Elsevier

Chemosphere

Volume 112, October 2014, Pages 362-369
Chemosphere

Estrogenic and androgenic activities of TBBA and TBMEPH, metabolites of novel brominated flame retardants, and selected bisphenols, using the XenoScreen XL YES/YAS assay

https://doi.org/10.1016/j.chemosphere.2014.04.080Get rights and content

Highlights

  • Carboxylic acid metabolites TBBA and TBMEPH are ER and AR antagonists.

  • TBMEPH exhibited an IC50 value of 265 nM in the ER antagonist YES assay.

  • BADGE and BisGMA demonstrated weak anti-estrogenic and anti-androgenic activities.

Abstract

The present study investigated and compared the estrogenic and androgenic activities of the three different classes of environmental pollutants and their metabolites using the XenoScreen XL YES/YAS assay, which has advantages compared with the original YES/YAS protocol. Contrary to the parent brominated flame retardants TBB and TBPH, which demonstrated no or very weak (anti)estrogenic or (anti)androgenic activities, their metabolites, TBBA and TBMEPH, exhibited anti-estrogenic (IC50 for TBBA = 31.75 μM and IC50 for TBMEPH = 0.265 μM) and anti-androgenic (IC50 for TBBA = 73.95 μM and IC50 for TBMEPH = 2.92 μM) activities. These results reveal that metabolism can enhance the anti-estrogenic and anti-androgenic effects of these two novel brominated flame retardants. Based on the activities of BPAF, BPF, BPA and MBP, we can conclude that the XenoScreen XL YES/YAS assay gives comparable results to the (anti)estrogenic or (anti)androgenic assays that are reported in the literature. For BPA, it was confirmed previously that the metabolite formed after an ipso-reaction (hydroxycumyl alcohol) exhibited higher estrogenic activity compared with the parent BPA, but this was not confirmed for BPAF and BPF ipso-metabolites, which were not active in the XenoScreen YES/YAS assay. Among the substituted BPA analogues, bis-GMA exhibited weak anti-estrogenic activity, BADGE demonstrated weak anti-estrogenic and anti-androgenic activities (IC50 = 13.73 μM), and the hydrolysed product BADGE·2H2O demonstrated no (anti)estrogenic or (anti)androgenic activities.

Introduction

A potential endocrine-disrupting chemical (EDC) is an exogenous substance or mixture that possesses properties that might be expected to lead to endocrine disruption in an intact organism, its progeny, or (sub) populations (WHO, 2002). EDCs encompass a variety of chemical classes, including compounds used in the plastics industry and in consumer products, which can act via receptor-mediated and/or non-receptor-mediated mechanisms to influence endocrine system function (Henley and Korach, 2006, Schmidt and Peterlin Mašič, 2012). Most of the reported health effects of EDCs result from their interference with nuclear receptor-mediated hormonal signalling (Molina-Molina et al., 2013).

Brominated flame retardants are classified as potential EDCs (Legler and Brouwer, 2003), and several of them are environmentally persistent, can be bioaccumulated, and are capable of long-range atmospheric transport (Wit et al., 2011). 2-Ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB) and bis(2-ethylhexyl)2,3,4,5-tetrabromophthalate (TBPH) (Fig. 1) are novel brominated flame retardants that are widely used as a replacement for polybrominated diphenyl ethers in several commercial flame retardant mixtures, such as Firemaster 550, Firemaster BZ-54, and DP-45™, as marketed by Chemtura (New Materials International, 2003, Covaci et al., 2011). They have also been identified in indoor dust at concentrations that have often exceeded 1 μg g−1 (Stapleton et al., 2008, Stapleton et al., 2009). Atmospheric concentrations of TBB and TBPH are increasing, and the US. EPA has listed them both as high production volume chemicals (Ma et al., 2012). Recently, TBPH has been found in human serum, while TBB levels in human tissues have not been evaluated yet (He et al., 2013). However, TBB transplacental and/or lactational transfer was observed in rats (Patisaul et al., 2013). The ratio of TBB to TBPH in the Firemaster 550 commercial flame retardant mixture is 4:1 (Stapleton et al., 2008); therefore, Roberts et al. (2012) emphasized the importance of TBB metabolism, which is consistently and rapidly metabolized by porcine carboxylases to the TBBA metabolite (Fig. 1). While rapid TBBA formation might reduce TBB bioaccumulation in mammals, the toxicity of this brominated benzoic acid metabolite (Fig. 1) is unknown. Conversely, TBPH metabolism to its monoester metabolite TBMEHP (Fig. 1) is slower and, as proposed by Roberts et al. (2012), might not be rapid enough to affect TBPH bioaccumulation. In a recent study, Patisaul et al. (2013) demonstrated that the TBPH molecule can be absorbed and that it bioaccumulates in rat liver after oral administration, although they were not able to detect the TBMEPH metabolite in rat tissues. The TBMEPH metabolite was toxicologically evaluated by Springer et al. (2012), and they reported that TBMEPH elicited maternal thyrotoxic and hepatotoxic effects and induced multinucleated germ cells in the rat foetal testes. Patisaul et al. (2013) described that exposure to the Firemaster 550 mixture could lead to endocrine disruption, while Saunders et al. (2013) recently studied in vitro endocrine disruption of the parent brominated flame retardants TBB and TBPH using the yeast YES/YAS reporter assay.

