Toxicology in Vitro 27 (2013) 1320–1346

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Toxicology in Vitro

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Review Mechanism-based testing strategy using in vitro approaches for identification of thyroid hormone disrupting chemicals ⇑ AlberTinka J. Murk a, , Eddy Rijntjes b, Bas J. Blaauboer c, Rebecca Clewell d, Kevin M. Crofton e, Milou M.L. Dingemans f, J. David Furlow g, Robert Kavlock h, Josef Köhrle b, Robert Opitz i, Theo Traas k, Theo J. Visser j, Menghang Xia l, Arno C. Gutleb m a Wageningen University, Sub-department of Toxicology, Tuinlaan 5, 6703 HE Wageningen, The Netherlands b Institut für Experimentelle Endokrinologie, Charité-Universitätsmedizin Berlin, Berlin, Germany c Doerenkamp-Zbinden Chair, Institute for Risk Assessment Sciences, Division of Toxicology, Utrecht University, The Netherlands d The Hamner Institutes for Health Sciences, Research Triangle Park, NC, USA e National Center for Computational Toxicology, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC, USA f Neurotoxicology Research Group, Toxicology Division, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands g Department of Neurobiology, Physiology, and Behavior, University of California, Davis, USA h Office of Research and Development, US Environmental Protection Agency, Washington, DC, USA i Institute of Interdisciplinary Research in Molecular Human Biology, Université Libre de Bruxelles, 1070 Brussels, Belgium j Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands k Bureau REACH, National Institute of Public Health and the Environment, Bilthoven, The Netherlands l National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA m Department Environment and Agro-biotechnologies, Centre de Recherche Public – Gabriel Lippmann, Belvaux, Luxembourg article info abstract

Article history: The thyroid hormone (TH) system is involved in several important physiological processes, including Received 10 November 2012 regulation of energy metabolism, growt hand differentiation ,development and maintenance of brain Accepted 18 February 2013 function, thermo-regulation, osmo-regulation, and axis of regulation of other endocrine systems, sexual Available online 27 February 2013 behaviou rand fertility and cardiovascular function. Therefore, concern about TH disru ption (THD)has resulted in strategies being developed to identify THD chemicals (THDCs). Information on potential of Keywords: chemicals causing THD is typically derived from animal studies. For the majority of chemicals, how- Endocrine disruption ever, this information is either limited or unavaila ble. It is also unlikely that animal experiments will Thyroid hormone be performed for all THD relevant chemicals in the near future for ethical, financial and practical rea- Alternatives to animal testing In vitro assays sons. In add ition, typical animal experimen ts often do not provide information on the mech anism of Toxicokinetics action of THDC, making it harder to extrapolate results across species. Relevant effects may not be Testing strategy identified in animal studies when the effects are delayed, life stage specific, not assessed by the exper- Battery imental paradigm (e.g., behaviour) or only occur when an organism has to adapt to environmental fac- Functional assay tors by modulating TH levels. Therefore, in vitro and in silico alternatives to identify THDC and quantify Metabolism their potency are needed. THD Chave many potential mechanisms of action, including altered hormone produc tion, transport, metabolism, receptor activation and disruption of several feed-back mecha- nisms. In vitro assays are availabl efor many of these endpoints, and the application of modern ‘-omics’ technologies, applicable for in vivo studies can help to reveal relevant and possibl ynew endpoints for inclus ion in a targeted THDC in vitro test battery. Within the framew ork of the ASAT initiative (Assur- ing Safety without Animal Testing), an international group consisting of experts in the areas of thyroid endocrinology , toxicology of endocrine disruption, neurotoxicology, high-throughput screening, com- putational biology, and regulatory affairs has reviewed the state of science for (1) known mechanisms for THD plus examples of THDC; (2) in vitro THD tests currently available or under development related to these mechanisms; and (3) in silico methods for estimating the blood levels of THDC. Based on this scientific review, the panel has recommen ded abattery of test methods to be able to classify chemicals as of less or high concern for further hazard and risk asses sment for THD. In addition, research gaps and needs are identified to be able to optimize and validate the targeted THD in vitro test battery for a mechanism-based strategy for a decision to opt out or to proceed with further testing for THD. Published by Elsevier Ltd.

⇑ Corresponding author. E-mail address: [email protected] (A.J. Murk).

