Thyroid Disruption by Bisphenol S Analogues via Thyroid Hormone

May 15, 2018 - College of Environment, Zhejiang University of Technology, Hangzhou ... The global usage of BPS and its analogues (BPSs) resulted in th...
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Ecotoxicology and Human Environmental Health

Thyroid Disruption by Bisphenol S Analogues via Thyroid Hormone Receptor #: in Vitro, in Vivo and Molecular Dynamics Simulation Study Liping Lu, Tingjie Zhan, Mei Ma, Chao Xu, Jingpeng Wang, Chunlong Zhang, Weiping Liu, and Shulin Zhuang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00776 • Publication Date (Web): 15 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018

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Thyroid Disruption by Bisphenol S Analogues via Thyroid

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Hormone Receptor β: in Vitro, in Vivo and Molecular Dynamics

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Simulation Study

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Liping Lu†, Tingjie Zhan†, Mei Ma‡,||, Chao Xu§, Jingpeng Wang†, Chunlong Zhang⊥,

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Weiping Liu†, Shulin Zhuang†,*

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College of Environmental and Resource Sciences, Zhejiang University, Hangzhou

310058, China. ‡

Key Laboratory of Drinking Water Science and Technology, Research Center for

Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. ||

College of Resources and Environment, University of Chinese Academy of Sciences,

Beijing 100085, China. §

College of Environment, Zhejiang University of Technology, Hangzhou 310032,

China. ⊥

Department of Biological and Environmental Sciences, University of Houston-Clear

Lake, 2700 Bay Area Blvd., Houston, Texas 77058, USA.

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* Address correspondence to E-mail: [email protected] (S. Zhuang).

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ABSTRACT Bisphenol S (4-hydroxyphenyl sulfone, BPS) is increasingly used as BPA

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alternatives. The global usage of BPS and its analogues (BPSs) caused the frequent

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detection of their residues in multiple environmental media. We investigated their

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potential endocrine disrupting effects toward thyroid hormone receptor (TR) β. The

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molecular interaction of BPSs toward TRβ ligand binding domain (LBD) was probed

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by fluorescence spectroscopy and molecular dynamics (MD) simulations. BPSs

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caused the static fluorescence quenching of TRβ LBD. The 100 ns MD simulations

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revealed that the binding of BPSs caused significant changes of the distance between

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residue His435 at helix 11(H11) and residue Phe459 at H12 in comparison with no

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ligand-bound TRβ LBD, indicating relative repositioning of H12. The recombinant

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two-hybrid yeast assay showed that tetrabromobisphenol S (TBBPS) and

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tetrabromobisphenol A (TBBPA) have potent antagonistic activity toward TRβ with

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IC10 of 10.1 nM, 21.1 nM, respectively. BPS and BPA have the antagonistic activity

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with IC10 of 312 nM, 884 nM, respectively. BPSs significantly altered the expression

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level of mRNA of TRβ gene in zebrafish embryos. BPS and TBBPS at

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environmentally relevant concentrations have antagonistic activity toward TRβ,

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implying that BPSs are not safe BPA alternatives in many of the BPA-free products.

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Future health risk assessment of TR disruption and other adverse effects should focus

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more on the structure-activity relationship in designing environmentally benign BPA

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alternatives.

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Keywords: Bisphenol S; Molecular Interaction; Fluorescence Spectroscopy; Molecular modeling; Endocrine disruption

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1. INTRODUCTION Bisphenol S (4-hydroxyphenyl sulfone, BPS) is one of the commercial

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alternatives for bisphenol A (BPA) due to its enhanced heat and light resistance. BPS

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and its analogues (BPSs) such as tetrabromobisphenol S (TBBPS) and TBBPS bis

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(2,3- dibromopropyl ether) (OBBPS, TBBPS-BDBPE) are used commonly in epoxy

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resins, thermal receipt papers, foodstuff containers, electronic devices, baby bottles

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and medical appliance1-3. The increasing global usage of BPSs resulted in frequently

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detected residues of BPSs in foodstuff, sewage sludge, sediment, surface water, and

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indoor dust4-7. The relatively high lipophilicity of BPSs also contributes to their

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bioaccumulation in different fish and wildlife species, mollusks and avian eggs

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through various food chains and trophic levels3, 8-10. BPSs were detected also in

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human urine, breast milk, blood and cord serum via oral and other exposure pathways,

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especially for individuals living near e-waste facilities11-15. BPSs showed a detection

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frequency of 81% and a mean concentration of 0.65 µg/L in urine samples collected

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from the United States and Asian countries16.

