In Vitro and in Silico Competitive Binding of Brominated Polyphenyl

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In Vitro and In Silico Competitive Binding of Brominated Polyphenyl Ether Contaminants With Human and Gull Thyroid Hormone Transport Proteins Katie L. Hill, Åse-Karen Mortensen, Daniel Teclechiel, William G. Willmore, Ingebrigt Sylte, Bjorn M. Jenssen, and Robert J. Letcher Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04617 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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In Vitro and In Silico Competitive Binding of Brominated Polyphenyl Ether Contaminants

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With Human and Gull Thyroid Hormone Transport Proteins

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Katie L. Hilla,b,c, Åse-Karen Mortensend, Daniel Teclechiele, William G. Willmoreb, Ingebrigt

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Syltef, Bjørn M. Jenssend, and Robert J. Letchera,b*

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a

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National Wildlife Research Centre, Carleton University, Ottawa, Ontario, K1A 0H3, Canada

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b

Department of Biology, Carleton University, Ottawa, Ontario, K1S 5B6, Canada

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c

Intrinsik Corp., Ottawa, Ontario, K1S 5R1, Canada

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d

Department of Biology, Norwegian University of Science and Technology, Trondheim, NO-

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7491, Norway

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e

AccuStandard, New Haven, Connecticut, 06513, U.S.A.

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f

Department of Medical Biology, UiT - The Arctic University of Norway, Tromsø, NO-9037,

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Norway

Ecotoxicology and Wildlife Health Division, Environment and Climate Change Canada,

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*Corresponding author. Phone: 613-998-6696; Fax: 613-998-0458; E-mail:

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[email protected]


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ABSTRACT

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Tetradecabromo-1,4-diphenoxybenzene (TeDB-DiPhOBz) is a highly brominated additive flame

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retardant (FR). Debrominated photodegradates of TeDB-DiPhOBz are hydroxylated in vitro in

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liver microsomal assays based on herring gulls (Larus argentatus), including one metabolite

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identified as 4"-OH-2,2',2",4-tetrabromo-DiPhOBz. Chemically related methoxylated tetra- to

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hexabromo-DiPhOBzs are known contaminants in herring gulls. Collectively, nothing is

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currently known about biological effects of these polybrominated (PB) DiPhOBz-based

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compounds. The present study investigated the potential thyroidogenicity of 2,2',2",4-

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tetrabromo-(TB)-DiPhOBz along with its para-methoxy (MeO)- and hydroxy-(OH)-analogues,

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using an in vitro competitive protein binding assay with the human thyroid hormone (TH)

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transport proteins transthyretin (hTTR) and albumin (hALB). This model para-OH-TB-

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DiPhOBz was found to be capable of competing with thyroxine (T4) for the binding site on

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hTTR and hALB. In silico analyses were also conducted using a 3D homology model for gull

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TTR, to predict whether these TB-DiPhOBz-based compounds may also act as ligands for an

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avian TH transport protein despite evolutionary differences with hTTR. This analysis found all

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three TB-DiPhOBz analogues to be potential ligands for gull TTR, and with similar binding

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efficacies to THs. Results indicate structure-related differences in binding affinities of these

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ligands, and suggest there is potential for these contaminants to interact with both mammalian

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and avian thyroid function.



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Introduction

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Polybrominated diphenyl ethers (PBDEs) were until recently some of the most widely used

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brominated flame retardants (BFRs) worldwide.1,2 Concerns of persistence, long-range transport,

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exposure of humans3,4 and wildlife e.g., fish and herring gulls from the Great Lakes5,6 and even

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more remote regions such as the Arctic,7 as well as toxicological effects, led to a worldwide

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phase-out of all three commercial mixtures (penta, octa, and deca-BDE) from commerce over the

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last several years.2 In 2009 penta- and octa-BDE formulations were added to Annex A of the

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Stockholm convention on persistent organic pollutants (POPs), and deca-BDE was added in

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early 2017.8 With the phase-out and regulation of the various PBDE formulations, several

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replacement flame retardants are now being produced including the deca-BDE replacement

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tetradecabromo-1,4-diphenoxybenzene (TeDB-DiPhOBz; CAS 58965-66-5). TeDB-DiPhOBz is

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a highly brominated additive flame retardant applied to polyesters, resins, wires and cables.9

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There are several Asian suppliers of TeDB-DiPhOBz-containing products and commercial

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formulations,10,11 however, no regulations exist for TeDB-DiPhOBz production and little

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information is available on the magnitude and global range of its use.