0887-2333/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tiv.2013.02.012 AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346 1321

Contents

1. Introduction ...... 1321 1.1. Possible physiological consequences of thyroid hormone disruption ...... 1321 1.2. Environment al chemicals and THD ...... 1322 1.3. Aim of the workshop...... 1327 2. Thyroid hormone system and endpoints for THD ...... 1327 2.1. Box 1. Central regulation ...... 1327 2.1.1. Endpoint: TRH receptor signalling ...... 1327 2.1.2. In vitro assays for TRH receptor signalling ...... 1327 2.1.3. Endpoint: TSH receptor signalling ...... 1328 2.1.4. In vitro assays for TSH receptor signalling ...... 1328 2.2. Box 2. TH synthesis and secretion by the thyroid gland ...... 1328 2.2.1. Endpoint: NIS-mediated iodide uptake ...... 1329 2.2.2. In vitro assays for NIS-mediated iodide uptake ...... 1329 2.2.3. Endpoint: TPO inhibition ...... 1329 2.2.4. In vitro assays for TPO inhibition ...... 1329 2.3. Box 3. Transport of thyroid hormones ...... 1330 2.3.1. Endpoint: Binding to transport proteins TTR and TBG ...... 1330 2.3.2. In vitro assays for binding to transport proteins TTR and TBG ...... 1330 2.4. Box 4. Metabolism and excretion ...... 1330 2.4.1. Endpoint: Deiodination ...... 1331 2.4.2. Endpoint: Alanine side chain modification ...... 1331 2.4.3. Endpoint: Sulfation ...... 1331 2.4.4. Endpoint: Glucuronidation ...... 1331 2.4.5. Inhibition of enzyme functioning ...... 1331 2.4.6. In vitro assays for induction and inhibition of enzymes for TH metabolism ...... 1332 2.5. Box 5. Cellular concentrations and transporters ...... 1332 2.5.1. Endpoint: cellular TH uptake ...... 1332 2.6. Box 6. Cellular responses ...... 1332 2.6.1. Endpoint: TR binding and transcriptional activity ...... 1333 2.6.2. In vitro assays for detecting disruption of TR activity ...... 1333 3. Chemical bio-activation and availability in vitro ...... 1334 4. Suggested testing strategy ...... 1334 4.1. Tier 1...... 1335 4.2. Tier 2...... 1335 5. Advantages of in vitro testing ...... 1337 6. Further outlook: using computational modelling to interpret in vitro assays for in vivo effects ...... 1337 7. Recommendations for assay use and development ...... 1337 8. Summarizing conclusions ...... 1338 Conflict of interest statement ...... 1338 Disclaimer ...... 1338 Acknowledgements ...... 1338 References ...... 1338

1. Introduction ‘permissive hormone action’. This indicates that the TH status of cells, tissues, and organisms provides the background and platform Endocrine disruption (ED) by chemicals is not restricted to the for other biological signals – hormonal, neural, immunological, sex hormone system, but also includes thyroid hormone (TH) dis- nutritive and environmental – that are critical for maintenance of ruption (THD). THD is defined herein as a change in hormone pro- both development and homeostasis of the organism as a whole duction, transport, function or metabolism resulting in impaired (Lopez-Juarez et al., 2012; Pascual and Aranda, 2012; Sirakov homeostasis. When the homeostasis is not impaired it is called a et al., 2012). hormone modulator. THD can be induced by a variety of causes The predominant TH in the circulation in the euthyroid situa- 0 0 including diet, disease, and exposure to environmental chemicals. tion is 3,3 ,5,5 -tetraiodothyronine (thyroxine, T4), which is the TH are involved in several important physiological processes precursor for the most active form of TH (3,30,5-triiodothyronine; such as regulation of energy metabolism (Cheng et al., 2010), T3). Most of the known functions of TH are mediated by the inter- growth and differentiation, development and maintenance of brain action of T3 with the nuclear T3-receptors (TRs), which act as li- function and the sympathetic nervous system (Bernal, 2007; Horn gand-modulated transcription factors. While almost none of the and Heuer, 2010; Reinehr, 2010; Warner and Mittag, 2012 ), ther- genes regulated by T3 are exclusively responsive to T3, virtually mo-regulation (Ribeiro, 2008), osmo-regulation and renal function all molecular, cellular and metabolic events are more or less sensi- (Vargas et al., 2006), regulation of onset and proper function of tive to TH (Grimaldi et al., 2012; König and Moura Neto, 2002; other endocrine systems including the estrogen system, sexual Oetting and Yen, 2007). behaviour and fertility, and cardiovascular functioning (Danzi and Klein, 2012; Krassas et al., 2010; Wagner et al., 2008). Whether 1.1. Possible physiological consequences of thyroid hormone disruption during development of the organism, differentiation of cells and tis- sues, maintenance or alteration of physiological functions of adult Lessons learned from decades of biomedical studies of iodide individuals, in many cases TH effects can best be characterized as deficiency, congenital hypothyroidism, genetic diseases related to 1322 AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346

Table 1 Physiological and related pathological process es related to the thyroid hormone system.