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The exposure to BPSs has been reported to cause multiple adverse effect to

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human health and animals, such as cytotoxicity, genotoxicity, immunotoxicity and

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teratogenic effects17-21. TBBPA and TBBPS stimulated neural differentiation of

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mouse embryonic stem cell via different disruption of the positive regulator22. BPSs

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can cause endocrine disruption such as estrogenic, androgenic, anti-androgenic

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disruption1, 23, 24. BPS was shown to significantly increase progestagens levels through

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steroidogenic pathway25. Structural differences in the bridging moiety and benzene

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ring of bisphenol analogues lead to distinct estrogenic potencies in cell proliferation

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and MVLN (MCF-7-p-Vit-tk-Luc-Neo) cell-based assays26, which may relate to their

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preferential binding toward estrogen receptor subtype20. BPS was revealed to disrupt

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the membrane-initiated estradiol-induced cell signaling18 and caused adipocyte

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differentiation more directly than BPA via the activation of peroxisome

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proliferator-activated receptor gamma (PPARγ)27.

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Thyroid hormones (TH) play vital role in metabolism, energy expenditure,

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growth and development in vertebrates and TRs represent potential target of various

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endocrine disrupting chemicals (EDCs)28, 29. TRβ is the major TR isoform in the

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thyroid responsible for the regulation of target gene transcription30. Exogenous

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ligands may induce conformational changes of TRβ ligand binding domain (LBD),

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thereby affecting transcriptional activity of TRβ31. Increasing reports focus on the

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adverse health effect of thyroid hormone disrupting chemicals29, 32, 33. BPA and it

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analogues could regulate TRβ gene and key genes related to

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hypothalamic-pituitary-thyroid (HPT) axis, thus influencing thyroid hormone

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homeostasis and inducing thyroid disruption34-36. They may also bind to transthyretin

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(TTR) or TRs37 and interfere with transcriptional regulation of target genes38,

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disrupting relevant signaling pathway39. The investigation on how BPSs affect

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conformations of TRβ LBD at molecular level and whether structural analogues of

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BPSs bear different TR disruption is of significance for the evaluation of TRβ

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disruption by BPSs.

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In the present study, we evaluated the potential risk of BPS analogues toward

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TRβ (Table 1). We purified the TRβ LBD protein and performed in vitro fluorescence

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spectroscopy together with molecular dynamics (MD) simulations to decipher the

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molecular mechanism of TR disruption induced by BPSs. The recombinant human

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TRβ two-hybrid yeast assay combined with the zebrafish assay was used to evaluate

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the TRβ disruption of BPSs. Our results provided the first in vitro, in vivo and in

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silico evidence on the binding between TRβ LBD and BPSs. Such information is of

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significance for understanding physiological effects and endocrine activities of the

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BPSs in the consumer products and facilitates future design of environmentally

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benign BPA and BPS substitutes.

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2. MATERIALS AND METHODS

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2.1 Materials.

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BPSs (Table 1), dimethyl sulfoxide (DMSO) and 4-hydroxytamoxifen (4-OHT)

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with a purity ≥ 98.0% were purchased from Sigma Aldrich (St. Louis, MO, USA).

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3,3',5-Triiodo-l-thyronine (T3) with a purity of 98% was purchased from J&K

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Chemical Ltd. (Shanghai, China). SD/-Leu/-Trp medium (Catalogue: 4823-6) was

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purchased from Mobitec company (Goettingen, Germany). Power-SF DNA

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polymerase (Catalogue: HG1001) and In-Fusion Enzyme (Catalogue: HG3001) were

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supplied by HAOGENE Biotech Co., Ltd (Hangzhou, China). The Restriction enzyme

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FastDigest EcoRI and FastDigest Xho I were purchased from Fermentas China Co.,

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Ltd (Shanghai, China). Other chemicals were of analytical grade. All chemicals were

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dissolved in DMSO and diluted using distilled water (18.2 MΩ, Millipore, Bedford, 6

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MA) for toxicity evaluation and also were diluted in Tris-HCl buffer (0.2 M Tris, 0.1

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M NaCl, pH 7.4) for spectroscopic measurement. The prepared solutions were stored

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in amber glass vials at 4 oC.