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TeDB-DiPhOBz has been found to undergo photolytic debromination including to degradation

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products of Br4- to Br7-DiPhOBzs.12–14 Recently, methoxylated (MeO) Br4- to Br6-DiPhOBz

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congeners were identified for the first time in herring gull (Larus argentatus) eggs, and upon

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retrospective analysis, these compounds were shown to be present in gull eggs for at least the last

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30 years and in concentrations up to 100 ng/g ww.15,16 An in vitro metabolism study using

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herring gull and rat microsomes that had been administered UV-degraded TeDB-DiPhOBz

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fractions containing Br4- to Br8-PB-DiPhOBzs, confirmed the formation of several Phase I

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biotransformed Br3- to Br5-OH-PB-DiPhOBz metabolites.17 In the same study, two hydroxylated


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tetra-brominated congeners were synthesized for the purpose of identifying specific metabolite

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products, i.e. 4"-OH-2,2',2",4-tetrabromo-DiPhOBz and 4"-OH-2,2”,3’,4-tetrabromo-DiPhOBz,

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and these congeners were identified as metabolites formed in vitro in both the gull and rat

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microsomal assays.17 The MeO-Br4- to Br6-DiPhOBz contaminants that were found in Great

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Lakes herring gulls and eggs are thus suspected degradation products of TeDB-DiPhOBz, where

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the MeO-PB-DiPhOBzs are potentially phase II conjugation products of OH-PB-DiPhOBz

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metabolites.

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Virtually no toxicological data exist for TeDB-DiPhOBz or its potential degradation products,

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apart from one in vitro study that identified alterations in CYP1A4 expression of chicken

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embryonic hepatocytes.13 Effects are expected to be similar to structurally analogous PBDEs

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including lower brominated hydroxylated (OH) PBDE metabolites, which are capable of

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perturbing thyroid homeostasis. Thyroid hormones (THs) are involved in several important

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functions including the regulation of metabolism in adult organisms, and tissue differentiation

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during fetal and embryonic development.18 One potential mechanism of action of thyroid

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disruption is competition for TH binding sites on serum transport proteins, as THs triiodo-L-

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thyronine (T3) and its precursor thyroxine (T4) rely on binding to TH transport proteins to be

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delivered to target tissues. There are three major TH transport proteins in vertebrates including

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transthyretin (TTR), albumin (ALB), and thyroid binding globulin (TBG), where TTR and ALB

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are consistently important in mammalian and avian species.19,20

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The objectives of the present study were to investigate the interactions between selected and

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model tetrabrominated (TB) DiPhOBz congeners with human and avian TH transport proteins as

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per the following approaches. Firstly, in vitro competitive binding interactions of the test set of

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TB-DiPhOBz congeners with T4 and human TTR and ALB (hTTR and hALB) were studied



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using a recently optimized radioligand binding assay.21 Secondly, we used in silico molecular

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homology modeling of glaucous gull (Larus hyperboreus) TTR (gTTR) developed by Mortensen

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(2015),22 to investigate competitive binding interactions of the test set of the TB-DiPhOBz

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congeners with T4 and T3. As we reported in Ucán-Marín et al.,23 glaucous gull and herring gull

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TTR were shown to have identical amino acid and nucleotide sequences, and thus the protein

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structure and TH binding pocket of TTR are likely to be the same in both species.