Thyroid-hormone physiological Related diseases References systems and processes Cellular metabolism Metabolic disease Brent (2012) and Song et al. (2011) Cell cycle/apoptosis Neoplasia Kim and Cheng (2012) and Piekielko-Witkowska and Nauman (2011) Cancer Kim and Cheng (2012) and Pascual and Aranda (2012) Angiogenesis Cancer; cardiac hypertrophy; neurological Berg et al. (2009), Cheng et al. (2010), Davis et al. (2009), Judson et al. (2010), deficit (brain) Luidens et al. (2010) and Zhang et al. (2010a) Thyroid gland Goitrogenesis Paschke (2011a,b) Metabolism Obesity; metabolic syndrome Baxter and Webb (2009), Brent (2012), Cioffi et al. (2010), Liu and Brent (2010) and Reinehr (2010) Nervous system Learning; memory; IQ Patel et al. (2011b) Neurobehavioral disorders (e.g. Chakrabarti (2011) and de Cock et al. (2012) hyperactivity; bipolar disorder) Sensory development (incl. hearing) Crofton and Zoeller (2005), Forrest and Swaroop (2012) and Sharlin et al. (2011) Depression in adults Alzheimer’s disease van de Ven et al. (2012) Immune system Autoimmunity Chang (2012) and Hodkinson et al. (2009) Skin Myxedema Aamir et al. (2010) and Safer (2011) Cardiovascular system Heart rhythm problems Donangelo and Braunstein (2011) and Klein and Danzi (2007) Reproductive system Reduced fertility Krassas et al. (2010) and Unuane et al. (2011) Impaired reproductive development Mann and Plant (2010) (onset of puberty) Ca-homeostasis, skeleton Osteoporosis; fracture risks Bassett et al. (2010), Greenspan and Greenspan (1999) and Waung et al. (2012) Brown adipose tissue Adaptation to cold stress; obesity Bianco (2011), Himms-Hagen (1989), Obregon (2008) and Silva (2011)

defective TH function as well as data from various animal models concentrations, which may give an overall in vivo effect at blood corroborate evidence for the hypothesis that transient or persistent concentrations that are lower than in vitro studies might suggest. THD could alter maintenance of homeostasis within the hypothal- This is expected to be the case with polychlorinated biphenyls amus–pituitary–thyroid-periphery (HPTP) axis and modulate the (PCBs) and polybrominated diphenyl ethers (PBDEs) in the blood peripheral thyroid hormone dependent functions. This might lead of mothers and infants that have been associated with changes in to temporary loss of homeostasis or even alter set points leading thyroid hormone status and developmental endpoints and fertility to long-term TH dysregulation and physiological consequences, (Chevrier et al., 2010; Harley et al., 2010; Koopman-Esseboom including thyroid pathology and altered metabolism and perinatal et al., 1994; Langer, 1998; Langer et al., 1998; Morse et al., 1993). development. Alterations of the TH system are associated with sev- In addition to the direct impact of chemicals on the TH system, eral serious human diseases (Table 1). biological and environmental stressors may also increase the TH are particularly important in perinatal development. They sensitivity of a number of physiological