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2.2 Expression of TRβ LBD.

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The coding DNA sequence of a full length Homo sapiens thyroid hormone

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receptor β (TRβ) (Genebank ID: GI: 358001055) with 1,386 base pairs was amplified

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by polymerase chain reaction (PCR) method with Power-SF DNA polymerase and

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specified primers (Table S1) and the DNA fragment was validated by agarose gel

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electrophoresis (Figure S1). The fragment of TRβ LBD was further amplified based

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on the full length TRβ. The vector pET-32a (Hangzhou Newbay Biological

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Technology Co., Ltd., Hangzhou, China) was digested with the enzyme FastDigest

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EcoRI and FastDigest XhoI. The DNA fragment of TRβ LBD was recominanted into

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the digested pET-32a using In-Fusion Enzyme. The recominant plasmid containing

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TRβ LBD was further amplified by Colony PCR using T7 promoter primer and T7

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terminator. TRβ LBD was expressed in E. coli BL21(DE3) and purified by

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Ni2+-affinity chromatography (Catalogue: 30210, Qiagen, Germany) and further

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eluted in 50 mM Tris HCl buffer (pH 8.0).

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2.3 Fluorescence Spectroscopy.

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The steady-state fluorescence spectra were measured with FluoroMax®-4

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spectrofluorometer (Horiba Jobin Yvon IBH) in a 1-mm quartz cell following a

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reported protocol40, 41. The emission spectra of TRβ LBD were recorded from 320 nm

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to 400 nm with an excitation wavelength of 280 nm at 303 K and 310 K. The

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fluorescence of a buffer solution was recorded to correct the background noise and

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eliminate the inner-filter effect. Each assay was performed in triplicate.

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2.4 Molecular Dynamic Simulations.

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The tertiary structure of TRβ LBD in complex with BPSs was constructed on the

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basis of X-ray crystal structure of TRβ LBD (PDB ID: 2J4A, 2.2 Å) by molecular

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docking using MVD 4.2 program (Text S2). The conventional MD simulations were

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further performed by Sander module implemented in Amber 14 with Amber ff12

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force field42. The atomic partial charges of BPSs were derived by R.E.D. Server

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Development with restrained electrostatic potentials (RESP) method and were used to

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build the parameters and libraries compatible with Amber ff12 force field. The

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complexes were solvated by 10 Å TIP3P waters in a periodic rectangular cubic box

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and were neutralized by 10 Na+ by AmberTools 1.542. The generated complex

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systems containing 40,000 atoms were minimized for 30 ps and then heated from 0 K

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to 300 K for 50 ps. The equilibration run of MD simulations were carried out using

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isothermal-isobaric (NPT) ensemble with unconstrained MD simulations for 100 ns

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with a time interval of 2 fs following a reported protocol43, 44. The MD trajectories

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were saved every 10 ps. The particle mesh Ewald (PME) method with a nonbonded

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cutoff of 10 Å was used to calculate the long range electrostatic interactions. The

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hydrogen atoms of the complex system were constrained by the SHAKE algorithm.

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The cpptraj module implemented in AmberTools 1.5 was used for conformational

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changes and the molecular mechanics generalized Born/surface area (MMGB/SA)

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with a single trajectory method was used to evaluate the binding free energies.

