In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors

Highlights

• We investigated the agonist/antagonist activities of BPS, BPF, BPA, TCBPA and TBBPA.

• The direct interaction of these compounds with hERα, hERβ, hAR and hPXR was studied.

• BPA congeners and derivatives were found to disrupt multiple NRs.

• Further evaluation of their role as endocrine-disrupting chemicals is needed.

Abstract

Bisphenols are a group of chemicals structurally similar to bisphenol-A (BPA) in current use as the primary raw material in the production of polycarbonate and epoxy resins. Some bisphenols are intended to replace BPA in several industrial applications. This is the case of bisphenol-S (BPS), which has an excellent stability at high temperature and resistance to sunlight. Studies on the endocrine properties of BPS have focused on its interaction with human estrogen receptor alpha (hERα), but information on its interaction with other nuclear receptors is scarce. The aim of this study was to investigate interactions of BPS, BPF, BPA and its halogenated derivatives, tetrachlorobisphenol A (TCBPA), and tetrabromobisphenol A (TBBPA), with human estrogen receptors (hERα and hERβ), androgen receptor (hAR), and pregnane X receptor (hPXR), using a panel of in vitro bioassays based on competitive binding to nuclear receptors (NRs), reporter gene expression, and cell proliferation assessment. BPS, BPF, and BPA efficiently activated both ERs, while TCBPA behaved as weak hERα agonist. Unlike BPF and BPA, BPS was more active in the hERβ versus hERα assay. BPF and BPA were full hAR antagonists (BPA > BPF), whereas BPA and BPS were weak hAR agonists. Only BPA, TCBPA, and TBBPA, were hPXR agonists (TCBPA > TBBPA > BPA). These findings provide evidence that BPA congeners and derivatives disrupt multiple NRs and may therefore interfere with the endocrine system. Hence, further research is needed to evaluate the potential endocrine-disrupting activity of putative BPA substitutes.

Introduction

Over the past few decades, concerns have grown about the possible health threat posed by endocrine-disrupting chemicals (EDCs), i.e., substances in our environment, food, and consumer products that interfere with hormone biosynthesis, metabolism, or activity and produce a deviation from normal homeostatic control or reproduction (Diamanti-Kandarakis et al., 2009). The direct interaction of chemicals, acting as receptor agonists or antagonists, with nuclear receptors (NRs), is a well-known mechanism of endocrine disruption. NRs are members of the steroid receptor superfamily, a large family of ligand-dependent transcriptional factors (Germain et al., 2006). Most research on EDCs has focused on their deleterious effects on sexual development and reproduction caused by interference with steroid signaling via human estrogen (hER) and androgen (hAR) receptors, because the outcome is readily identifiable and represents a sensitive health issue for a wide public (Henley and Korach, 2006). However, more recent reports have shown that several environmental chemicals can also affect hormone metabolism and synthesis by regulating their related enzymes, e.g., cytochrome P450, as activators of other NRs (Tabb and Blumberg, 2006), such as the human pregnane X receptor (hPXR). Indeed, activation of hPXR and up-regulation of their target genes by numerous compounds can increase the levels of endocrine-disrupting metabolites while at the same time altering the local bioavailability of endogenous androgens and estrogens. This provides a pathway for EDCs to alter steroid receptor activity without directly binding to steroid receptors. The problem posed by EDCs was addressed by European regulation (EU, 2006) on Registration, Evaluation, Authorization and Restriction of Chemicals (REACH), which set out the steps for authorizing their use and called for the development of safer alternatives. Subsequently, the European Commission (EC) published a new Directive (EU, 2011) that amended Directive 2002/72/EC to restrict the use of bisphenol-A (BPA) in plastic infant feeding bottles. Currently, a law banning the use of BPA in food packaging has passed its final stage in the French Senate and is set to be implemented in 2013 for packaging for children under the age of three and for all food packaging in France in 2015. The National Assembly of France has asked the government to submit a report on the potential toxicity of possible alternatives to BPA before 1 July 2014, six months before the ban takes effect.

BPA [2,2-bis(4-hydroxyphenyl)propane], one of the highest production volume chemicals worldwide (Vandenberg et al., 2010), is an industrial chemical used to make a hard clear plastic known as polycarbonate (Fiege et al., 2000), a component of numerous consumer products. BPA is also found in epoxy resins, which act as a protective lining on the inside of metal-based food and beverage cans. BPA has been detected in the environment (Kang et al., 2007) and in human fluids and tissues (Calafat et al., 2008, Jiménez-Díaz et al., 2010), and its toxicity has been intensively studied since the 1970s. Despite possessing only modest estrogenic activity in comparison to the natural estrogen 17β-estradiol (E₂), BPA has produced a range of adverse effects in laboratory animals, and major concerns have been raised about its impact on reproductive systems (Richter et al., 2007). Further receptor-mediated biological activities have been reported in different model systems, e.g., binding to the orphan estrogen-related receptor gamma (ERRγ) (Okada et al., 2008), thyroid hormone disruption (Moriyama et al., 2002), altered pancreatic β-cell function (Ropero et al., 2008), and obesity promotion (Newbold et al., 2008). However, although BPA is a well-known EDC, the effects of low doses remain controversial (Vandenberg et al., 2012).

