AOP-Wiki

AOP ID and Title:

AOP 152: Interference with thyroid serum binding protein transthyretin and subsequent adverse human neurodevelopmental toxicity

Short Title: Transthyretin interference

Authors

Erik R. Janus; M3 Technical & Regulatory Services; Shepherdstown, WV; <erik@mcubedservices.com>

Kristie Sullivan; Physicians Committee for Responsible Medicine; Washington, DC; <ksullivan@pcrm.org>

Katie Paul-Friedman; US Environmental Protection Agency; Research Triangle Park, NC

Mary Gilbert; National Health and Environmental Effects Research Laboratory; US Environmental Protection Agency; Research Triangle Park, NC; <gilbert.mary@epa.gov>

Kevin M. Crofton; National Center for Computational Toxicology; US Environmental Protection Agenc; Research Triangle Park, NC; <crofton.kevin@epa.gov>

Status

Author status	OECD status	OECD project	SAAOP status
Open for adoption	Under Development	1.41	Included in OECD Work Plan

Abstract

This AOP describes adverse neurodevelopemental effects that may result from xenobiotic interference with thyroid serum binding protein transthyretin (TTR). Binding of TTR by a xenobiotic (the MIE) during certain developmental windows may disrupt the normal neurodevelopment of mammals through a transient increase in free thyroxine (T4) levels, permitting increased tissue uptake of thyroid hormone (TH), followed by a decrease in both serum and neuronal tissue concentrations. Due to the highly conserved nature of the TTR protein, birds, reptiles, fish and amphibians can also express TTR and be impacted by interference by xeniobiotics. The adverse consequences of TH insufficiency depend both on the severity and developmental timing, indicating that exposure to thyroid toxicants may produce different effects at different developmental windows of exposure. This AOP discusses the potential for developmental TTR interference to adversely impact hippocampal anatomy, function, gene expression and, ultimately, cognitive function.

Background

Transthyretin is one of three ancient, highly conserved serum binding proteins that collectively act to transport thyroid hormone (TH) and thus help maintain normal homeostasis via modulation of the hypothalamic/pituitary/thyroid axis. In addition to TTR, albumin (ALB) and thyroxine-binding globulin (TBG) also serve to transport TH in serum and the relative contribution of each binding protein differs across species. In man, TBG has the greatest affinity for thyroxine (T4), followed by TTR and ALB shows the lowest affinity for T4 while prevalence in serum is the opposite, while in rat, TTR is the major serum transport protein (as rats lack TBG). Interference with TH serum binding proteins is one of several mechanisms through which xenobiotics and environmental contaminants can disrupt normal thyroid endocrine function ("thyroid disruptors") and development of this AOP is expected to contribute towards a fuller understanding of the mechanism of TTR interference and how it may be measured in vitro as part of a larger screening battery for thyroid toxicants.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Sequence	Type	Event ID	Title	Short name
1	MIE	957	Binding, Transthyretin in serum	Binding, Transthyretin in serum

2	KE	958	Displacement, Serum thyroxine (T4) from transthyretin	Displacement, Serum thyroxine (T4) from transthyretin
3	KE	959	Increased, Free serum thyroxine (T4)	Increased, Free serum thyroxine (T4)
4	KE	960	Increased, Uptake of thyroxine into tissue	Increased, Uptake of thyroxine into tissue
5	KE	961	Increased, Clearance of thyroxine from tissues	Increased, Clearance of thyroxine from tissues
6	KE	281	Thyroxine (T4) in serum, Decreased	T4 in serum, Decreased
7	KE	280	Thyroxine (T4) in neuronal tissue, Decreased	T4 in neuronal tissue, Decreased
8	KE	756	Hippocampal gene expression, Altered	Hippocampal gene expression, Altered
9	KE	757	Hippocampal anatomy, Altered	Hippocampal anatomy, Altered
10	KE	758	Hippocampal Physiology, Altered	Hippocampal Physiology, Altered

11	AO	402	Cognitive Function, Decreased	Cognitive Function, Decreased

Key Event Relationships

There are no Relationships associated with this AOP

Stressors

Name	Evidence
Halogenated phenols	Low
Polychlorinated biphenyl	High
Polychlorinated dibenzodioxins	Not Specified
Polybrominated diphenyl ethers	High
Isoflavones	High
Perflourinated chemicals	Moderate
Phthalates	Low
Tetrabromobisphenol A	Not Specified
Clonixin	Not Specified
Meclofenamic acid	Not Specified
2,6-dinitro-p-cresol	Not Specified
Triclopyr	Not Specified
2,2',4,4'-Tetrahydroxybenzophenone	Not Specified

Halogenated phenols

Pentachlorophenol is measured in mammals as parent chemical, hexachlorobenzene, and can bind to TTR, like other halogenated phenols (Marchesini et al 2008; van den Berg 1990). Halogenated phenols, such as PCP, have been implicated as thyroid toxicants (Miller et al 2009); however, it appears only 2,4,6-tribromophenol and pentabromophenol can competitively bind TTR (Weiss et al 2015).

Marchesini, G. R., Meimaridou, A., Haasnoot, W., Meulenberg, E., Albertus, F., Mizuguchi, M., … Murk, A. J. (2008). Biosensor discovery of thyroxine transport disrupting chemicals. Toxicology and Applied Pharmacology, 232(1), 150–160. http://doi.org/10.1016/j.taap.2008.06.014

Miller, M. D., Crofton, K. M., Rice, D. C., & Zoeller, R. T. (2009). Thyroid-disrupting chemicals: Interpreting upstream biomarkers of adverse outcomes. Environmental Health Perspectives, 117(7), 1033–1041. http://doi.org/10.1289/ehp.0800247

van den Berg KJ. 1990. Interaction of chlorinated phenols with thyroxine binding sites of human transthyretin, albumin and thyroid binding globulin. Chem Biol Interact 76(1):63–75.

Weiss, J. M., Andersson, P. L., Zhang, J., Simon, E., Leonards, P. E. G., Hamers, T., & Lamoree, M. H. (2015). Tracing thyroid hormone-disrupting compounds: database compilation and structure-activity evaluation for an effect-directed analysis of sediment. Analytical and Bioanalytical Chemistry, 5625–5634. http://doi.org/10.1007/s00216-015-8736-9

Polychlorinated biphenyl

A number of PCBs and metabolites have been found to bind to TTR (Lans et al 1993; Grimm et al 2013; Marchesini et al 2008) and have been frequently found to be associated with interference in thyroid signaling (Boas et al 2012; Gore et al 2015; Miller et al 2009; Murk et al 2013; Preau et al 2015). Overall, the hydroxylated metabolites of PCBs competitively bind T4 more than the parent compounds (Weiss et al 2015). Depending on the congener and/or type of metabolite (i.e. hydroxylate, sulfate, etc.), there are multiple mechanisms by which this class of environmental contaminants can affect thyroid hormone levels in many tissues, including through interfering with TTR-T4 binding and transport of T4 into the brain (Morse et al 1996; Martin and Klaassen 2010; Martin et al 2012). Multiple epidemiological studies in humans have reported associations between exposure to PCBs and/or metabolites and serum thyroid hormone concentrations (Dallaire et al 2009a, 2009b; Eguchi et al 2015), with hydroxylated metabolites demonstrating the greatest potential for TTR interference (Cheek et al 1999; Dirinck et al 2016).