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2.5 Recombinant Two-hybrid Yeast Assay. The reported recombinant human TRβ two-hybrid yeast assay, which is highly

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specific to TRβ ligand without cross-talk to other receptors was used to screen the

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potential TR disruption of BPSs45, 46. The yeast cells were cultured in SD/-Leu/-Trp

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medium at 30oC overnight. BPSs solutions (5 µL) at varying concentrations were

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added to 995 µL medium with the OD600 value of approximately 0.75. For

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antagonistic activity of BPSs, a series of 5 µL BPSs (0.005 nM to 50 µM) and 5 µL

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T3 (10-4 M) were co-incubated with 990 µL yeast cells. This test culture (200 µL) was

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further cultured for 2.5 hr at 30oC. After the lysis of the cultured yeast cells using

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chloroform, the enzyme reaction was initiated by adding 40 µL

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o-nitrophenyl-β-D-galactopyranoside (13.3 mM). This reaction continued for 60 min

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and was terminated by adding 100 µL sodium carbonate (1.0 M). The OD420 was

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measured by Infinite 200 PRO NanoQuant Multimode Reader (Tecan Group Ltd.,

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Switzerland) and the β-galactosidase activity (U) was calculated following a reported

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protocol47-49. All assays were repeated three times and triplicate samples were

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measured in each assay.

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2.6 Determination of BPSs Concentration in ZebraFish Exposure Water.

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To measure actual exposure concentrations of BPSs, water samples were

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collected and determined in triplicate following the reported protocol20 with some

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modifications (Text S4). The solid-phase extraction (SPE) was performed after the

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conditioning of an Oasis HLB cartridge (6 mL, 200 mg, Waters, Massachusetts, USA)

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with 5 mL methanol and ultrapure water, respectively. Water samples of 100 mL

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containing BPSs analogues and 0.2% DMSO were extracted through the Oasis HLB.

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The compounds were eluted with 20 mL methanol after washed with 5 mL ultrapure

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water. Extracts were dried with a stream of nitrogen and was then solvated in 1 mL

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methanol. The water samples were injected in ultra-high-performance liquid

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chromatography tandem mass spectrometry (UHPLC-MS/MS) (Acquity UPLC,

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Waters, USA) after passed through 0.22 centrifuge filters. The detection limits were

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determined by 3/1 signal-to-noise ratio (S/N) and the method detection limits (MDLs)

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were determined on concentration of detection limits in water sample (n=3). A five

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point calibration curve was established between the peak area of compounds and their

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concentrations. The measured water concentrations and qualitative and quantification

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of BPSs were provided (Table S7-S9).

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2.7 Transcription of TRβ Gene in ZebraFish.

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The transcription profile of TRβ gene was evaluated using zebrafish (Danio rerio)

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by the quantitative real-time polymerase chain reactions (qPCR). The adult zebrafish

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were maintained following a recently reported protocol35. After spawning, the live

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embryos were collected and exposed to BPSs at 0.01, 0.1, and 1.0 µM for 72 h. The

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total RNA was then extracted from the zebrafish larvae using Trizol reagent (Takara

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Biochemicals, Dalian, China). The reverse-transcription of cDNA of TRβ was

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performed by the reverse transcriptase kit (Takara Biotechnology Co. Ltd., Dalian,

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China) using 500 ng total RNA. The cDNA (GenBank ID: GI: 358001055) was

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amplified in a 10-µL SYBR reaction mixture by the Mastercycler® ep realplex

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(Eppendorf, Hamburg, Germany) using designed primers (Table S1). Solvent control

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was constructed by equivalent DMSO concentration in ultrapure water. Triplicate

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samples were analyzed in each assay.

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2.8 Statistical Analysis.

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The data obtained from recombinant two-hybrid yeast assay was fitted by

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Levenberg-Marquardt algorithm with IBM SPSS Statistics 20. Data were analyzed by

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one-way analysis of variance (ANOVA), followed by LSD’s multiple comparisons. A

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p value of < 0.05 was considered as significant difference.

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3. RESULTS AND DISCUSSION

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3.1 BPSs Altered Conformational Changes of TRβ LBD.

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The binding of cognate ligands to TRβ LBD enables TRβ to activate

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transcription of target genes50, 51. The disturbed conformations of TRβ LBD by the

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binding of contaminants may consequently affect transcriptional activity of TRβ,

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leading to TR disruption. Studies on how BPSs with different structural moieties bind

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to TRβ LBD and induce conformational changes are therefore essential for the

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evaluation of TR disruption. We expressed TRβ LBD and probed the potentially

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induced conformational changes of TRβ LBD upon binding of BPSs by steady-state

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fluorescence spectroscopy. The fluorescence spectroscopic data on TRβ LBD, the first

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in vitro evidence reported to date, was further supported by MD simulations below to

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elucidate the intrinsic changes of TRβ LBD at the atomic level.