processes to THDCs. are involved in several critical processes for neurodevelopment: Iodine deficiency, which is commonly observed worldwide neuronal proliferation, migration, synaptogenesis, synaptic plastic- (Andersson et al., 2012; de Benoist et al., 2008; Walker et al., ity and myelination processes (Horn and Heuer, 2010; Howdeshell, 2007; Zimmermann and Andersson, 2012), is a well-known risk 2002). In humans, TH production starts at approximately 11 gesta- factor for increased sensitivity to adverse effects from thyroid-dis- tional weeks and increases with the development of the fetal HPTP rupting chemicals (Blount et al., 2006; Pearce and Braverman, axis (Howdeshell, 2002). Trans-placental transfer of maternal TH to 2009). Such effects are known as well from co-exposure to goitro- the fetus is critical for neurodevelopment (Bernal, 2007; Zoeller gens originating from the diet (e.g., (iso-)flavones like genistein in and Rovet, 2004), as impaired psychomotor development, behav- soy, goitrin contained in millet and cassava) in areas of iodine defi- ioural changes and effects on visuo-spatial processing have been ciency (Delange, 1994; Doerge and Sheehan, 2002; Köhrle, 2008 ). observed in children born to mothers with (subclinical) hypothy- roidism (de Cock et al., 2012; Haddow et al., 1999; Pop et al., 1.2. Environmental chemicals and THD 1999). Because alterations in TH balance can lead to altered devel- opment, even temporary THD during the perinatal period can have Various environmental contaminants have been shown to dis- long-term consequences on human health (Zoeller and Rovet, rupt thyroid homeostasis via several mechanisms( Brucker-Davis, 2004). But also later in life alterations in the TH system have been 1998; Capen, 1997; Cavalieri and Pitt-Rivers, 1981; Crofton, shown to play a role in several mental illnesses such as Alzheimer’s 2008; Hurley, 1998; Jugan et al., 2010; Köhrle, 2008; Surks and disease, bipolar disorder and major depressive disorder (Bauer Sievert, 1995; Zoeller, 2007). Disruption of the TH system has been et al., 2008; Carta et al., 2002; Cooper-Kazaz and Lerer, 2008; de shown as one of the major toxic effects for chemicals ranging from Jong et al., 2009; Hogervorst et al., 2008; Lovell et al., 2008; Tan halogenated aromatic chemicals such as PCBs to inorganic anions and Vasan, 2009). such as perchlorate and nitrate (Brouwer et al., 1998; Brucker- THDCs may interact with a number of molecular components of Davis, 1998; Hallgren and Darnerud, 2002; Wolff, 1998 ), arsenic the HPTP axis and the functioning of the peripheral tissues: TH (Ciarrocca et al., 2012) or perfluorinated chemicals (Lopez-Espin- synthesis, TH storage and release by the thyroid gland, feedback osa et al., 2012). This results in alteration of important biological mechanisms within the HPT, protein-binding and TH distribution, processes under control of THs (Lopez-Juarez et al., 2012; Pascual cellular TH uptake, intracellular TH metabolism, catabolism of and Aranda, 2012; Sirakov et al., 2012).