Boas, M., Feldt-Rasmussen, U., & Main, K. M. (2012). Thyroid effects of endocrine disrupting chemicals. Molecular and Cellular Endocrinology, 355(2), 240–248. http://doi.org/10.1016/j.mce.2011.09.005

Cheek, A. O., Kow, K., Chen, J., & McLachlan, J. a. (1999). Potential mechanisms of thyroid disruption in humans: Interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin. Environmental Health Perspectives, 107(4), 273–278. http://doi.org/10.1289/ehp.99107273

Dallaire, R., Muckle, G., Dewailly, É., Jacobson, S. W., Jacobson, J. L., Sandanger, T. M., … Ayotte, P. (2009a). Thyroid hormone levels of pregnant inuit women and their infants exposed to environmental contaminants. Environmental Health Perspectives, 117(6), 1014–1020. http://doi.org/10.1289/ehp.0800219

Dallaire, R., Dewailly, É., Pereg, D., Dery, S., & Ayotte, P. (2009b). Thyroid function and plasma concentrations of polyhalogenated compounds in inuit adults. Environmental Health Perspectives, 117(9), 1380–1386. http://doi.org/10.1289/ehp.0900633

Dirinck E, Dirtu AC, Malarvannan G, Covaci A, Jorens PG, Van Gaal LF. A Preliminary Link between Hydroxylated Metabolites of Polychlorinated Biphenyls and Free Thyroxin in Humans. Int J Environ Res Public Health. 2016 Apr 13;13(4):421. doi: 10.3390/ijerph13040421.

Eguchi, A., Nomiyama, K., Minh Tue, N., Trang, P. T. K., Hung Viet, P., Takahashi, S., & Tanabe, S. (2015). Residue profiles of organohalogen compounds in human serum from e-waste recycling sites in North Vietnam: Association with thyroid hormone levels. Environmental Research, 137, 440–449. http://doi.org/10.1016/j.envres.2015.01.007

Gore, a. C., Chappell, V. a., Fenton, S. E., Flaws, J. a., Nadal, a., Prins, G. S., … Zoeller, R. T. (2015). Executive Summary to EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals. Endocrine Reviews, (October), er.2015-1093. http://doi.org/10.1210/er.2015-1093

Grimm, F. a., Lehmler, H. J., He, X., Robertson, L. W., & Duffel, M. W. (2013). Sulfated metabolites of polychlorinated biphenyls are high-affinity ligands for the thyroid hormone transport protein transthyretin. Environmental Health Perspectives, 121(6), 657–662. http://doi.org/10.1289/ehp.1206198

Lans MC, Klasson-Wehler E, Willemsen M, Meussen E, Safe S Brouwer A. 1993. Structure-dependent, competitive interaction of hydroxy-polychlorobiphenyls, -dibenzo-p-dioxins and -dibenzofurans with human transthyretin. Chem Biol Interact 88(1):7–21.

Martin, L., & Klaassen, C. D. (2010). Differential effects of polychlorinated biphenyl congeners on serum thyroid hormone levels in rats. Toxicological Sciences : An Official Journal of the Society of Toxicology, 117(1), 36–44. http://doi.org/10.1093/toxsci/kfq187

Martin, L. A., Wilson, D. T., Reuhl, K. R., Gallo, M. A., & Klaassen, C. D. (2012). Polychlorinated biphenyl congeners that increase the glucuronidation and biliary excretion of thyroxine are distinct from the congeners that enhance the serum disappearance of thyroxine. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 40(3), 588–95. http://doi.org/10.1124/dmd.111.042796

Miller, M. D., Crofton, K. M., Rice, D. C., & Zoeller, R. T. (2009). Thyroid-disrupting compounds: Interpreting upstream biomarkers of adverse outcomes. Environmental Health Perspectives, 117(7), 1033–1041. http://doi.org/10.1289/ehp.0800247

Morse DC, Seegal RF, Borsch KO, Brouwer A. Long-term alterations in regional brain serotonin metabolism following maternal polychlorinated biphenyl exposure in the rat. Neurotoxicology. 1996 Fall-Winter;17(3-4):631-8.

Murk, A. J., Rijntjes, E., Blaauboer, B. J., Clewell, R., Crofton, K. M., Dingemans, M. M. L., … Gutleb, A. C. (2013). Mechanism-based testing strategy using in vitro approaches for identification of thyroid hormone disrupting chemicals. Toxicology in Vitro, 27(4), 1320–1346. http://doi.org/10.1016/j.tiv.2013.02.012

Préau, L., Fini, J. B., Morvan-Dubois, G., & Demeneix, B. (2014). Thyroid hormone signaling during early neurogenesis and its significance as a vulnerable window for endocrine disruption. Biochimica et Biophysica Acta - Gene Regulatory Mechanisms, 1849(2), 112–121. http://doi.org/10.1016/j.bbagrm.2014.06.015

Polychlorinated dibenzodioxins

Both dioxins and furans have been reported to bind to TTR (Lans et al 1993) and, similar to PCBs, have been reported as thyroid toxicants (Boas et al 2012; Miller et al 2009)

Lans, M. C., Klasson-Wehler, E., Willemsen, M., Meussen, E., Safe, S., & Brouwer, A. (1993). STRUCTURE-DEPENDENT, COMPETITIVE INTERACTION OF HYDROXY-POLYCHLOROBIPHENYLS, -DIBENZO-p-DIOXINS AND -DIBENZOFURANS WITH HUMAN TRANSTHYRETIN. Chemico-Biological Interactions, 88, 7–21.

Polybrominated diphenyl ethers

It has been shown that many PBDE congeners and/or metabolites (including hydroxylated metabolites of PBDE congeners) can bind to TTR, in some cases more strongly than T4 (Hallgren and Darnerud 2002; Marchesini et al 2008; Ren and Guo 2012; Weiss et al 2015), and this class of congeners has been implicated as a thyroid toxicant (Boas et al 2012; Gore et al 2015; Miller et al 2009; Murk et al 2013). In rats, certain PBDEs have been found to impact learning and memory ability via damage to hippocampal neurons, perhaps through a TTR-mediated transport process as described in this AOP (Driscoll et al 2009; Kato et al 2009; Sun et al 2017). In humans, PBDEs and/or metabolites affect TH during vulnerable windows (i.e. pregnancy) and have been associated with pediatric neurobehavioral development and the developing nervous system, with particular emphasis on the hydroxylated metabolites that have been found to bioaccumulate in serum in children (Athanasiadou et al 2008; Chevrier et al 2010; Dingemans et al 2011; Eskenazi et al 2013; Preau et al 2015).

Athanasiadou, M., Cuadra, S. N., Marsh, G., Bergman, A., & Jakobsson, K. (2008). Polybrominated diphenyl ethers (PBDEs) and bioaccumulative hydroxylated PBDE metabolites in young humans from Managua, Nicaragua. Environmental Health Perspectives, 116(3), 400–408. http://doi.org/10.1289/ehp.10713

Chevrier, J., Harley, K. G., Bradman, A., Gharbi, M., Sjödin, A., & Eskenazi, B. (2010). Polybrominated diphenyl ether (PBDE) flame retardants and thyroid hormone during pregnancy. Environmental Health Perspectives, 118(10), 1444–1449. http://doi.org/10.1289/ehp.1001905

Dingemans, M. M. L., van den Berg, M., & Westerink, R. H. S. (2011). Neurotoxicity of brominated flame retardants: (In)direct effects of parent and hydroxylated polybrominated diphenyl ethers on the (Developing) nervous system. Environmental Health Perspectives, 119(7), 900–907. http://doi.org/10.1289/ehp.1003035

Driscoll, L. L., Gibson, A. M., & Hieb, A. (2009). Chronic postnatal DE-71 exposure: Effects on learning, attention and thyroxine levels. Neurotoxicology and Teratology, 31(2), 76–84. http://doi.org/10.1016/j.ntt.2008.11.003

Eskenazi, B., Chevrier, J., Rauch, S. A., Kogut, K., Harley, K. G., Johnson, C., … Bradman, A. (2013). In utero and childhood polybrominated diphenyl ether (PBDE) exposures and neurodevelopment in the CHAMACOS study. Environmental Health Perspectives, 121(2), 257–262. http://doi.org/10.1289/ehp.1205597

Hallgren, S., & Darnerud, P. O. (2002). Polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and chlorinated paraffins (CPs) in rats—testing interactions and mechanisms for thyroid hormone effects. Toxicology, 177(2–3), 227–243. http://doi.org/10.1016/S0300-483X(02)00222-6

