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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] ACS Paragon Plus Environment
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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:);