TH, classical ‘nuclear’ T3 receptor binding, as well as other target Historically, information on the potency of THDCs has been de- proteins (‘receptors’) in the cell membrane, mitochondria and rived from animal studies, mostly using rodents or amphibians other subcellular structures (Brix et al., 2011; Brucker-Davis, (Biegel et al., 1995; Christenson et al., 1996; Hallgren and 1998; Capen, 1994; Cheng et al., 2010; DeVito et al., 1998; Köhrle, Darnerud, 2002; Davey et al., 2008; Grimaldi et al., 2012). How- 2008; Miller et al., 2009). THDCs may also react with more than ever, this information is limited to a small number of the one component of the TH system, possibly at different internal 10,000+ chemicals that need assessments of potential risk (EPA, AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346 1323

Fig. 1. Schematic representation of relevant aspects of thyroid hormone (TH) regulation and action (boxes 1-6), aspects of regulation of availability of TH disrupting compounds, and physiological consequences and related diseases of TH disruption. For further explanation see text.

2012; Judso n et al., 2009; NRC, 1984, 2007; Wagner, 2000 ). Perform - not all of these methods are currently part of the standard informa- ing in vivo experi me nts to asse ss possib le THD for such a large num- tion requirements of the above mentioned regulations. Considering ber of chemi ca ls is unlik ely to happ en in the near future for practi ca l, the large number of substances that might need to be prioritized as well as ethical and financial reaso ns. In additi on , a scie ntific argu- for endocrine-related endpoints, both in the US and in the EU, there ment to replace animal experi me nts is that apica l endpoin ts provid e is a need for an efficient, less-animal intensive screening method to litt le insig ht in underl yin g mechanis ms (NIEHS, 2007 ), may not be identify potential THDCs. pred ic tiv e for the human situa tio n or may not identif y releva nt Over the last two decades there has been a growing interest in life -st ag e specific effects, espe cia ll y since unexp ec ted specie s differ- defining the mechanisms of toxicity in in vitro systems (Eisenbrand ences may exist, and current rode nt exper im ents are not optim ize d et al., 2002). Furthermore, there has been an explosion of technol- for the identi fica tio n of THD. In additio n, the fact that THDC could ogy that has greatly increased not only the breadth of information react with more than one compon ent of the TH syst em, possi bly at that can be extracted from biological samples, but also the diff ere nt intern al concentr at ion s, could result in confusi ng non- throughput by which samples can be analysed and interpreted. monoto nic in vivo dose –re sp onse curve s. A more mechani st ic all y Thus, genomics, proteomics and metabolomics are becoming com- driv en in vitro appro ac h can help to discri mi nate between chemicals monplace tools in biological research, as are genetically modified withou t indi cat io n for THD, with a stron g indi cat io n for THD and cells, transgenic animal models, model organisms such as zebra with weak or not conclusi ve indi ca tio n for THD. fish, and stem cell models. Many of these approaches have been The relevance of such an approach is illustrated by the fact that adapted to the process of drug discovery (Houck and Kavlock, the U.S. EPA is considering screening a universe of approximately 2008), which has facilitated their adoption in toxicological studies 10,000 chemicals for endocrine disrupting properties as part of including endocrine disruption, based on knowledge of the interac- the endocrine disruptor screening program. To facilitate rapid tion of chemicals with biological pathways. While there has been screening of endocrine-related endpoints, further priority setting much attention to develop pathway-based assays for estrogen will be done using high throughput in vitro assays and in silico and androgen function, approaches to the assessment of thyroid models (EPA, 2012). In Europe, different regulations contain spe- function have lagged behind because of the multiplicity of ways cific provisions regarding endocrine disruptors: REACH (EU, in which TH function can (and has) been altered by environmental 2007), plant protection products (EU, 2009a), cosmetics (EU, chemicals( OECD, 2006 ). This paper focuses on the implementation 2009b) and biocides (EU, 2012). The OECD has developed several of these approaches in testing strategies to the assessment of the test methodologies and testing strategies relating to EDCs but effect of chemicals on the thyroid system. 1324

Table 2 This table describes potential targets for thyroid hormone disrupting chemicals plus available non-vertebrate test systems. The numbers of the different sub-sections of the table refer to the Boxes with relevant aspects of TH regulation and action as depicted in Fig. 1.

Target Assay Model References Comments Box 1. Central regulation of the thyroid system via the hypothalamic–pituitary–thyroid axis TRH production hypothalamus Genomic array indicating TRH Human glioblastoma–astrocytoma (U373MG) cells Garcia et al. (2000) Validation of assay and HTS application to be production developed TRH receptor activation pituitary TRH receptor binding and calcium Stable ValiScreen TRH (human) cell line www.perkinelmer.com/ 96-well and 384-well plate format measurement (functional assay) GPCRcomplete (2012) TSH expression and secretion Mouse pituitary (T aT1) cells Zatelli et al. (2010) Validation of assay and HTS application to be developed TSH receptor activation thyroid TSH-mediated cAMP production Human embryonic kidney (HEK293) cells stably Titus et al. (2008) Published HTS assay in 1536-well plate, requires a gland cAMP measurement via CNG expressing TSH receptor coupled to a cyclic specific cell line with expression of CNG channel. channel-coupled cAMP assay nucleotide gated ion channel as a biosensor Assay kit available at Cisbio, www.htrf.com