Kato, Y., Haraguchi, K., Kubota, M., Seto, Y., Ikushiro, S. I., Sakaki, T., … Degawa, M. (2009). 4-Hydroxy-2,2’,3,4’,5,5’,6-heptachlorobiphenyl-mediated decrease in serum thyroxine level in mice occurs through increase in accumulation of thyroxine in the liver. Drug Metabolism and Disposition, 37(10), 2095–2102. http://doi.org/10.1124/dmd.109.028621

Ren, X. M., & Guo, L. H. (2012). Assessment of the binding of hydroxylated polybrominated diphenyl ethers to thyroid hormone transport proteins using a site-specific fluorescence probe. Environmental Science and Technology, 46(8), 4633–4640. http://doi.org/10.1021/es2046074

Sun W, Du L, Tang W, Kuang L, Du P, Chen J, Chen D. PBDE-209 exposure damages learning and memory ability in rats potentially through increased autophagy and apoptosis in the hippocampus neuron. Environ Toxicol Pharmacol. 2017 Mar;50:151-158. doi: 10.1016/j.etap.2017.02.006.

Isoflavones

Soy isoflavones, such as genistein, have been found to bind to TTR (Radovic et al 2006) and have been noted as thyroid toxicants (Miller et al 2009; Murk et al 2013). Synthetic flavonoids, such as EMD 21388 have also been found to interfere with TH transport via TTR binding and have been well-studied in rats (Leuprasitsakul et al 1990; Mendel et al 1992; Pedraza et al 1996; Schröder-van der Elst et al 1997; Schröder-van der Elst et al 1998). This early work in rats helped refine knowledge of the TTR interference pathway and its important to brain and fetal thyroid function.

Lueprasitsakul, W., Alex, S., Fang, S. L., Pino, S., Irmscher, K., Köhrle, J., & Braverman, L. E. (1990). Flavonoid administration immediately displaces thyroxine (T4) from serum transthyretin, increases serum free T4, and decreases serum thyrotropin in the rat. Endocrinology.

Mendel, C. M., Cavalieri, R. R., & Kohrle, J. (1992). Thyroxine (T4) transport and distribution in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from transthyretin. Endocrinology, 130(3), 1525–1532.

Pedraza, P., Calvo, R., Obregón, M. J., Asuncion, M., Escobar Del Rey, F., & Morreale De Escobar, G. (1996). Displacement of T4 from transthyretin by the synthetic flavonoid EMD 21388 results in increased production of T3 from T4 in rat dams and fetuses. Endocrinology, 137(11), 4902–4914. http://doi.org/10.1210/en.137.11.4902

Radović, B., Mentrup, B., & Köhrle, J. (2006). Genistein and other soya isoflavones are potent ligands for transthyretin in serum and cerebrospinal fluid. The British Journal of Nutrition, 95(6), 1171–1176. http://doi.org/10.1079/BJN20061779

Schröder-Van Der Elst, J. P., Van Der Heide, D., Rokos, H., Köhrle, J., & Morreale De Escobar, G. (1997). Different tissue distribution, elimination, and kinetics of thyroxine and its conformational analog, the synthetic flavonoid EMD 49209 in the rat. Endocrinology, 138(1), 79–84. http://doi.org/10.1210/en.138.1.79

Schröder-van der Elst, J. P., van der Heide, D., Rokos, H., Morreale de Escobar, G., & Köhrle, J. (1998). Synthetic flavonoids cross the placenta in the rat and are found in fetal brain. The American Journal of Physiology, 274(2 Pt 1), E253-6. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9486155

Perflourinated chemicals

Some PFCs have been found to bind to TTR more strongly than T4 and been implicated as thyroid toxicants that could act through transport protein displacement (Boas et al 2012; Coperchini et al 2017; Gutshall et al 1989; Ren et al 2016; Weiss et al 2015; Zhang et al 2015). Zhang et al (2016) determined the X-ray structures of TTR-PFOA and TTR-PFOS. Evidence of interference with thyroid function and/or TH concentrations has been reported in both animals (Chang et al 2008; Yu et al 2009) and human (Ballesteros et al 2016; Dallaire et al 2009; Preau et al 2014; Wang et al 2014), including evidence of transplacental transfer (Yang et al 2016).

Ballesteros, V., Costa, O., Iñiguez, C., Fletcher, T., Ballester, F., & Lopez-Espinosa, M.-J. (2016). Exposure to perfluoroalkyl substances and thyroid function in pregnant women and children: A systematic review of epidemiologic studies. Environment International. http://doi.org/10.1016/j.envint.2016.10.015

Chang SC, Thibodeaux JR, Eastvold ML, Ehresman DJ, Bjork JA, Froehlich JW, Lau C, Singh RJ, Wallace KB, Butenhoff JL. Thyroid hormone status and pituitary function in adult rats given oral doses of perfluorooctanesulfonate (PFOS). Toxicology. 2008 Jan 20;243(3):330-9.

Coperchini F, Awwad O, Rotondi M, Santini F, Imbriani M, Chiovato L. Thyroid disruption by perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA). J Endocrinol Invest. 2017 Feb;40(2):105-121. doi: 10.1007/s40618-016-0572-z. Review.

Dallaire, R., Dewailly, É., Pereg, D., Dery, S., & Ayotte, P. (2009). Thyroid function and plasma concentrations of polyhalogenated compounds in inuit adults. Environmental Health Perspectives, 117(9), 1380–1386. http://doi.org/10.1289/ehp.0900633

Gutshall, D. M., Pilcher, G. D., & Langley, A. E. (1989). Mechanism of the serum thyroid hormone lowering effect of perfluoro-n-decanoic acid (PFDA) in rats. Journal of Toxicology and Environmental Health, 28(0098–4108; 1), 53–65. http://doi.org/10.1080/15287398909531328

Ren XM, Qin WP, Cao LY, Zhang J, Yang Y, Wan B, Guo LH. Binding interactions of perfluoroalkyl substances with thyroid hormone transport proteins and potential toxicological implications. Toxicology. 2016 Jul 29;366-367:32-42. doi: 10.1016/j.tox.2016.08.011. Epub 2016 Aug 12.

Wang Y, Rogan WJ, Chen PC, Lien GW, Chen HY, Tseng YC, Longnecker MP, Wang SL. Association between maternal serum perfluoroalkyl substances during pregnancy and maternal and cord thyroid hormones: Taiwan maternal and infant cohort study. Environ Health Perspect. 2014 May;122(5):529-34. doi: 10.1289/ehp.1306925.

Yang L, Li J, Lai J, Luan H, Cai Z, Wang Y, Zhao Y, Wu Y. Placental Transfer of Perfluoroalkyl Substances and Associations with Thyroid Hormones: Beijing Prenatal Exposure Study. Sci Rep. 2016 Feb 22;6:21699. doi: 10.1038/srep21699.

Yu WG, Liu W, Jin YH, Liu XH, Wang FQ, Liu L, Nakayama SF. Prenatal and postnatal impact of perfluorooctane sulfonate (PFOS) on rat development: a cross-foster study on chemical burden and thyroid hormone system. Environ Sci Technol. 2009 Nov 1;43(21):8416-22. doi: 10.1021/es901602d.

Zhang, J., Kamstra, J. H., Ghorbanzadeh, M., Weiss, J. M., Hamers, T., & Andersson, P. L. (2015). In Silico Approach To Identify Potential Thyroid Hormone Disruptors among Currently Known Dust Contaminants and Their Metabolites. Environmental Science and Technology, 49(16), 10099–10107. http://doi.org/10.1021/acs.est.5b01742

Zhang J, Begum A, Brännström K, Grundström C, Iakovleva I, Olofsson A, Sauer-Eriksson AE, Andersson PL. Structure-Based Virtual Screening Protocol for in Silico Identification of Potential Thyroid Disrupting Chemicals Targeting Transthyretin. Environ Sci Technol. 2016 Nov 1;50(21):11984-11993. Epub 2016 Oct 10.

