA induced a great expression of progesterone receptors and increased their proliferation rate [3, 15]. From this period to date, numerous investigations have reported the potential risk that continuous exposure to BPA implies for human and animal health and ecosystems [3]. This evidence has contributed to consider BPA as one of the main xenoestrogens of ubiquitous environmental distribution. In response to these effects, the industry has sought alternatives to traditional BPA, generating a variety of new bisphenols, such as bisphenol S (BPS), bisphenol AF (BPAF), bisphenol E (BPE), bisphenol B (BPB), and bisphenol F (BPF) among other phenolic molecules, some of which have also been related to estrogenic activity and are considered physiological disruptors of varying degrees in humans [3, 16].

### **2.2 Routes of BPA exposure and metabolism**

The continuous presence of BPA in our environment suggests that several routes of exposure may exist. Oral ingestion seems to be the main route, given the storage of food and liquids in plastic containers that include BPA among its major constituents, which also diffuses into the environment after exposure to high temperatures or frequent washing. The US EPA has established a safe daily intake of 50 g BPA/kg of body weight per day based on the assumption that the main source of exposure to BPA is through food ingestion [17]. Not only in humans but also in primates, ingested BPA is rapidly absorbed (5–15 min later) by the intestinal wall and is transformed into BPA glucuronide following its first passage through the intestine and liver; in addition, a small fraction of BPA is also transformed into a sulfate conjugate [4, 16, 18, 19]. Conjugated forms of BPA are estimated to have no endocrine activity [19, 20]. In murine models, and after oral administration of nanomolar doses of BPA, oxidation products of this compound have been found, suggesting the formation of secondary metabolites with greater estrogenic activity than the parent molecule [5]. BPA has a half-life between 4 and 5 h, and most of the conjugated forms are finally excreted through the urine [4, 5, 16]. Inhalation seems to be another route of entry, inducing cough, bronchospasm, and asthmatic attacks; similarly, eye exposure may cause conjunctivitis, itching, and periorbital edema whereas skin contact usually produces localized redness and inflammation [19].

BPA has been detected in all biological fluids, including serum, urine, cerebrospinal fluid, and milk, in most of today's human populations. In fetal tissues, BPA has been found in concentrations similar to those present in maternal blood, showing that it can cross the transplacental barrier [19]. Furthermore, toxicological data indicates that human embryos and neonates, unlike adults, cannot conjugate BPA increasing its possibility to exert toxic effects [5]. Epidemiological and experimental studies suggest that embryonic exposure to BPA is in the long term related to the occurrence of a series of disorders, such as precocious puberty, infertility, metabolic disorders, and a series of hormone-dependent tumors, like breast cancer [1, 5, 17, 20, 21].

### **2.3 Pathophysiological effects of bisphenols: endocrine, metabolic, and carcinogenic disruptors**

To date, only a few studies have explored the effects produced by exposure to BPs, mainly BPA, during intrauterine or postnatal life together with their effects on general human health. Multiple metabolic disorders, polycystic ovary syndrome, spontaneous abortion, infertility, endometrial hyperplasia, hormone-dependent tumors, immunity alterations, cardiovascular pathologies, neurodegenerative disorders and obesity have so far been reported among their deleterious effects on human health [1]. Dumitrascu et al. in 2019 [1] highlighted that women suffering from polycystic ovary syndrome exhibited higher circulating levels of BPA and testosterone than healthy women and that high androgen levels decreased BPA clearance. Furthermore, they pointed out that women with endometriosis showed high levels of BPA in serum, suggesting an association between this compound and the disease. It has also been suggested that patients with high urinary levels of BPA have a high probability of implantation failure during *in vitro* fertilization procedures. In young people, an early exposure to BPA has been associated with high percentage of body fat, elevated body mass index, and abdominal circumference and numerous neurological implications, such as anxious or depressive behavior, all conditions that have been suggested to increase the risk of developing cancer. Comparative studies between BPA and its analogs (BPB, BPF, and BPS) show that they have toxic effects on the testes and spermatogenesis that are mediated by an increase in the levels of oxidative stress and a decrease in the levels of enzymes with antioxidant activity [22]; some of them may also have a neuroendocrine disrupting activity [6] (see **Figure 1**). Examples of neurobehavioral disorders associated with BPA in different experimental models (rodents, zebrafish, and *Caenorhabditis elegans*) range from cognitive deficit, increased anxiety, socio-sexual deficiencies to hyperactivity or autism spectrum disorders. It is postulated that neurological effects may be due to the weak estrogenic effect of BPA or its analogs by binding to estrogen receptors in different areas of the brain [6, 23].

With regard to the risk of developing malignant neoplasms, the greatest association has been observed with breast, ovarian, and prostate cancer though the studies have not been conclusive [1]. To overcome the effects of BPA associated with an increased public concern about the risk of developing endocrine-related cancer due to exposure to BPA [24], the industry has replaced it with analogs such as BPS, BPB, BPF, or BPAF, which are now parts of products labeled as BPA-free [25]. Nevertheless, *in vitro* assays have demonstrated that BPAF has a stronger binding affinity for estrogen receptors when compared with BPA [26].