TSH-mediated cAMP production Chinese hamster ovary (CHO) cells stably Santini et al. (2003), Van Sande et al. Sensitive and rapid assays using novel (colorimetric, 1320–1346 AlberTinka J.Murketal. in Vitro27 Toxicology (2013) / (direct cAMP measurement) expressing TSH receptor (2003) and Zimmermann-Belsing luminometric, dye, etc.) readouts not requiring et al. (2002) isotopes or immunoassays for cAMP detection TSH-dependent cell proliferation Rat thyroid (FRTL5) cell with endogenous TSH Jomaa et al. (2013) Validation of assay is needed; non-TSH mediated cell receptor proliferation cannot be excluded Box 2. Thyroid hormone production and biokinetics NIS-mediated iodide uptake Radioiodide uptake assay Rat thyroid (FRTL5) cells Arturi et al. (2002) and Lecat-Guillet Assay is usable for MTS (96-well) and has been applied et al. (2008) for many chemicals, disadvantage: radio iodide use Iodide uptake assay (non- Rat thyroid (FRTL5) cells Waltz et al. (2010) and Renko and Assay is usable for MTS, not tested in HTS radioisotope) Köhrle (unpublished) Iodide uptake assay (use of halide- Rat thyroid (FRTL5) cells stably expressing YFP- Di Bernardo et al. (2011) and Rhoden Not yet validated as HTS; changes in YFP fluorescence sensitive fluorescence of yellow H148Q/I152L et al. (2007) not strictly specific for cellular iodine fluorescent protein (YFP)) Iodide retention and efflux via NIS Transfected FTC-133 (human thyroid cancer cell Schroder-van der Elst et al. (2003) FTC-133 transfected with NIS; not validated for HTS line) TPO enzyme activity TPO-mediated oxidation of iodide Cell-free enzymatic assay Schmutzler et al. (2007a) Oxidative activity of TPO is determined by or alternative substrates (e.g., spectrophotometric detection systems. Iodide guaiacol) oxidation assays are likely to capture more inhibitors than guaiacol-based assays. Not yet validated as HTS TPO-mediated iodination of Cell-free enzymatic assay Freyberger and Ahr (2006) and Tyrosine iodination by TPO is determined tyrosine Zebrafish eleutheroembryos AntonicaSchmutzler et al. et (2012), al. (2007a) Hornung et al. Thisspectr assayophoto-metrically. covers effects on Not all yetfactors validated involved as HTS in TH Thyroid hormone production Measuriesement production of TH with in follicle (2010), Kunisue et al. (2011) and synthesis, e.g. NIS, Tg, TPO and DUOX. It requires intact cellsantibod LC-MS/MS analysis of TH Raldua et al. (2008) thyroid follicles and still is ex vivo , not in vitro. The LC- production MS/MS method determines several TH metabolites simultaneously from a single sample of thyroid tissue or follicles in vitro , Not suitable for HTS, for Tier 2 DUOX-mediated H2O2 Determination of H 2O2 generation PCCl3 rat thyroid cells, CHO cells stably transfected Massart et al. (2011) H 2O2 generation is determined spectrophotometric production by DUOX/DUOXA with DUOX2/DUOXA2 analysis of cell medium (conversion of homovanillic acid). Not yet validated as HTS Box 3. Systemic distribution and transport of thyroid hormones 4 bindingT proteins TTR and TBG Competition with T 4 for TTR and Cell-free biosensor assay (Biacore) with T 4 bound Marchesini et al. (2008) Performance of Biacore chips variable TBG binding to chip Fluorescent microtiter method Purified human TTR and TBG Cao et al. (2011) Assay is disrupted by autofluorescent chemicals TTR or TBG Anilinosulfonic acid Purified human TTR and TBG Montano et al. (2012) Microtiter method; ANSA, potent ligand, substitute for (ANSA) displacement assay T 4, fluorescent when bound to TTR or TBG TH membrane transport Transfer over cell monolayer in Human placental choriocarcinoma (JEG) cells; Kovo and Golan (2008), Patel et al. This system is developed for transport of drugs; placenta and blood brain transwell system human choriocarcinoma (JAR) cells; bovine brain (2011a), Powell et al. (2000) and applicability for THs and THDs to be determined barrier cells Vandenhaute et al. (2011) Box 4. Metabolism and excretion of thyroid hormones (De-)Glucuronidation Inhibition or upregulation of Tests are performed in intact (transfected) cells Martin et al. (2012), Tong et al. (2007), This also includes effects on enzyme production, cellular enzyme activity of: (over-)expressing the enzymes; Human hepatoma Wu et al. (2005) unless the cells overexpress the enzymes; in that case – Glucuronidases (HepaRG) cells only inhibition of enzyme activity can be determined – UDP-glucuronosyltransferases Inhibition of: Human recombinant enzymes; or enzymes Hamers et al. (2006), Schuur et al. Can be performed with recombinant enzymes or prepared from human and mouse livers, purified (1998a,b,c) and Wu et al. (2005) microsomal preparations, not yet validated as HTS – Glucuronidases enzyme preparation – UDP-glucuronosyltransferases (De-)Sulfation Inhibition of enzymatic activities Human recombinant enzyme Ekuase et al. (2011) Human cell lines with the relevant enzyme activities related to (de)sulfation: remain to be developed – Sulfotransferases – Sulfatases Deiodination Upregulation of Dio1/2/3 activities Human, rat, mouse cell lines Dentice and Salvatore (2011), Köhrle Performed in cellular systems; sensitivity of non- (2002, 2007, 2008) and Leonard and radioactive alternatives to be determined Rosenberg (1980) Inhibition of Dio1/2/3 activities Human recombinant enzyme; human, rat, mouse Dentice and Salvatore (2011), Hotz Can be performed in tissue homogenates or cellular cell lines; purified enzyme preparation et al. (1996), Köhrle (2002, 2007, systems; sensitivity of non-radioactive alternatives to