Phthalates

Some phthalates have been found to competitively bind to TTR (Ishihara et al 2003; Weiss et al 2015) and have been implicated as thyroid toxicants (Boas et al 2012; Gore et al 2015; Miller et al 2009). While both animal and human data is scarce, there is some evidence for dose-dependent biochemical and morphological changes in animals (Howarth et al 2001; Liu et al 2015; O'Connor et al 2002) as well as in humans (Huang et al 2007; Meeker et al 2007).

Howarth JA, Price SC, Dobrota M, Kentish PA, Hinton RH. Effects on male rats of di-(2-ethylhexyl) phthalate and di-n-hexylphthalate administered alone or in combination. Toxicol Lett. 2001 Apr 8;121(1):35-43.

Huang PC, Kuo PL, Guo YL, Liao PC, Lee CC. Associations between urinary phthalate monoesters and thyroid hormones in pregnant women. Hum Reprod. 2007 Oct;22(10):2715-22.

Ishihara A1, Nishiyama N, Sugiyama S, Yamauchi K. The effect of endocrine disrupting chemicals on thyroid hormone binding to Japanese quail transthyretin and thyroid hormone receptor. Gen Comp Endocrinol. 2003 Oct 15;134(1):36-43.

Liu, C., Zhao, L., Wei, L., & Li, L. (2015). DEHP reduces thyroid hormones via interacting with hormone synthesis-related proteins, deiodinases, transthyretin, receptors, and hepatic enzymes in rats. Environmental Science and Pollution Research. http://doi.org/10.1007/s11356-015-4567-7

Meeker JD, Calafat AM, Hauser R. Di(2-ethylhexyl) phthalate metabolites may alter thyroid hormone levels in men. Environ Health Perspect. 2007 Jul;115(7):1029-34.

O'Connor JC, Frame SR, Ladics GS. Evaluation of a 15-day screening assay using intact male rats for identifying steroid biosynthesis inhibitors and thyroid modulators. Toxicol Sci. 2002 Sep;69(1):79-91.

Tetrabromobisphenol A

Tetrabromobisphenol A (TBBPA) has been found to competitively bind to TTR (Meerts et al 2000; Weiss et al 2015) and other bisphenols have been implicated as thyroid toxicants (Boas et al 2012). Iakovleva et al (2016) recently reported the crystal structure of TTR-TBBPA

Iakovleva, I., Begum, A., Brännström, K., Wijsekera, A., Nilsson, L., Zhang, J., … Olofsson, A. (2016). Tetrabromobisphenol a is an efficient stabilizer of the transthyretin tetramer. PLoS ONE, 11(4), 1–16. http://doi.org/10.1371/journal.pone.0153529

Meerts, I. a, van Zanden, J. J., Luijks, E. a, van Leeuwen-Bol, I., Marsh, G., Jakobsson, E., … Brouwer, a. (2000). Potent competitive interactions of some brominated flame retardants and related compounds with human transthyretin in vitro. Toxicological Sciences : An Official Journal of the Society of Toxicology, 56(1), 95–104. http://doi.org/10.1093/toxsci/56.1.95

Clonixin

Similar structure and binding affinity as meclofenamic acid.

Meclofenamic acid

2,6-dinitro-p-cresol

Triclopyr

Major metabolite 3,5,6-trichloro-2-pyridinol has been reported to disrupt TH levels via TTR binding.

2,2',4,4'-Tetrahydroxybenzophenone

Overall Assessment of the AOP

Essentiality of the Key Events

Molecular Initiating Event Summary, Key Event Summary
Provide an overall assessment of the essentiality for the key events in the AOP. Support calls for individual key events can be included in the molecular initiating event, key event, and adverse outcome tables above.

In vivo evidence for MIE

Kohrle et al (1989) added 10 μmol/L 3-methyl-4’,6-dihydroxy-3’,5-dibromo-flavone (EMD 21388) to pooled rat serum and measured displacement of [125I]-T4 from TTR. EMD21388 was synthesized using “molecular drug design” (and resembles T4) to help confirm previous findings that certain flavonoid deiodinase inhibitors also displaced thyroxine (T4) for TTR (or T3-binding prealbumin). Displacement of [125I] from TTR in rat serum was analyzed by gel electrophoresis (PAGE) and individual serum samples were assayed for T3 and T4 content by RIA and % free TH by equilibrium dialysis (lower limit of detectability 0.3 ug/dL for T4). There was a significant increase in % free T4 (0.031 to 0.124), which was dose-dependent and resulted in complete inhibition of [125I]-T4/TTR at 8-10 umol (radiolabeled TH were displaced primarily to albumin).

insert Fig 2 from Kohrle et al 1989

One to 4 hours following ip delivery of 2 μmol/100 g BW to euthyroid Sprague-Dawley rats (a dose that is 1000x higher than daily T4 production in rat), inhibition of [125I]-T4/TTR binding was observed. T4 decreased from 5.6 to 2.3 ug/dl after 1 hour and remained low while % free T4 increased from 0.035 to 0.091 and remained high; however, free T4 did not change. TSH decreased to very low values after 2 hours and increased slightly, despite no change in the free TH concentration (hypothyroid rats did not show changes in serum TSH following EMD 21388 administration). Lueprasitsakul et al (1990) performed a series of experiments with Sprague-Dawley rats using smaller doses of EMD 21388 (up to 2 μmol /100 g BW) and the same measurement methods (RIA, equilibrium dialysis). Administration of 2 μmol of EMD 21388 inhibited [125I]-T4/TTR binding within a few minutes, displacing [125I] to albumin to a greater degree of magnitude, due to slight differences in preparing the EMD 21388 solutions. Dose-dependent decreases in displacement were found with decreasing dose.

insert Figure 1 & Figure 2

Following a single dose of 2 μmol, a significant decrease was seen in total serum T4 after 10 minutes that persisted, % free T4 also increased immediately (peaked after 10 minutes) and stayed elevated and a significant increase in free T4 was observed within three minutes that stayed elevated for 60 minutes. Following a single dose of 0.3 μmol, decreased [125I]-T4/TTR binding was observed reaching a nadir after 10 minutes and slowly recovering over the 180-minute experiment. The % free T4 and serum free T4 both increased and returned to normal after 180 minutes as well while total serum T4 hit a nadir after 10 minutes and mostly recovered. Serum TSH decreased after 20 minutes, significantly at the nadir hit after 60 minutes.

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Appendix 1

List of MIEs in this AOP

Event: 957: Binding, Transthyretin in serum

Short Name: Binding, Transthyretin in serum

Key Event Component

Process	Object	Action
binding	transthyretin	increased

AOPs Including This Key Event

AOP ID and Name	Event Type
Aop:152 - Interference with thyroid serum binding protein transthyretin and subsequent adverse human neurodevelopmental toxicity	MolecularInitiatingEvent
Aop:366 - Competitive binding to thyroid hormone carrier protein transthyretin (TTR) leading to altered amphibian metamorphosis	MolecularInitiatingEvent

Stressors

Name
Halogenated phenols
Polychlorinated biphenyl

Biological Context

Level of Biological Organization
Molecular

Organ term

Organ term
serum

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

Rickenbacher et al (1986) provided initial direct evidence of competition for the T4 binding site using molecular modeling and binding assays using radiolabeled TH. Brouwer and van den Berg (1986) reported preferential binding of a metabolite of radiolabeled tetrachlorobiphenyl to TTR in rats (15 mg/kg, ip), using gel electrophoresis followed by HPLC analysis. Van den Berg (1990) used a competitive binding assay to assess the ability of hydroxylated chlorinated aromatic compounds to bind to radiolabeled T4. Van den Berg et al (1991) extended this work to 65 compounds from 12 different chemical groups in rats treated via a single ip dose and competitive binding assay. Chlorophenols were found to have higher affinity relative to other chlorinated aromatics, particular at higher levels of chlorination, and the combination and position of hydroxyl & chlorine atoms. {insert Figure 2/Van den Berg 1990}

Lans et al (1993) described the ability of hydroxylated metabolites of PCBs, PCDDs and PCDFs to act as competetive ligands at the TTR-T4 binding site using an in vitro binding assay. Many of the hydroxylated PCBs examined were more potent ligands than T4, as opposed to those PCDFs and PCDDs that lacked chlorine atoms substituted adjacent to hydroxyl groups. When the hydroxyl group was in the meta or para positions, a 35- to 136-fold greater potency was found relative to ortho substitutions. These results were confirmed through later competitive binding work published by Cheek et al (1999) and Chauhan et al (2000).