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3.1.1 BPSs Caused Fluorescence Quenching of TRβ LBD

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Fluorescence spectroscopy is widely used to study the interaction of various xenobiotic contaminants with biomacromolecules. The intrinsic tryptophan

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fluorescence may change due to the microenvironmental disruption of proteins upond

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the binding of contaminants52, 53. Our in vitro spectroscopic spectra revealed that the

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ligand free form of TRβ LBD (apo TRβ LBD) has the maximum fluorescence

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emission at 350 nm (Figure 1). This maximum emission wavelength of TRβ LBD

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shifted from 350 nm to 351 nm, 352 nm, 352 nm, 357 nm, 355 nm and 352 nm after

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titrating with BPA, BPS, BPS-DAE, TBBPA, OBBPS and TBBPS, respectively,

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indicating a slight red-shift (Figure 1). This red-shift of maximum emission with λmax

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to longer wavelengths was caused by the affected positively charged residues upon

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the binding, suggesting the alteration of microenvironment in TRβ LBD.

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Along with the red-shift, the intensity of fluorescence from TRβ LBD was

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significantly decreased after the successive addition of TBBPS, BPA and TBBPA

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with the increasing concentration from 1 to 20 µM, implying the

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concentration-dependent fluorescence quenching. Further examination indicated that

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BPS and BPS-DAE has a similar extent of small fluorescence quenching, whereas

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TBBPA led to a more marked quenching of fluorescence in comparison with BPA,

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BPS, and BPS-DAE (Figure 1). An increasing concentration of OBBPS caused the

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increase of fluorescence intensity of TRβ LBD. It is likely that the high

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hydrophobicity of OBBPS as revealed by the relatively large LogKow value (Table 1)

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may partly contribute to the increased fluorescence intensity. It was reported that

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perfluorodecanoic acid (PFDA) with a longer side chain bears higher hydrophobic

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interaction and enhances the fluorescence quantum yield, which in turn increases the

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fluorescence intensity upon binding with bovine hemoglobin and myoglobin54, 55.

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Fluorescence spectra at 303 K and 310 K were analyzed following the

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Stern-Volmer equation (Text S2) to further characterize the interaction of these

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chemicals with TRβ LBD. As detailed in Text S2, BPSs, BPA and TBBPA caused

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static quenching for TRβ LBD. Moreover, TBBPS and TBBPA have markedly higher

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values of quenching constants (Ksv and Kq in Table S2), implying that both

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brominated bisphenols induced more significant microenvironment change.

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3.1.2 BPSs Induced Cα RMSD Changes of TRβ LBD

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Whether BPSs can induce conformational changes of TRβ LBD was studied by

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performing MD simulations on ligand free (apo) form and ligand-bound form of TRβ

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LBD. The root-mean-square deviation of α carbon atoms (Cα RMSD) was used as an

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important indicator to monitor conformational changes and hence the structural

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stability of the complexes with TRβ LBD39. MD simulations were reportedly

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performed 50 ns and 10 ns for interactions of TRβ with household dust contaminants

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and its agonists, respectively29, 56. In our study, each simulation lasted for 100 ns and

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this timescale enables us to probe local conformational changes. We sampled 10,000

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conformations from 100 ns MD trajectories with an interval of 10 ps to measure Cα

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RMSD in reference to the initial minimized structures (Figure S2). The averaged Cα

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RMSD for apo TRβ LBD is 1.78 Å. Upon binding, the averaged Cα RMSD for the

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complex of TRβ LBD with BPS, BPS-DAE, TBBPS, OBBPS, BPA and TBBPA was

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1.96 Å, 1.77 Å, 1.90 Å, 1.98 Å, 1.99 Å and 2.22 Å, respectively, suggesting the

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induced conformational changes of TRβ LBD. As revealed by the Cα RMSD

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monitored along 100 ns MD trajectories (Figure S2), the binding of BPSs affects the

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structural stability of TRβ LBD in comparison with apo TRβ LBD, generally in

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agreement with the in vitro fluorescence quenching of TRβ LBD upon binding with

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BPSs, further confirming the disturbed conformational changes of TRβ LBD.

