Elsevier

Toxicology

Volume 277, Issues 1–3, 9 November 2010, Pages 20-28
Toxicology

Structure-based investigation on the binding interaction of hydroxylated polybrominated diphenyl ethers with thyroxine transport proteins

https://doi.org/10.1016/j.tox.2010.08.012Get rights and content

Abstract

Polybrominated diphenyl ethers (PBDEs) have been shown to alter thyroid hormone level in experimental animals. One of the possible mechanisms for hormone disruption is the competitive binding of hydroxylated PBDEs (OH-PBDEs) with hormone transport proteins. In this study, binding interaction of 14 diversely structured OH-PBDEs with two thyroxine transport proteins was investigated by fluorescence displacement assay, circular dichroism, and molecular docking. Binding affinity of the 14 OH-PBDEs with transthyretin (TTR) and thyroxine-binding globulin (TBG) was measured by competitive fluorescence displacement assay. The binding constant was found to fall in the range of 1.4 × 107 M−1 and 6.9 × 108 M−1 for TTR, and between 6.5 × 106 M−1 and 2.2 × 108 M−1 for TBG. Binding affinity increased significantly with bromination number from 1 to 4, whereas 5- and 6-brominated diphenyl ethers did not show any further increase. Protein secondary structural change of TTR and TBG upon binding with 5-OH-BDE-047 was investigated by circular dichroism. The spectral change displayed a pattern similar to the one with thyroxine, suggesting that the environmental chemical binds to the two proteins at the same sites as the hormone. In molecular docking analysis, a ligand-binding channel in TTR was revealed for OH-PBDEs binding, which appeared to be mostly hydrophobic inside but guarded by positively charged residue Lys15 at the entrance. Binding affinity of the 14 OH-PBDEs with TTR could be rationalized reasonably well by their pocket binding mode and hydrophobic characteristics. Based on the binding constant obtained in this work, possibility of in vitro competitive displacement of thyroid hormones from the transport proteins by OH-PBDEs was evaluated.

Introduction

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants used in a large number of consumer products. Before they were regulated, approximately 70,000 t was produced every year worldwide (De Wit, 2002, Tanabe, 2004). In 2004 the European Union began to phase out the use of two PBDE commercial mixtures, PentaBDE and OctaBDE, and in 2005 the US manufacturer stopped manufacturing these two PBDEs. Currently, decabromodiphenyl ether (BDE-209), a fully brominated BDE congener, is the only unregulated PBDE in production. Due to their widespread use and high stability, PBDEs have become ubiquitous environmental contaminants with the concentration increasing in the past 30 years. They have been detected in almost all environmental media including soil, air, sediments, birds, fish, human tissues, blood and breast milk (Darnerud et al., 2001, Rahman et al., 2001). Five tetra-, penta- and hexa-BDE congeners (BDE-47, BDE-99, BDE-100, BDE-153, BDE-154) are the most commonly found PBDEs in human samples (Sjodin et al., 1999). It has been suggested that these lower brominated congeners are the degradation products of BDE-209 in the environment. In addition, hydroxylated PBDE metabolites have also been detected in human samples (Athanasiadou et al., 2008, Qiu et al., 2009).