Weiss et al (2009) first confirmed competitive binding of TTR with perfluorinated compounds (PFCs)

Domain of Applicability

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Homo sapiens	Homo sapiens	High	NCBI
Bubalus bubalis	Bubalus bubalis	High	NCBI
Rattus norvegicus	Rattus norvegicus	High	NCBI
Mus musculus	Mus musculus	High	NCBI
Gallus gallus	Gallus gallus	High	NCBI
Sus scrofa	Sus scrofa	High	NCBI
Pan troglodytes	Pan troglodytes	High	NCBI
Monodelphis domestica	Monodelphis domestica	High	NCBI
Xenopus tropicalis	Xenopus (Silurana) tropicalis	High	NCBI
Bos taurus	Bos taurus	High	NCBI
Macaca mulatta	Macaca mulatta	High	NCBI
Meleagris gallopavo	Meleagris gallopavo	High	NCBI
Canis lupus familiaris	Canis lupus familiaris	High	NCBI
Ovis aries	Ovis aries	High	NCBI
Erinaceus europaeus	Erinaceus europaeus	High	NCBI
Xenopus laevis	Xenopus laevis		NCBI

The transthyretin protein has been found to be highly conserved among vertebrates, as supported by X-ray crystallography studies in man, rat, chicken and fish as well as comparison of amino acid sequence (Richardson 2007). Over time, evolution has driven the nature of the N-terminal region of TTR from a more hydrophobic and longer region (as found in fish and amphibians) to a shorter and more hydrophilic region (as found in the placental mammals). The consequence of this biochemical change in the TTR protein was to change the primary function of TTR from binding and carrying T3 in serum to carrying T4 in serum (as it does almost exclusively in rats, as rats lack TBG during adult life)(Alshehri et al. 2015). Thus, in the placental mammals, TTR operates to carry T4 to specific tissues, where it is displaced, transported across the cell membrane by a different ligand-mediated process, and then converted to T3 via deiodinase enzymes within the cell (where it activates the nuclear receptor to cause downstream effects).

TTR is encoded by a single gene on human chromosome 18 (18q11.2-12.1) (LeBeau and Geurts van Kessel 1991).

TTR is synthesized in the adult liver only by eutherians (birds and placental mammals) and herbivorous marsupials; however, it is also synthesized in the choroid plexus of reptiles, birds and mammals. TTR is one of three thyroid hormone serum transport proteins, along with albumin (ALB) and thyroid-binding globulin (TBG). Over evolutionary time, the N-terminal section of TTR has become shortened and more hydrophilic such that T3 is more tightly bound in birds and reptiles but T4 is more tightly bound to TTR in mammals (Schrieber 2002). They all differ in binding affinity and dissociation rate for both T4 and T3 and together form a buffered system that seeks to keep circulating T4 within a certain range: between the normal concentration of free T4 in serum (30 pM) and solubility limit in serum (2 uM) (Schreiber 2002). Given these differences, more T4 available for cellular uptake likely comes from ALB-bound T4 in capillaries with fast moving blood but TTR-bound T4 is likely more important in slow moving fluid (like CSF)(Schrieber 2002).

Mammalian TTR has a greater affinity for T4 (and lower affinity for T3) in mammals relative to birds and reptiles, neither of which express a high-affinity TBG protein in serum (Schweizer and Kohrle 2013); while Larsson reported the presence of TTR (i.e. thyroxine-binding prealbumin) in all vertebrate species investigated and failed to detect the presence of TBG in cat, rabbit, rat, chicken, frog and salmon. Binding capacity of serum TTR in female rats is lower relative to males (Emerson et al 1990 in Zoeller et al 2007).

Key Event Description

The key event that initiates this AOP (i.e. the MIE) is binding of a xenobiotic to the thyroxine (T4)-binding site of transthyretin (TTR), displacing T4 from the binding site(s) of TTR and adding this to the pool of free T4 in serum, which is normally roughly 0.02% (20 pM) of total T4 (100 nM) [in CSF, free T4 is roughly 1.4% (70 pM) of total T4 (2-3 nM)](Schweizer and Kohrle 2013).

TTR has been found to bind with a number of ligands, including pharmacologic agents as well as flavonoids and halogenated aromatic compounds, with differing strengths with some xenobiotics found to be possessing a binding affinity equal to or stronger than T4 (i.e. "competitive binder"). Studies with other xenobiotics with similar structural characteristics have found that a higher degree of halogen substitution, as well as placement on the ring relative to the hydroxyl group, increases binding affinity (Chauhan et al. 2000, Lans et al. 1993, Lans et al. 1994, Ren and Guo 2012, van den Berg 1990, van den Berg et al. 1991, Weiss et al 2015). Weiss et al (2015) compiled a database of almost 150 compounds found to bind to TTR and 52 of these were found to have higher affinity than T4 and thus, could be considered competitive binders in serum.

The function of the serum protein TTR is to deliver T4 to target cells in the liver, tight junctions, etc. where it is facilitated across the membrane via specific receptors and converted to the active form T3, where it can activate nuclear receptors. TTR facilitates passage across key tight junctions, such as the blood-brain barrier, the CSF barrier and transplacentally, and the interruption of thyroid serum protein-assisted transport during certain developmental windows can lead to profound developmental neurotoxicity (i.e. cretinism). It should be noted, though, that in mammals with all three functional serum transport proteins (TTR, albumin and TBG), substantial reductions in total T4 can be observed with little to no adverse effect due to overall redundancy of this system. In this scenario, roughly 75% of serum T4 is bound to TBG, 15% to TTR and up to 5% for to albumin (OECD DRP 2012). That being said, TTR is the sole transport protein in the CSF and T4 is not biosynthesized by the fetus until the second trimester - thus, the mother is the sole source of T4 for the fetus during early gestation and this appears to be the developmental window of greatest concern to human physiology (for example, see Loubiere et al 2010).

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Transthyretin is a 55-kDa tetramer protein composed of two identical monomer subunits, each of which is formed by two four-stranded beta sheets, which are assembled around the central channel of the protein (Blake et al. 1978). This central channel contains two identical binding sites; however, there is a 100-fold difference in binding constants between the first and second T4 molecule bound and this "negative cooperativity" results in a 1:1 relationship between T4 and TTR in terms of protein transport in serum (Ferguson et al. 1975). The phenolic hydroxyl and iodine substitutions on the phenolic ring of T4 are important structural characteristics that differentiate ability to bind to TTR, as opposed to other serum binding proteins like TBG (Andrea et al. 1980). T4 binds to TTR in one of two ways: "forward" and "reverse" mode. In forward mode, the phenolic ring of T4 is buried deeply in the TTR binding site while this ring interacts with residues at the entrance of the binding channel in reverse mode (Lans et al 1993). Detailed structural analysis of the protein is available from X-ray crystallography (Rerat and Schwick 1967; Blake et al 1977).