The bisphenols BPA, BPAF, and BPF (Supplementary material Fig. S1) are known for their estrogenic and anti-androgenic effects (Matsushima et al., 2010, Grignard et al., 2012, Molina-Molina et al., 2013). Yoshihara et al., 2001, Yoshihara et al., 2004 discovered that incubation of BPA with the rat liver S9 fraction increased BPA estrogenicity by several times, and the active estrogenic metabolite was 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene (MBP) (Fig. S1). Moreover, Nakamura et al. (2011) observed an enhancement of the estrogenic activity of the BPA metabolite hydroxycumyl alcohol (HCA) that was formed via an ipso-substitution reaction and catalysed by cytochrome P450. In addition to BPA, Schmidt et al. (2013) extended the hypothesis of Nakamura et al. to bisphenols BPAF and BPF, where both bisphenols can be metabolized in vitro similar to BPA via an ipso-substitution reaction that was catalysed by P450 (Fig. S1).

Bisphenol A diglycidyl ether (BADGE), which is used as an interior coating material for food cans, can migrate from epoxy-based packing materials into food and can be easily hydrolysed to bisphenol A bis(2,3-dihydroxypropyl) ether (BADGE·2H2O) (Supplementary material Fig. S2) (Satoh et al., 2004, Miguez et al., 2012). Both are common contaminants in indoor dust that were found in all of the analysed urine samples from the US and China so far (Wang et al., 2012). Bisphenol A glycerolate dimethacrylate (bis-GMA) is used in dental materials and can also be hydrolysed to BADGE·2H2O, and both compounds have been observed in guinea pig urine samples (Seiss et al., 2009, Fleisch et al., 2010). BADGE and bis-GMA are both BPA analogues where both phenolic groups are substituted with ether moieties. The impact of the elimination and modification of both phenolic groups on estrogenic activity has been confirmed (Nakazawa et al., 2002, Nomura et al., 2003), but some studies suggest possible anti-androgenic and anti-estrogenic effects of these compounds (Satoh et al., 2004, Terasaki et al., 2006).

The purpose of this investigation was to determine and compare the (anti)estrogenic and (anti)androgenic activities for (i) the carboxylic acid metabolites TBBA and TBMEPH (Fig. 1) and their parent brominated flame retardants TBB and TBPH, (ii) the metabolites BPAF-M and BPF-M that are formed after an ipso-reaction catalysed by P-450 of BPAF and BPF, and BPA, MBP, BPAF and BPF that have well-known estrogenic or anti-androgenic activities, and (iii) bisphenol ethers bis-GMA, BADGE, and BADGE·2H2O that possess alkyl groups attached to both phenolic groups. Agonistic and antagonistic activities were measured using the yeast reporter assay (XenoScreen XL YES/YAS assay). In vitro yeast-based reporter assays for detecting chemicals with potential endocrine-disrupting activity have been firmly established and are commonly used for detecting chemicals with potential endocrine activity (Sanseverino et al., 2009). Advantages of the assay used in this study compared with the original YES/YAS protocol (Routledge and Sumpter, 1996) include greatly reduced assay time, and easier handling because of ready-to-use components. The main differences are the shorter incubation time (18 h vs. 3 d), and therefore the shorter time needed to conduct and complete the assay, which is due to use of a lyticase/detergent based lysis step, and the absence of the chlorophenol red-β-D-galactopyranoside (CPRG) substrate during the exposure phase, which eliminates any possible interference of CPRG with the induction of the response (Vanderperren et al., 2001).

Section snippets

Chemicals

BPA (>99% pure, CAS # 80-05-7), MBP (92% pure, CAS # 13464-24-9), BPAF (97% pure; CAS # 1478-61-1), BPF (98% pure, CAS # 620-92-8), bis-GMA (⩾99% pure, CAS # 1565-94-2), and BADGE·2H2O (⩾97% pure, CAS # 5581-32-8) were obtained from Sigma (St. Louis, MO, USA). TBB, 95% pure, CAS # 183658-27-7) and TBPH (97% pure, CAS # 26040-51-7) were from TRC (North York, Ontario, Canada). TBBA, ⩾98% pure) and TBMEPH (⩾98% pure) were synthesised in our laboratory (for synthesis see Supplementary material).

Screening

First, all tested compounds were screened in a concentration range of 300 μM to 30 nM (or from 30 μM to 3 nM for compounds that were insoluble at 300 μM) (Table S1). For nine tested compounds that were positive at at least one non-cytotoxic concentration we observed (i) estrogen-like activity for BPA, MBP, BPAF and BPF; (ii) androgen-like activity for BPF; (iii) an anti-estrogenic effect for bis-GMA, BADGE, TBBA, TBPH and TBMEPH; and (iv) an anti-androgenic effect for BPA, MBP, BPAF, BPF, BADGE,

Discussion

Our investigation elucidated and compared the endocrine disrupting properties of three different classes of environmental pollutants and their metabolites (Figs. 1, S1, S2) using the XenoScreen XL YES/YAS assay and among the tested compounds, special attention was focused on two carboxylic acid metabolites TBBA and TBMEPH of the two novel brominated flame retardants TBB and TBPH, which are components of several flame retardant technical mixtures that have been discovered in numerous

Conclusion

In conclusion, this is one of the first studies to demonstrate that metabolism can enhance the anti-estrogenic and anti-androgenic effects of two novel brominated flame retardants, TBB and TBPH. The mono ester metabolite TBMEPH of the parent flame retardant TBPH had an IC50 value of 265 nM and was the most active among all of the tested compounds in the ER antagonist YES assay. Among bisphenol ethers, bis-GMA exhibited weak antagonistic activity only on estrogen, BADGE demonstrated weak

Acknowledgements

The authors would like to acknowledge the gift of the XenoScreen XL YES/YAS assay kits from Xenometrix AG, Allschwil, Switzerland. This work was supported by the Slovenian Research Agency (Grant No. Program P1-0208 and Program P1-0245).

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