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3.1.3 BPSs Induced Repositioning of H12 of TRβ LBD

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The crucial C terminal H12 constitutes important part of the binding site and the

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dynamics of H12 are vital to the regulation of the transcriptional activity of nuclear

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receptors57, 58. Upon the binding of contaminants, H12 of TRβ LBD may undergo

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repositioning, hindering the recruitment of co-activators and transcriptional activity of

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transactivation function 2(AF-2)39. Figure 2 and 3 show the distance between the

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nitrogen atom NE2 of residue His435 at helix H11 and nitrogen atom N of residue

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Phe459 at H12 during 100 ns MD simulation in the presence and absence of BPSs.

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The changing distance means the relative repositioning of H12. This averaged

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distance for apo TRβ LBD was 4.41Å and increased significantly to 12.62, 7.66,

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10.53, 13.74, 8.00 and 8.22 Å upon the binding of BPA, TBBPA, BPS, BPS-DAE,

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TBBPS and OBBPS, respectively. Ligands can be locked into binding site and the

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helices shield them from escape at the binding pocket59. The altered position of H12

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induced by the binding of BPSs could be related to their potentional disruption of

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transcriptional activity of TR. Since TR and retinoid X receptor (RXR) can form a

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heterodimer through their LBD interface60, the disruption of conformation of TR LBD

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may also affect the heterodimerization of TR with RXR, altering ligand-dependent

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signaling pathways between dimer partners and consequently exerting adverse

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physiological effects.

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3.2 Different Binding Characteristics of BPSs

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3.2.1 Distinct Binding Mode to TRβ LBD

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The ligand competitive binding assay using T3 as a fluorescence probe revealed

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that BPSs and T3 have the same binding site within TRβ LBD (Text S3, Table S4).

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The complex of TRβ LBD with BPSs was constructed by molecular docking and was

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optimized by 100 ns MD simulations. The hydrogen bonds between BPSs and TRβ

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LBD were calculated based on 10,000 conformations (Table S5). BPSs exhibited

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distinct binding mode to TRβ LBD. BPA can form hydrogen bonds with residues

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Gly344, Phe272, Ser331, Ile275 and His435, with a corresponding occupancy of

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32.52%, 26.39%, 55.99%, 22.59% and 6.42%, respectively. In contrast, BPS forms

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hydrogen bonds with Thr273 and His435, having a corresponding occupancy of 83.44%

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and 92.60%. BPA also has a distinct hydrogen bond mode with important amino acid

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residues of TRβ in comparison with BPS.

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BPS-DAE, TBBPA and TBBPS form hydrogen bonds with Ser331. The

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occupancy for the hydrogen bonds with Ser331 was 49.18% (BPS-DAE), 73.36%

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(TBBPA), and 53.41% (TBBPS), respectively. OBBPS forms hydrogen bond mainly

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with His435 with occupancy of 14.38%. It was reported that Gly344, Thr273, Ile275

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and His435 were the critical residues for the interactions of contaminants with TRβ

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LBD29, 31, 61. These key residues of TRβ LBD can accommodate different moieties of

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xenobiotic chemicals. For example, His435 and Thr273 of TRβ LBD can be shifted to

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cater the large moiety of chemicals, whereas Ile275 and Gly344 can reposition to

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hydroxyl group-related chemicals31, 61.

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3.2.2 Varying Binding Potencies to TRβ LBD The total binding free energies (∆Gcalc) of BPSs were evaluated by MMGB/SA at

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300 K (Table 2). The van der Waals interactions (∆Evdw), electrostatic interactions

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(∆Eele) and the hydrophobic interactions (∆Esurf) are favourable for the binding. ∆Evdw

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constitutes the major component of the ∆Gcalc, suggesting van der Waals interactions

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are most important for the binding between TRβ LBD and BPSs. TBBPA and TBBPS

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have more negative ∆Gcalc, compared with BPA and BPS, in line with the determined

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association constant ( ) from the competitive binding assay (Table S4). The more

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potent binding of the brominated TBBPA and TBBPS induced stable conformational

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changes, resulting in higher antagonistic activity toward TRβ. For BPS and BPA, the

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less potent binding caused less alternation of H12 reposition than TBBPS and TBBPA,

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consequently causing less TR disruption.