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Materials and Methods

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Standards and chemicals

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Tris-EDTA, thyroxine (T4; ≥ 98 % purity), and the lyophilized plasma proteins human

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transthyretin (hTTR; ≥ 95 % purity) and albumin (hALB; ≥ 99 % purity) were obtained from 125

I-labelled T4 (125I- T4; 50 µCi) was

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Sigma-Aldrich (Oakville, ON, Canada). The radioligand

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obtained from MP Biomedicals (Solon, OH). The PBDE metabolite 4’-hydroxy-2,2’,4,5’-

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tetrabromo diphenyl ether (4-OH-BDE-49; 97.8 % purity) was obtained from Chromatographic

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Specialties Inc. (Brockville, ON, Canada). Pure standards of the diphenoxybenzene-based

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chemicals of interest for this study were developed and provided by Daniel Teclechiel at

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AccuStandard® Inc. (New Haven, CT, USA). These included a TB-DiPhOBz as well as para-

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OH- and MeO-TB-DiPhOBz associated congeners with the same Br- positioning: 2,2’,2’’,4-

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tetrabromo-diphenoxybenzene (BDPB-402; 98.1 % purity), 4"-OH-2,2',2",4-tetrabromo-

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diphenoxybenzene

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diphenoxybenzene (MOBDPB-401; 99.5 % purity) (Figure 1).

(HBDPB-401;

100

%

purity),

and

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4"-MeO-2,2',2",4-tetrabromo-

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Competitive thyroid hormone binding assay

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A full and highly detailed account of the procedure, including reagent preparation, sample set

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up and separation are described in Hill et al. (2017).21 The assay is based on previously described

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methods23–27 with some important modifications. That is, a reduction in sample volume, a

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reduction in the amount of radioisotope required, and a streamlined sample processing

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procedure. Briefly, the 100 µL incubation mixtures were prepared in triplicate and comprised of

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55.5 nM T4 / 125I-T4 (ratio of 99.5 : 0.5, in Tris-EDTA), 30 nM hTTR (or 600 nM hALB), and a

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competitor (in a two-fold serial dilution series). Negative controls were included for each

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competitor ligand series using DMSO in place of the competitor. Calibrations were conducted in

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duplicate using 5 to 5,120 nM T4 as a competitor ligand in a homologous assay, as well as a

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negative control (DMSO) sample and a positive control sample (the IC50 of 4-OH-BDE-49,

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which is a known TTR competitor ligand). The PBDE metabolite 4-OH-BDE-49 has been

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previously identified as having a higher affinity for both human and recombinant gull TTR and

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ALB over T3 and T4 using a similar assay.20 Samples were prepared in polypropylene test tubes

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with caps. The initial radioactivity of each sample was recorded, followed by overnight

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incubation at 4 ˚C to allow mixtures to reach binding equilibrium. Protein-bound and free ligands

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were then separated by size exclusion chromatography using Bio-Rad Bio-Spin® P-6 gel

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columns as per Bio-Rad instructions. Samples were centrifuged at 4 ˚C for 4 mins at 1000 x g,

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and the eluate (containing only protein-bound ligand) was measured for radioactivity. The

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residual radioactivity remaining in the incubation tube and transfer pipette was also measured to

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account for nonspecific binding and transfer loss.

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Analysis of in vitro data



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The percent of

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I-T4-hTTR or -hALB protein binding was calculated for each sample by

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dividing the radioactivity of eluate by the initial radioactivity, minus transfer loss. Results were

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expressed by plotting the logarithmic concentration of the competitor ligand on the X-axis, and

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mean ± standard deviation of the percent binding compared to controls on the Y-axis. The 50 %

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inhibition concentration (IC50) was calculated for each competitor in GraphPad Prism 7.02

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(GraphPad Software, Inc., La Jolla, CA, USA), using the sigmoidal dose-response equation

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“log[inhibitor] vs. response – variable slope”, defined by Equation 1: ( ) (()× ! "# )

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= +

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The inhibition constant (Ki) for each competitor was determined based on Cheng and Prusoff

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(1973),28 and following GraphPad guidance.29 In a homologous assay (i.e., with T4 as the

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competitor) the cold ligand and radioligand can be assumed to have the same binding affinities.

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As such, the Ki and the equilibrium dissociation constant (Kd) for T4 was determined with the

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IC50 of T4 and the concentration of radioligand (denoted as T4*) in samples using Equation 2:

(1)

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($% ) & ()* &∗ = ($* ) & ()* &∗ = (,-50) & − [24∗ ]

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With these defined parameters, the Ki of other competitors were determined using Equation 3:

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($% )5 6% 7 =

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Note that at low concentrations of radioligand, the Ki for a competitor approximates the

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(89:);

In Vitro and in Silico Competitive Binding of Brominated Polyphenyl

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