There are three main in vitro approaches to measuring binding affinity between TTR and its ligands: radioligand-binding assays (RLBAs), surface plasmon resonance (SPR) and fluorescence displacement. Biochemical (in chemico) approaches using GC/MS or LC/MS also exist that use recombinant (rTTR) and display limits of detection in the low nM range, including a multi-mode, ultra-high-performance LC method and anon-radioactive label, [¹³C₆]-T4 (Aqai et al 2012).

Van den Berg et al (1991) used an in vitro competitive radioligand binding assay with gel-filtration procedure modified from Somack et al (1982) as well to screen 65 compounds from 12 chemical classes for evidence of interference with the T4 binding site of TTR using a radioligand binding assay and reported results semi-quantitatively, while previous work using the same method on chlorinated phenols found that affinity rose with degree of chlorination (pentachlorophenol displayed an IC50 of 2.3 x 10⁸ M; derived K_a = 1.2 x 10⁸ M^-1) versus the natural ligand (T4; 4 x 10⁸ M)(Van Den Berg, 1990).

Lans et al (1993, 1994) used purified TTR and T4 [¹²⁵I] as a displaceable radioligand and gel-filtration procedure modified from Somack et al (1982) to study hydroxylated PCBs PCDDs and PCDFs and found many competitive binders in all classes; again, dependent on degree of chlorination, presence and placement of hydroxyl atom relative to chlorine placement (IC50 in 6.5-25 nM range; K_a = 0.78-3.95 x 10⁸ M^-1) versus the natural ligand (T4, K_a = 2.05 x 10⁷M^-1). Meerts et al (2000) studied PBDEs and related compounds (pentabromophenol, Tetrabromobisphenol A) and found multiple competitive binders (IC50 range 7.7 - 67 nM; Ka = 4.3 - 38 x 10⁷M^-1) versus the natural ligand (T4; IC50 = 80.7 nM; Ka = 3.5 x 10⁷M^-1).

Cao et al (2010) used a novel fluorescence displacement method using a protein-binding probe that fluoresces when bound, and the intensity of which dips following displacement for a ligand. Analyte titration curves are used to derive IC50 and binding constant values. They used an ANSA probe to screen 14 hydroxylated PBDEs and found competitive binding with TTR (K_a = 1.4 x 10⁷M^-1 to 6.9 x 10⁸ M^-1). Molecular docking analysis revealed a ligand-binding channel in TTR for OH-PBDE binding. Ren and Guo (2012) reported use of a non-radioactive, fluorescin-T4 conjugate designed as a fluorescent probe (vs ANSA probe which is less TTR-specific) to study the binding interaction of multiple hydroxylated PBDEs with differing degrees of bromination and differing hydroxy positions and TTR. Competitive binders were found (IC50 range = 110-219 nM) relative to the natural ligand (T4; IC50 = 260 nM) and Kd values for the competitive binders ranged from 101-210 nM versus T4 (Kd = 239 nM). Weiss et al (2009) initially determined the competitive binding of TTR with PFCs via radioligand-binding assay of Hamers et al (2006) and Lans et al (1993). Ren et al (2016) used a fluorescence displacement assay to study 16 PFCs and found T4 to have an IC50 of 31 nM (Kd = 5 nM), as compared to PFOS (the strongest binder) TC50 of 130 nM (Kd = 20 nM).

The radioligand methods have been criticized for use of hazardous and costly radio-labeled material and physical separation of bound and free T4-[¹²⁵I] is required. Marchesini et al (2006, 2008) reported a surface plasmon resonance biosensor-based method using rTTR, a surface-based measurement which may not fully characterize the binding reaction of a homogenous solution or matrix. Optimized SPR is more sensitive, amenable to high-throughput screening and easier to perform than RLBAs. Furthermore, hydroxy-metabolites do not endure destructive clean-up/extraction methods and differences in physicochemical qualities from parent compounds complicates separation.

Montano et al (2012) introduced a new method (using a modified method of Murk et al 1994 for in vitro bioactivation of PCBs and PBDEs with an ANSA-TTR displacement assay) to selectively extract metabolites (and limit co-extraction of interferants, like free fatty acids) and derive an IC50 dose-response curve for OH-PBDEs, OH-PCBs, etc. using bioactivated parent compound in a 96-well plate system. IC50 for the natural ligand (T4) was 0.26 uM while the IC50s for the hydroxy metabolites ranged from 0.23-0.63 uM.

References

Andrea et al. 1980

Blake et al 1978

Cao, J., Y. Lin, L.H. Guo, A.Q. Zhang, Y. Wei, and Y. Yang. 2010. Structure-based investigation on the binding interaction of hydroxylated polybrominated diphenyl ethers with thyroxine transport proteins. Toxicology 277(1-3):20-8.

Ferguson et al. 1975

Loubiere et al 2010

Richardson, S. J. (2007). Cell and molecular biology of transthyretin and thyroid hormones. International Review of Cytology, 258(January), 137–93. http://doi.org/10.1016/S0074-7696(07)58003-4

Schreiber, G. (2002). The evolutionary and integrative roles of transthyrein in thyroid hormone homeostasis. Journal of Endocrinology, 175(1), 61–73. http://doi.org/10.1677/joe.0.1750061

Schweizer and Kohrle 2013

Weiss et al 2009

List of Key Events in the AOP

Event: 958: Displacement, Serum thyroxine (T4) from transthyretin

Short Name: Displacement, Serum thyroxine (T4) from transthyretin

Key Event Component

Process	Object	Action
	thyroxine	increased

AOPs Including This Key Event

AOP ID and Name	Event Type
Aop:152 - Interference with thyroid serum binding protein transthyretin and subsequent adverse human neurodevelopmental toxicity	KeyEvent

Stressors

Name
2,4-Dinitrophenol

Biological Context

Level of Biological Organization
Molecular

Key Event Description

Despite the two binding sites for T4 on the TTR serum binding protein, each molecule of TTR only carries a single T4 molecule due to the negative cooperativity displayed by these binding sites (Ferguson et al 1975). As such, xenobiotics and pharmacologic agents can displace T4 from TTR and early on, this was demonstrated for ethnacrynic acid, salicylates, penicillin and 2,4-dinitrophenol (Munro et al 1989). More recently, work with the flavonoid EMD 21388 (and other compounds structurally similar to thyroxine) showed competitive binding and displacement of T4 from the TTR carrier protein.

Rickenbacher et al (1986) provided initial direct evidence of competition for the T4 binding site using molecular modeling and binding assays using radiolabeled TH. Brouwer and van den Berg (1986) reported preferential binding of a metabolite of radiolabeled tetrachlorobiphenyl to TTR in rats (15 mg/kg, ip), using gel electrophoresis followed by HPLC analysis. Van den Berg (1990) used a competitive binding assay to assess the ability of hydroxylated chlorinated aromatic compounds to bind to radiolabeled T4. Van den Berg et al (1991) extended this work to 65 compounds from 12 different chemical groups in rats treated via a single ip dose and competitive binding assay. Chlorophenols were found to have higher affinity relative to other chlorinated aromatics, particular at higher levels of chlorination, and the combination of hydroxyl chlorine atoms in the ortho position. {insert Figure 2/Van den Berg 1990}

Kohrle et al (1989) showed complete displacement (via gel electrophoresis) of radiolabeled T3 and T4 by EMD 21388 in pooled rat serum followed by increase in the percent free TH as measured by equilibrium dialysis. Complete inhibition occurred at 10 umol and displaced T4 from TTR to serum albumin and thyroxine-binding globulin (TBG), which normally serve a lesser role in thyroid hormone transport in humans. {insert Figure 2}

Kohrle et al (1989) administered EMD 21388 via ip route to rats at 2 umol/100 g BW and observed displacement of radiolabeled T3 and T4 from TTR followed by a decrease of T3 and T4 in serum while the percent free TH remained unchanged. {insert Figures 3-5}

Lueprasitsakul et al (1990) repeated this protocol and found that inhibition of binding occurred within 3 minutes followed by a decrease in serum T4 concentration and an increase in both serum percent free T4 as well serum total T4. {insert Figures 1 and 4}

Mendel et al (1992) demonstrated in rats dosed via IP with EMD 21388 (2 μmol/100 g BW) both displacement of radiolabeled T4 from TTR (as assessed via electrophoresis of serum proteins) and susbsequent increase of free T4 in serum.