2008), Leonard and Rosenberg (1980), be determined 1320–1346 AlberTinka J. Murk et al. Toxicologyin Vitro 27 (2013) / Renko et al. (2012) and Schmutzler et al. (2007b) TH deiodination profile (mainly Enzyme preparation; can be pooled human liver Butt et al. (2011) Mass spectrometry-based method for measuring the Dio1) microsomes activity of Dio, allows for analysis of TH profile Box 5. (Sub-)Cellular distribution of thyroid hormones by membrane transporters and deiodinases TH membrane transporters TH transport assay via TH Transfected COS, HEK293, MDCK, JEG, Hela, GH3 Cavalieri et al. (1999), Kinne et al. Stably transfected cells expressing individual TH membrane transporters and other cells (over) expressing membrane (2009), Mitchell et al. (2005), van der transporters are available. Not yet validated as HTS. transporters MCT8/10, LAT1/2, OATP1c1. MDRs Deure et al. (2010) and Westholm Transporters cell type specific. Not yet validated as also play a role in rat FRTL-5 thyroid cells and et al. (2009, 2010) HTS mouse NIH-3T3 cells Up- or down regulation of OATPs Rat hepatoma (H4) cells Chalmers et al. (1993) and Westholm Can only be tested in cells not over-expressing OATPs. et al. (2009) Needs further development TH membrane transport Transfer over cell monolayer in Human placental choriocarcinoma (JEG) cells; Kovo and Golan (2008), Patel et al. This system is developed for transport of drugs; placenta and blood brain transwell system human choriocarcinoma (JAR) cells; bovine brain (2011a), Powell et al. (2000) and applicability for THs and THDs to be determined barrier cells Vandenhaute et al. (2011) Peripheral deiodination Inhibition of peripheral Dio1/2/3 Recombinant enzymes, tissue homogenates, Hotz et al. (1996), Köhrle (2002), Radioligand, well-characterized deiodinases are enzyme activity subcellular fractions, cellular systems Leonard and Rosenberg (1980), Renko tissue-specific not yet fully in vitro (ex vivo ) et al. (2012), Schuur et al. (1998a,b,c) Upregulation of peripheral Dio1/2/ In vivo (non-vertebrate) or in cellular system andKöhrle Visser (2002), et al. Leonard (1983) and Rosenberg T 3 is a strong inducer of hepatic Dio1 and also Dio3 3 enzymes activity (1980), Renko et al. (2012) and Visser activity in several tissues. T 4 inactivates Dio2 by et al. (1983) posttranslational mechanism, sensitivity of non- radioactive alternatives to be determined Box 6. Cellular responses to thyroid hormones Nuclear TR binding Molecular docking/ in silico In silico approach Schapira et al. (2003) >250,000 chemicals were screened, validation by modelling of binding to nuclear TR transfection and ligand binding 125 TH–TR competitive binding assay Whole cell uptake, nuclear extracts of TH Kitamura et al. (2002, 2005a,b), Disadvantages: Radioligand ( I) based, potential for (radioligand) responsive cells, overexpressed TRs Schapira et al. (2003) and You et al. high rate of false negatives; cannot distinguish (2006) between agonists and antagonists; folding of TR in vitro. Advantages: HTS possible; solid-state binding assays available Nuclear TR activation Stable luciferase reporter gene Rat GH3 cell line based reporter line (GH3.TRE- Freitas et al. (2011) and Sugiyama Both TRa /b endogenously expressed. Published HTS assay for endogenous TR- Luc); Lentivirus transduced Xenopus reporter line et al. (2005b) (1536 well); not clear if line can be propagated as a activation and antagonism (XL58-TRE-LUC) stable integrant, specific for amphibian cell conditions Stable reporter gene assay for Rat PC12 cells expressing chicken TR a1 (PC-DR- Jugan et al. (2007, 2009) A variety of cell lines available, may need specifica TRor TR b receptor LUC) Human (TRa, HeLa-Luc cells) NR1A1 and optimization/validation for HTS; with overexpressed activation and antagonism NR1A2 indigobiosciences.com TRs reporter gene Stable co-transfected gal4 TR Human embryonic kidney (HEK293T) cells stably Huang et al. (2011) and Invitrogen TR b-specific; Human based published HTS 1325 expression vectors and UAS transfected with gal4 TRb fusion, UAS b lactamase (2012) (upstream activating sequence) reporter (GeneBLAzer cell, Invitrogen, Inc.) (continued on next page) 1326 Table 2 (continued)