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TBBPA and TBBPS with four Br atoms at 3, 5,3’,5’-positions in phenolic rings

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possess more negative ∆Evdw value and less negative ∆Eele in comparison with the

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other three chemicals without any bromination, suggesting that bromination may

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contribute favourable to van der Waals force. The number of bromination was

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reported to positively correlated with van der Waals force and negatively related to

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the electrostatic interactions between BPA analogues and PPARγ and ERα LBD43.

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The sulfone moiety and alkyl moiety linking two aromatic rings contribute to the

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different binding free energies, however, bromination appears to contribute

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predominantly to the ∆Gcalc.

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3.3 BPSs Showed Antagonistic Activity toward TRβ.

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The TR disruption of BPSs was evaluated by the recombinant two-hybrid TRβ

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gene yeast assay. This established yeast assay is well suited for a rapid evaluation of

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various thyroid disrupting chemicals45, 46. T3 was chosen as the positive control and

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the β-galactosidase activity of this endogenous TRβ agonist was determined. The

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standard dose-response curve of T3 (Figure S3) revealed a maximum activity at 5 µM,

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in line with values reported previously46. The β-galactosidase activities of BPSs at

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environmentally relevant concentrations (5×10-7 µM to 50 µM) were measured. No

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significant changes of β-galactosidase activity were observed in comparison with the

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solvent control (Figure S4), suggesting that BPSs showed no agonistic activity toward

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human TRβ.

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We further determined whether BPSs present antagonistic activity toward TRβ.

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BPSs were co-incubated with T3 and the ratio of β-galactosidase activity (U) induced

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by BPSs and T3 to the β-galactosidase activity induced by T3 was calculated. As

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shown in Figure 4, BPSs significantly suppressed β-galactosidase activity in a

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dose-dependent manner, suggesting their antagonistic effect toward TRβ. The

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corresponding IC10 and relative potency (RP) values were obtained for each BPS in

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comparison with T3 (Table S6). TBBPS showed the strongest antagonistic activity to

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TRβ with IC10 value at 10.1 nM and the largest RP at 87.5, followed by TBBPA (IC10:

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21.1 nM, RP: 41.9) and BPS (IC10: 312 nM, RP: 2.8). The brominated TBBPS and

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TBBPA showed much stronger antagonistic activity than their corresponding BPS and

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BPA. Some brominated phenol-related chemicals reportedly show thyroid disrupting

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activities62. BPS-DAE has a similar dose-response curve to that of its close BPS

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analogue. The brominated OBBPS has a very high LogKow of 9.19 and the low

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aqueous solubility may contribute partially to its non-detected antagonistic activity63,

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which may account for low correlation with the MD simulation. Despite the

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previously reported estrogenic and androgenic disruption23, we show that BPS

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exhibited weak TRβ disruption similar to BPA with a relatively higher RP, implying

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that BPS may not be safe substitute of BPA64.

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3.4 BPSs Altered mRNA Expression Level of TRβ in Zebrafish Larvae.

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TRβ gene is responsible for the resistance to thyroid hormones and decreasing

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sensitivity of T3 target tissues 65. We further evaluated the effect of BPSs on the

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expression level of mRNA of TRβ gene in zebrafish larvae. As revealed by qPCR, the

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exposure to BPSs at concentrations of 0.01, 0.1, and 1.0 µM for 72 h could

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significantly alter the expression level of mRNA of TRβ gene (Figure 5). TBBPS and

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OBBPS at 0.01 µM significantly up-regulated the mRNA expression levels of TRβ,

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approximately a two-fold increase in comparison with the DMSO control (Figure 5);

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however, BPS and BPS-DAE at 0.01 µM did not significantly change the mRNA

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expression levels. Compared with the DMSO control, OBBPS and BPS-DAE at 0.1

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µM significantly upregulated mRNA expression of TRβ, while BPS and TBBPS

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showed no significant effect. BPS, TBBPS and OBBPS with the concentration up to

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1.0 µM significantly up-regulated the mRNA expression level.

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BPA showed less disruption on mRNA expression of TRβ at 0.01 µM in

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comparison with TBBPS (P=0.018) and OBBPS (P=0.0061) and more disruption at

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0.1 µM in compared with BPSs (P