To initially evaluate the impact of EMD 21388 on maternal/fetal hormones, Pedraza et al (1996) administered 2.5 mg 21388/day subcutaneously in pregnant female rats which led to displacement of T4 from TTR, reduced total T4 and increased free T4 in maternal circulation.

Compounds that have been found to compete with TTR for binding to T4 (and thus lead to some degree of thyroid disruption) include pharmaceuticals and environmental contaminants, such as halogenated aromatic compounds. This latter category include PCBs, PBBs, PBDEs and perfluoro compounds and specifically, hydroxylated metabolites of all these compounds often display greater binding affinity than the natural ligand; however, this is a function of degree of halogenation as well as orientation of the halogens and hydroxyl functional group.

Gutshall et al 1989 treated male Wistar rats with a single IP dose of perfluorodecanoic acid (PFDA) and ¹²⁵I and then measured TH, uptake of ¹²⁵I, liver enzymes and binding of [¹²⁵I]-T4 to albumin. The authors did not observe increased conversion of T4 to rT3 but did note that PFDA displaced [¹²⁵I]-T4 from rat albumin with an affinity similar to T4. While this study involved albumin, it showed that perfluoro compounds may also have potential to interact with thyroid serum transport proteins.

Meerts et al 2000 investigated the affinity of several polybrominated flame retardants (including 17 PBDE congeners) to TTR using human TTR in an in vitro competitive binding assay and [¹²⁵I]-T4. Pentabromophenol and tetrabromobisphenol A were found to have affinities 7-10 fold that of T4; however, a microsomal enzyme mediated transformation was needed first (i.e. hydroxylation) for PBDEs. It should be noted that no reference OH-PBDEs were available at the time of this experiment. Like the PCB congeners, degree of bromination is a driver of binding potency as is the nature of the halogen substitution (as well as hydroxy substitution). In addition, brominated analogs are more potent in general relative to chlorinated ones. Hydroxylation of parent compound via CYP2B enzymes appears to be a prerequisite of binding to TTR.

Hallgren et al 2001 treated female Sprague Dawley rats and C57BL/6 mice daily with Aroclor 1254, PCB-105, Bromkal 70-5 DE (commercial PBDE mixture) or BDE-47 via gavage for 14 days and measured TH, induction of microsomal phase I enzymes (EROD, MROD, PROD) and UDP UGT activity. Free and total T4 was decreased in both species with no significant change to TSH and minimal impact on UDP UGT activity. Rats were found to be more sensitive than mice to the observed effects. The findings suggested that PBDEs may be metabolized by CYP2B, but also CYP1A to an extent, and that these induced enzymes increased the availability of hydroxylated metabolites in vivo and increase binding to T4 transport proteins.

Hallgren and Darnerud 2002 treated female Sprague Dawley rats with BDE-47, Aroclor 1254 and Witaclor 171P, alone or in combinations, daily via gastric intubation for 14 days. Microsomal enzyme (cytochrome P450 isozymes and UDP UGTs), ex vivo binding of [¹²⁵I]-T4 to plasma proteins and light microscopy morphology of the thyroid was examined. Aroclor 1254 and BDE-47 was observed to decrease T4, decrease [¹²⁵I]-T4 binding to TTR, induce several phase I enzymes as well as moderate elevation of UDP UGT activity. These data suggested that decreased plasma T4 is mainly due to interference with serum transport binding of parent and metabolites to TTR; however, there was clearly some role for glucuronidation in reducing T4 in this study. (PCB mixtures have been demonstrated to impact a number of different endpoints affecting normal thyroid homeostasis.)

Metabolites of BDE 47 formed by CYP2B6 include hydroxylated BDE 47 in addition to other hydroxylated congeners (Erratico et al 2013, Feo et al 2013) and it should be noted that CYP2B6 is also expressed in brain which has implications for formation of hydroxylates in that tissue (Miksys and Tyndale 2004).

Cao et al 2004 performed molecular docking analysis on the TTR and OH-PBDE interactions and confirmed the effect of degree of bromination.

Darnerud et al 2006 treated female Sprague Dawley rats with BDE 47 or Bromkal 70-5 DE at 2 doses via gavage daily for 2 weeks. Thyroid hormones were measured from plasma via radioimmunoassay and samples pooled for analysis for individual congeners (BDEs 28, 47, 66, 99, 100, 138, 153, 154) and internal plasma doses were calculated that corresponded with decreased free T4. This critical dose was estimated to be ~ 400 ug/g lipid BDE 47 based on significant reduction of free T4.

Hamers et al 2006, 2008 collectively found that OH-PBDEs act as agonists or antagonists at TH receptors, that OH-PBDEs with high affinity for T4 can be detected in human serum and all metabolites were found to be more potent than the natural ligand in vitro using rat liver microsomes (3-OH-BDE-47 was found to have the highest potency).

Lau et al 2007 and Chang et al 2008 reported that PFOS alters serum T4 via interference with binding proteins, leading to a transient increase in free T4 and decrease in TSH.

Weiss et al 2009 were the first to examine the potential of 24 perfluorinated compounds and 6 structurally-related fatty acids to compete with T4 for TTR via [125I]-T4 binding assay and HPLC-MS/MS analysis. From this analysis, 56 chemical descriptors to evaluate the structure-activity relationship (SAR) of binding potency of perfluorinated compounds to TTR. Binding potency was found to strengthen with degree of fluorination, with maximum potency found at a chain length of eight (8) carbons; however, in general, the perfluorinated compounds were found to have T4 binding potency at about 10% that of the natural ligand.

Cao et al 2010 looked at binding interactions for 14 OH-PBDEs with TTR and TBG using fluorescence probe & competitive binding assay, circular dichroism (spectroscopic measurement of a protein’s secondary structure) and molecular docking analyses. Binding constant data was generated for the first time and affinity was observed to increase with degree of bromination, until a peak at the 5- and 6-brominated diphenyl ethers was reached. CD analysis showed that the OH-PDBEs bind to TTR and TBG at the same sites as the natural ligand while the molecular docking studies revealed a ligand-binding channel in TTR that was mostly hydrophobic inside but characterized by a positive-charged Lys15 residue at the channel entrance. The novel binding constant allowed meaningful quantitative evaluation of competitive displacement, by assuming human serum levels reported in the literature (Athanasiadou et al 2008; Marchesini et al 2008) and choosing the congener with the highest potency (5-OH-BDE47). This evaluation suggested that competitive displacement would be insignificant, as serum levels of protein-bound 5-OH-BDE47 are at least two orders of magnitude lower than protein-bound T4 (suggesting ~10% T4 displacement by 5-OH-BDE47).

Cao et al 2011 generated binding constant data for the interactions of BPA and TH for TTR, TBG and human albumin via fluorescence probe, noting that concentrations of BPA commonly reported in human plasma are likely not high enough to interfere with T4 transport in serum. A large excess of TTR and albumin in plasma are found relative to T4 and BPA and there is no competition for binding, which is further supported by the fact that affinity of BPA for T4 is much weaker than the natural ligand (by 2-3 orders of magnitude).

Ren and Guo 2012 designed a fluorescin T4 conjugate for use as a fluorescence probe in binding assays to examine interaction between eleven OH-PBDEs and transport proteins TTR and TBG. 3-OH-BDE47 and 3’-OH-BDE154 were found to be competitive with T4.