Several studies have shown that in vivo exposure of experimental animals to PBDEs results in reduction of the thyroid hormone thyroxine (T4) level in serum (Fowles et al., 1994, Hallgren et al., 2001, Zhou et al., 2001, Zhou et al., 2002). T4 and triiodothyronine (T3) are essential for the modulation of the cellular metabolic rate and for the development and differentiation of brain tissues. There is a link between the decreased T4 concentration and the developmental neurotoxic and behavioral effects observed for rodents exposed to PBDEs (Eriksson et al., 2001, Branchi et al., 2002, Viberg et al., 2002). The contaminants may interfere with the thyroid hormone system by one of the following three mechanisms: (a) thyroid gland function and regulation; (b) thyroid hormone metabolism; and (c) thyroid hormone transport (Hallgren and Darnerud, 2002). Due to the close structural resemblance with T4, the bio-activated PBDE hydroxylate metabolites have been shown to bind with high affinity to the thyroid hormone transport proteins and displace the hormone. In human plasma, thyroxine-binding globulin (TBG) and transthyretin (TTR) are the two major thyroxine transport proteins, each carrying 75% and 20% of total T4. In plasma of rodents, TTR is the major T4 transport protein (Larsson et al., 1985, Savu et al., 1989). Meerts et al. investigated seventeen different PBDE congeners and three synthesized PBDE hydroxylates for their competitive binding to TTR in vitro, using 125I labeled T4 as the radio-tracer (Meerts et al., 2000). The results revealed for the first time that the three hydroxylated PBDEs bound to human TTR with extremely high potency (Ka  107 M−1) and yet none of the 17 PBDEs competed with T4-TTR binding. Similarly, 6-OH-2,2′,4,4′-tetrabromodiphenyl ether (6-OH-BDE-47), the metabolite found in human plasma, also bound to TTR with relatively high affinity (Hamers et al., 2006, Legler and Brouwer, 2003). More recently, the same research group identified six hydroxylated metabolites of BDE-47 after its incubation with rat liver microsomes (Hamers et al., 2008). All the metabolites showed higher binding potency for TTR than T4, with 3-OH-BDE-47 being the most potent. Lately, a surface plasmon resonance (SPR) biosensor-based screening method was developed for examining the binding of chemicals with thyroid hormone transport proteins TTR and TBG (Marchesini et al., 2008). The results confirmed previous finding in radio-labeled competition assays that the tetra- and penta-brominated hydroxylated BDEs had high affinity for TTR. Strikingly, the results also showed that the PBDE metabolites had the most potent binding with TBG in all the tested chemicals.

Overall, data on the binding of PBDEs and their metabolites with thyroid hormone transport proteins are still very limited. Previous studies focused almost exclusively on the hydroxylated metabolites of BDE-47. Given the complex pattern of environmental distribution, human exposure and in vivo metabolism, both the number and the structural variation of PBDE metabolites need to be expanded greatly for such studies. In a recent work, BDE-47 was found to be the dominant congener in human sample, but 4-OH-BDE-90 was the most abundant metabolite (Athanasiadou et al., 2008). Another problem is, in all the previous studies except one, the binding affinity of PBDEs with TTR and TBG was assessed with IC50 and relative potency, not binding constant. The lack of binding constant makes it difficult to evaluate quantitatively the potential disruption of thyroid hormone binding to the transport proteins by PBDEs if their concentration is different from that of the hormones. Thirdly, although some studies have identified the number of bromine atoms, the position of bromination, and hydrogen bonding function as important factors in the binding of PBDE to TTR (Legler and Brouwer, 2003), the structure–activity relationship has not been investigated extensively nor quantitatively.

With these questions in mind, we have investigated the binding of fourteen diversely structured PBDE hydroxylates with thyroid hormone transport proteins. Their binding constant with TTR and TBG was obtained by the fluorescence displacement measurement, and was then used for the quantitative evaluation of potential disruption on T3 and T4 binding with the two transport proteins. Molecular docking analysis coupled with Hansch model was performed to investigate the intrinsic structural basis of PBDE–protein binding.

Section snippets

Materials

All the fourteen mono-hydroxylated PBDEs (Fig. 1), 2′-hydroxy-4-monobromodiphenyl ether (2′-OH-BDE-003), 3′-hydroxy-2,4-dibromodiphenyl ether (3′-OH-BDE-007), 2′-hydroxy-2,4-dibromodiphenyl ether (2′-OH-BDE-007), 4′-hydroxy-2,2′,4-tribromodiphenyl ether (4′-OH-BDE-017), 3′-hydroxy-2,4,4′-tribromodiphenyl ether (3′-OH-BDE-028), 2′-hydroxy-2,4,4′-tribromodiphenyl ether (2′-OH-BDE-028), 3-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether (3-OH-BDE-047), 5-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether

Binding of fluorescent probe to thyroid hormone transport proteins

As mentioned above, although the binding interaction of a few OH-PBDEs with TTR and TBG has been investigated previously, the binding constant has not been obtained. We therefore set out to investigate the binding interaction by first measuring the binding constant with the use of a fluorescence displacement method. In our work, ANSA was employed as the fluorescence probe because it is a well established probe for proteins, and has been shown to act as a competitive inhibitor with thyroxine for

Conclusions

Binding interaction of 14 OH-PBDEs with two thyroid hormone transport proteins was investigated by fluorescence displacement assay, circular dichroism, and molecular docking analysis. The binding constant with TTR and TBG was obtained for the first time by competitive fluorescence displacement measurement. The binding affinity was highly dependent on the number of bromines, with 5-OH-BDE-047 possessing the strongest affinity for TTR. The structural dependence was rationalized by

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (20890112, 20921063, and 20825519). We thank Ms. Zhou Mei from Tri-ibiotech (Shanghai) Ltd. for providing the computational environment.

References (50)

  • S.F. Nilsson et al.

    Studies on thyroid hormone-binding proteins I the subunit structure of human thyroxine-binding globulin and its interaction with ligands

    J. Biol. Chem.

    (1975)
  • S.F. Nilsson et al.

    Studies on thyroid hormone-binding proteins II binding of thyroid hormones, retinol-binding protein, and fluorescent probes to prealbumin and effects of thyroxine on prealbumin subunit self-association

    J. Biol. Chem.

    (1975)
  • F. Rahman et al.

    Polybrominated diphenyl ether (PBDE) flame retardants

    Sci. Total Environ.

    (2001)
  • L. Savu et al.

    Thyroxine-binding globulin and thyroxine-binding prealbumin in hypothyroid and hyperthyroid developing rats

    Biochim. Biophys. Acta

    (1989)
  • S.M. Snyder et al.

    Binding of thyroid hormones and their analogues to thyroxine-binding globulin in human serum

    J. Biol. Chem.

    (1976)
  • S. Tanabe

    PBDEs, an emerging group of persistent pollutants

    Mar. Pollut. Bull.

    (2004)
  • J.T. Yang et al.

    Calculation of protein conformation from circular dichroism

    Methods Enzymol.

    (1986)
  • M. Athanasiadou et al.

    Polybrominated diphenyl ethers (PBDEs) and bioaccumulative hydroxylated PBDE metabolites in young humans from Managua, Nicaragua

    Environ. Health Perspect.

    (2008)
  • C.C.F. Blake et al.

    Protein–DNA and protein–hormone interactions in prealbumin: a model of the thyroid hormone nuclear receptor?

    Nature

    (1977)
  • D.L. Boger et al.

    A simple, high-resolution method for establishing DNA binding affinity and sequence selectivity

    J. Am. Chem. Soc.

    (2001)
  • L. Celik et al.

    Exploring interactions of endocrine-disrupting compounds with different conformations of the human estrogen receptor α ligand binding domain: a molecular docking study

    Chem. Res. Toxicol.

    (2008)
  • S.Y. Cheng et al.

    Analysis of thyroid hormone binding to human serum prealbumin by 8-anilinonaphthalene-1-sulfonate fluorescence

    Biochemistry

    (1977)
  • Y.C. Cheng et al.

    Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction

    Biochem. Pharmacol.

    (1973)
  • V. Cody et al.

    Molecular conformation of ammonium 8-anilino-1-naphthalenesulfonate hemihydrate. A fluorescent probe for thyroxine binding to thyroxine binding globulin

    J. Med. Chem.

    (1977)
  • P.O. Darnerud et al.

    Polybrominated diphenyl ethers: occurrence, dietary exposure, and toxicology

    Environ. Health Perspect.

    (2001)
  • Cited by (0)

    View full text