Several chemicals that are structurally similar to BPA are utilized in the manufacture of resins and plastics. They consist of two phenolic rings joined by a bridging carbon or other chemical structures (Fig. 1) and are designated BPA analogs, congeners or bisphenols. Some of these are considered candidates for the partial replacement of BPA in the industrial applications, including bisphenol-S [bis(4-hydroxyphenyl)sulfone (BPS)], whose two phenolic rings are linked by a sulfur dioxide (SO₂) group. BPS is of interest for the preparation of high temperature resistant thermosetting thermoplastic polymers (Spitsbergen et al., 1971). BPS-based epoxy resins resist deformation by heat and thermal stability and offer shorter gelling gel times, the more rapid development of mechanical properties in cured systems, improved resistance to organic solvent attack, increased dimensional stability, and better wetting of glass reinforcements (Rwei et al., 2003). As well as in epoxy resins, BPS is widely used as a monomer in the production of cyclic carbonates (Kim et al., 2001) and sulfonated poly(ether ketone ether sulfone) (Changkhamchom and Sirivat, 2010), and is a chemical additive in pesticides, dyestuffs, color-fast agents, leather tanning agents, dye dispersants, and fiber improvers. BPS replaced BPA as a developer in dyes for thermal paper in Japan (Watanabe et al., 2004) and China (Liu, 2005) and has been detected in canned food (Viñas et al., 2010) and in paper products and currency bills (Liao et al., 2012a). In fact, widespread exposure of the general population to BPS has been demonstrated in various countries, with the detection of BPS levels ranging from 0.02 to 21 ng/ml (0.8–84 nM) in urine samples from people living in the U.S. and seven Asian countries (Liao et al., 2012b). BPS is much less biodegradable than BPA (Danzl et al., 2009; Ike et al., 2006) and, given its annually increasing production, it is expected to become as widespread as BPA (Liao et al, 2012c). There has been less research on BPS than on BPA, but preliminary studies have shown that it also possesses hormone-mimicking properties (Chen et al., 2002, Delfosse et al., 2012, Grignard et al., 2012, Hashimoto et al., 2001; Kitamura et al., 2005; Kuruto-Niwa et al., 2005). However, studies on BPS as an endocrine disrupter have focused on its interaction with human estrogen receptor alpha (hERα), and much less is known about its interaction with other NRs.

Bisphenol-F, [bis(4-hydroxyphenyl)methane, (BPF)], which has no substituent at the bridging carbon (except with H atoms), has a broad range of industrial applications. The BPF monomer is polymerized to prepare epoxy resins and polycarbonates for use in the manufacture of lacquer, varnishes, coatings, adhesive plastics, and other products (Jana et al., 2005). Although no information is available on human exposure, BPF has been detected in the environment (Fromme et al., 2002, Stachel et al., 2003) and has demonstrated an estrogenic effect in various in vivo (Yamasaki et al., 2002) and in vitro studies (Cabaton et al., 2009, Hashimoto and Nakamura, 2000, Hashimoto et al., 2001). BPF has also shown anti-androgenic activity in several human recombinant cell lines carrying hAR (Cabaton et al., 2009, Satoh et al., 2004).

Halogenated derivatives of BPA, such as tetrabromobisphenol-A [2,2-bis(4-hydroxy-3,5-dibromophenyl)propane, (TBBPA)] and tetrachlorobisphenol-A [2,2-bis(4-hydroxy-3,5-dichlorophenyl)propane, (TCBPA)] are both widely used as flame-retardants for building material, paints, and epoxy resin-containing plastic products such as electronic circuit boards, and other electronic equipment. Like BPA, both compounds are considered environmental contaminants (De Wit et al., 2009, Fukazawa et al., 2001) and have also been reported in human fluids and/or tissues (Cariou et al., 2008, Fernández et al., 2007, Jiménez-Díaz et al., 2010, Johnson-Restrepo et al., 2008). Moreover, these compounds have been found to interact with and disrupt thyroid hormone receptor signaling (Kitamura et al. 2002). TBBPA and TCBPA are also potent peroxisome proliferator-activated receptor gamma (PPARγ) agonists (Riu et al., 2011a).

As noted, research has focused mainly on the endocrine disrupting activity of BPA, and much less attention has been paid to the toxicity of the other bisphenols proposed as substitutes, such as BPS. The present study was designed to develop a comprehensive NR interaction profile of five bisphenols in current use (BPS, BPF, BPA, TCBPA and TBBPA) in order to contribute additional information on their endocrine disruptive activity. For this purpose, we investigated the direct interaction of these compounds with hERα, hERβ, hAR, and hPXR, using a panel of steroid hormone receptor cell based assays to measure different endpoints at distinct levels of biological complexity.