Ren et al 2013 higher brominated OH-PBDEs act as antagonists (i.e. BDEs 154 and 188) while lesser bromination (i.e. BDEs 47) found to be agonists

Grimm et al 2013 used fluorescence probe displacement and molecular docking simulations to characterize the binding of sulfated PCB metabolites to TTR, and stability and reversibility of these complexes were characterized by HPLC. The hydroxylated PCB metabolites (OH-PCBs) are excellent substrates for sulfation (phase II conjugation) via sulfotransferases (SULTs) and thus could represent another mechanism through which clearance can occur. Of the five lower-chlorinated sulfates for which K_d values were generated and compared against T4, only one would be considered a competitive inhibitor (4’PCB 11 sulfate) and the only case where the sulfate displays higher affinity than its corresponding OH metabolite (4’ OH-PCB 11). Molecular docking simulation confirmed the affinity that PCB sulfates have for TTR, confirming previous reports showing higher affinity among those congeners with meta- and para-chlorination. These data demonstrate the toxicological relevance of PCB sulfates to TTR-mediated transport of thyroid hormones in serum for the first time. The generation of sulfates from OH-PCBs could be another mechanism through which PCBs may disrupt thyroid homeostasis.

Weiss et al 2015 compiled a database of 250 compounds and mixtures (including 33 never tested before), of which 144 were TTR binders and 36% (n=52) of these were found to be more potent than the natural ligand T4. The vast majority of these 52 (n=48) were aromatic, halogenated and hydroxylated. A subset of 220 compounds was further analyzed via PCA and a set of chemical descriptors to understand the chemical characteristics of TTR binders and four significant components were found to explain 85% of the variance.

Zhang et al 2015 developed a QSAR model and applied to a database of almost 500 dust contaminants taken from literature data and over 400 in silico derived metabolites, predicting 37 contaminants and 230 metabolites as potential TTR binders. Twenty-three (23) contaminants were than analyzed via radioligand binding assay which identified four novel TTR ligands that were then analyzed via molecular docking studies.

How it is Measured or Detected

In humans, approximately 0.03% of total serum T4 is present in unbound/free condition (Refetoff et al 1970). Of the bound T4, approximately 75% is bound to TBG, approximately 20% to TTR and the remainder to ALB and some high density lipoprotein carriers. ALB is present at roughly 100-fold the molar concentration of TTR and roughly 2,000-fold higher than TBG; however, the affinity of T4 to TBG is 50-fold higher than TTR and 7,000-fold higher than ALB (Refetoff 2015). TTR binds roughly 80% of the T4 circulating in ventricular CSF although it constitutes only 25% of protein found there (Herbert et al 1986). In serum, only about 0.5% of circulating TTR is bound to T4, average serum concentration is 25 mg/dL (which can bind up to 300 ug T4/dL (Refetoff 2015).

TTR can be measured by densitometry after its separation from other serum proteins via electrophoresis, hormone saturation and/or immunoassays (Refetoff 2015).

Total T4 is most often measured using human serum based diagnostic kits, but free T4 (and T3) is only directly measured through equilibrium dialysis and ultrafiltration (Midgley 2001). Large volumes of serum must be used due to the very low concentrations of free T4 normally found (0.1% of total T4), which requires pooling of samples taken from fetus or pup. Some researchers have tried to “micronize” this process through combining RIA to measure total TH and dialysis to estimate the free fraction (Zoeller et al 2007). Extracted materials can also be quantified by HPLC. The reference range for free T4 is 9.8 to 18.8 pM/L (Dirinck et al 2016).

T3 is found in similar plasma concentrations to T4 (i.e. 5-10 pM) with < 0.4% being in the unbound state. Measuring free serum T3 is labor intensive and requires equipment not available in many clinical reference laboratories and thus ultrafiltration is often used (Abdalla and Bianco 2014). Immunoassays and MS/MS are also used.

Measuring displacement of T4 from serum transport proteins is done mainly via one of three in vitro methods: radioligand binding assay, plasmon resonance-based biosensor, or fluorescence displacement.

Radioligand binding assays, using [¹²⁵I]-T4 as a label, were developed to demonstrate affinity for xenobiotics to human or rat TTR and TBG (Brouwer and van den Berg 1986, Lans et al 1994). The most commonly used method was first published by Somack et al 1982 and adapted by Hamers et al 2006, Lans et al 1993 and Ucan-Marin et al 2010. Similar assays have been developed using [¹²⁵I]-T3 as a label for affinity to chicken and bullfrog TTR (Yamauchi et al 2003). Radioligand methods suffer from having to use heavily regulated isotopes and lower throughput to provide free T4 measurements (due to the extra wash/separation procedure needed). The most well-known protocol uses TTR purified from human serum (which may not be as stable as recombinant) and performed in a pure aqueous solution, which may not be as stable for lipophilic compounds (Chauhan et al 2000 is an example using PCBs).

Purkey et al 2001 published a binding assay using polyclonal TTR antibodies covalently bound to sepharose resin which is then mixed with plasma pre-treated with compound of interest, washed and analyzed via HPLC.

Marchesini et al 2006 reported on the development of two surface plasmon resonance(SPR)-based biosensor assays using recombinant TTR and TBG, validated with known thyroid disruptors and structurally related compounds including halogenated phenols, polychlorinated biphenyls, bisphenols and a hydroxylated PCB metabolite (4-OH-CB 14). TH is covalently bound to a gold-layered chip and a mixture of the compound of interest and transport protein are injected in a flow cell passing over the bound TH. The authors found that these biosensor methods were more sensitive (IC50 of 8.6 ± 0.7 nM for rTTR), easier to perform and more rapid that radioligand binding assays and immunoprecipitation-HPLC.

Marchesini et al 2008 applied their biosensor-based screen to 62 chemicals of public health concern and found that hydroxylated metabolites of PCBs (particularly para-hydroxylated ones) and PBDEs (BDEs 47, 49 and 99) displayed the most potent binding to TBG and TTR, confirming many other previous studies. The authors conclude their optimized assays are suitable for high-throughput screening for potential thyroid disruption.

Cao et al 2010, Cao et al 2011 and Ren and Guo 2012 developed the FLU-TTR, based on a protein-binding fluorescent probe (ANSA, or 8-anilo-1-naphthalenesulfonic acid ammonium salt) that becomes highly fluorescent after binding to T4. When the compound of interest is introduced and displaces the ANSA-thyroxine probe, this fluorescence is reduced. This allows generation of binding constant (K) data as opposed to past efforts that generated IC₅₀ values. Cao et al 2011 developed a fluorescent microtiter method for pTTR and TBG tested with bisphenol A.

Montano et al 2012 developed a competitive T4-TTR fluorescence displacement assay in a 96-well format, modified from the original method (Nilsson and Petersen 1975) and using a new selective method to extract hydroxylated metabolites while reducing fatty acid interference (modified from Hovander et al 2000).

Aqai et al 2012 described a rapid and isotope-free (¹³C₆-T4) screening of thyroid transport protein ligands, using a competitive binding assay for rTTR using fast ultrahigh performance LC-electrospray ionization triple-quadrupole MS. The method involves the use of immunomagnetic beads followed by screening with flow cytometry and UPLC-MS. The high-throughput screening mode is capable of detecting T4 in water at the part-per-trillion level and in the part-per-billion level in urine.

Relevant Phase II enzymes that are responsible for TH metabolism include UGT1A1, UGT1A6 and SULT2A1 while relevant cellular import/export transport proteins include MCT8, OATP1A4 and MRP2. All contribute towards systemic clearance of TH and conjugates from serum whether increasing biliary excretion or moving TH into tissues and across the placenta and BBB. Enzyme induction can only be measured via in vitro cell-based assays and since these enzymes are all controlled by specific nuclear receptors, assays targeting these receptors might act as surrogate measurement (Murk et al 2013). Several methods measuring expression of UGT or SULT mRNA have been published; however, there have been limited efforts to develop higher-throughput methods. The EPA ToxCast Phase I efforts used quantitative nuclease protection assays (qNPA) to screen several hundred chemicals for UGT1A1 and SULT2A1 (Rotroff et al 2010, Sinz et al 2006).