Likewise, recent studies have shown that both BPA and BPS can contribute to breast cancer malignancy by disrupting the organization of acinar structures and by affecting the normal development of the mammary gland [9]. To date, the effects of BPA in eukaryotic cells have been reported to be mediated primarily by steroid receptors, including ERɑ and ERβ, estrogen-related receptors (ERR), androgen receptors (AR), and peroxisome proliferator-activated

### **Figure 1.**

*Structure of main bisphenols produced by industrial activity. BPA, bisphenol A; BPF, bisphenol F; BPAF, bisphenol AF; BPS, bisphenol S.*

*Interaction of Bisphenol A with G Protein: Coupled Receptors - New Paradigms in Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.101204*

receptors (PPAR) [24]. Other interactions include signaling by stimulation of angiotensin (AT), α-adrenergic (AR,) or chemokine (CXC) receptors.

## **3. Bisphenols and their role in breast cancer**

Although BPA does not possess the potency of estrogen, it is ubiquitously distributed in nature and its resistance to enzymatic or chemical degradation makes it even more dangerous. Breast cancer has a high mortality in women in many countries, and approximately 10% of these tumors are due to genetic influence whereas 90% are related to lifestyle or associated with negative elements present in the surrounding environment [1]. The most harmful effects attributed to EDCs would occur during breast development when this tissue is more susceptible to developing atypical differentiation. *In vivo* studies show that prolonged exposure (60 days) of breast cells to 400 μg of BPA/kg of body weight induces an increase in the density of mammary buds [27], in cell proliferation, and in the levels of oxidative stress without significant differences in the expression of estrogen receptors [1, 27, 28]. Previous studies have demonstrated that women who exhibit mutations in the tumor suppressor genes *BRCA1* and *BRCA2* in mammary gland cells have a greater risk of developing breast cancer and also a high susceptibility to the negative effects of environmental BPA [1, 27, 29]. In normal or cancerous adult breast tissue, BPA has been associated with an increased proliferation rate and with the induction of chemoresistance in ERα-positive cells [1, 30, 31]. BPA can also modify DNA repair, inactivate p53, and induce changes in genes associated with apoptosis to stop cell death through DNA methylation. By activating vascular endothelial growth factor, BPA can increase angiogenesis in breast tumors [32] and at the same time activate the MAPK and STAT pathways to modulate the proliferation kinetics of human mammary epithelial cells [1, 33].

Breast cell cultures developed in a 3D fashion are one of the most widely used models to gain a better understanding of the role of bisphenols in breast cancer. Using this approach and MCF-12A cells, which exhibit a typical luminal epithelial morphology, it was observed that low doses of BPA and BPS generated a disruption in the normal organization of the mammary acinus and promoted cell invasion [9]. Interestingly, mammospheres of MCF-7 cells (ERα-positive cell line) treated with 10 nM BPA displayed high expression of aldehyde dehydrogenase 1, a marker of breast stem cells, and SOX-2, a key transcription factor for cell pluripotentiality and self-renewal. That effects were not observed when MDA-MB-231 (ERα-negative cell line) was used instead of MCF-7 cells, suggesting that the receptors through which BPA modulates its signaling exhibit a different expression pattern in this kind of cancer, highlighting the implications of the heterogenicity of tumor mammary tissue when evaluating the effects of EDCs [34].

Evidence suggests that BPA may play an important role in the lifecycle and carcinogenesis of mammary epithelial cells and challenge us to continue studying its role in the origin and progression of breast cancer. One approach is to determine the relationship between BPA and G protein–coupled receptors (GPCRs) signaling, considering the recent evidence of the role of GPER-1 in breast cancer progression. Interestingly, approximately 20% of human neoplasms are related to some alteration in GPCRs [35]. The first relationship of GPCRs with tumorigenesis dates back to the 1980s, when a novel proto-oncogene called *MAS1* was described together with its ability to encode a hydrophobic protein of 325 amino acids with seven transmembrane domains. Today, it is known that the *MAS1* proto-oncogene generates a GPCR that binds to angiotensin (1–7), a metabolite of angiotensin II [35]; therefore, MAS1 receptor is part of the signaling cascade that supports the endocrine activity

### *Bisphenols*

of the renin-angiotensin-aldosterone system (RAS), which regulates the proliferative or antiproliferative effects produced by hormones participating in the RAS pathway [36]. At present, it has been shown that components of RAS are expressed in various types of cancer, including breast cancer [37]. Additionally, several members of the large family of GPCRs have been identified as promoters of carcinogenesis, interacting directly or indirectly with BPA and other phenolic compounds to generate disruptive effects not only during adulthood but also during intrauterine and early postnatal life.