Target Assay Model References Comments based reporters Transient transfection reporter Overexpressed TRs, Gal4-TR fusions or endogenous Hofmann et al. (2009), Ibhazehiebo Applicable to a variety of cell lines, several gene assay to investigate nuclear TRs in a variety of cell types co-transfected with et al. (2011), Kitamura et al. (2005a,b) overexpressed TRs, reporter gene not fully wrapped in TR transactivation and TRE or UAS based reporter genes and You et al. (2006) native chromatin which is important for part of the TR antagonism role in repression/activation Stable reporter gene assay in yeast Full length TR or gal4 TR Iwasaki et al. (2002), Kitamura et al. HTS possible; not mammalian; yeast cell wall (2005a), Kudo et al. (2006), Moriyama permeability to chemicals limited, high incidence of et al. (2002), Sugiyama et al. (2005a,b) false negatives Liquid chemiluminescent DNA Cell-free bead assay Ibhazehiebo et al. (2011) Novel endpoint; binding to TRE does not determine pull-down assay for association of transactivation or trans-repression TH receptor with TRE Co-activator or co-repressor PathHunter NHR co-activator CHO-K1 cells stably transfected with TR a or TR b Patel et al. (2009) HTS possible, cell-based in vitro assay available at binding assay assay based on b galactosidase and steroid receptor coactivator peptide (SRCP) DiscoveRx complementation (DiscoveRx) Human TR-alpha activation (yeast S. cervisiae (Y190) cells into which human TR a and Arulmozhiraja et al. (2005) HTS possible; chemical accessibility across yeast cell two-hybrid assay) coactivator TIF2 have been introduced wall a concern 1320–1346 AlberTinka J.Murketal. in Vitro27 Toxicology (2013) / LanthaScreen TR-FRET TRbeta Human recombinant TR ligand-binding domain D’Souza et al. (2008) HTS possible, in vitro assay available at Invitrogen coactivator assay (Invitrogen) CoA-BAP system; protein–protein Recombinant NR ligand-binding domain Kanayama et al. (2003) Completely in vitro assay, HTS possible; limited use for interactions detected via TRs to date (PPARs and other NRs) coactivator tagged alkaline phosphatase activity Coactivator or corepressor peptide Human TRb LBD protein, SRC2 derived peptide; Johnson et al. (2011) and Levy-Bimbot HTS adapted, >250,000 chemicals screened; cell free, interaction assay based on Human TRa 1 LBD protein, SRC1 or NCoR derived et al. (2012) robust assay; corepressor interactions, or coactivator fluorescence polarization peptides interactions acting via the amino terminal transactivation domain are not included; HTS possible, includes corepressor interaction assay, isoform specificity not included TR, coregulator stabilization and Western blot, e.g. Rat pituitary tumour (GH3) cells (or other cell Ball et al. (1997), Baumann et al. Not clear how reflective of in vivo situation, possible autoregulation immunoprecipitation, types expressing endogenous or tagged receptors (2001), Hong et al. (2001) and Misiti role for arsenic in SMRT localization. Research gap quantitative PCR to investigate TR and coregulators) et al. (1998) and coregulator modification, turnover, localization and autoregulation Mitochondrial TR- and non-TR Integrin binding CV-1 cells express alpahVbeta3 integrin but not Bergh et al. (2005), Blanchet et al. Importance to overall TH physiology in progress, mediated responses nuclear TR (2012) and Freindorf et al. (2012) needs action; radioligand assay; Mitochondrial thyroid hormone effects mediated by the p43 TR a1 form Cell type specific responses TH-dependent cell proliferation Rat pituitary tumour cell line (GH3) cells, others Gutleb et al. (2005), Kitamura et al. Can distinguish between agonist and antagonist; also (T-screen) (2005b), Medina and Santisteban non-TR-mediated TH-related effects included; non- (2000) and Schriks et al. (2006) TH-related proliferation cannot be excluded (false positive) AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346 1327

1.3. Aim of the workshop 2.1. Box 1. Central regulation

The current review is based on the outcome of a workshop or- The tight regulation of circulating TH levels, a prerequisite for ganized within the framework of the ASAT initiative (Assuring essential physiological control, is achieved by complex control Safety without Animal Testing) (Fentem et al., 2004). The general mechanisms along the hypothalamus–pituitary–thyroid (HPT) aim of the ASAT initiative is to develop ‘‘a radical new approach axis. Thyroid-stimulating hormone (TSH) is the primary physiolog- to assessing the risk posed by exposure to chemicals that would ical regulator of thyroid gland function and growth (Vassart and not involve testing of animals, taking advantage of the rapid ad- Dumont, 1992). The production and secretion of TSH by the thyro- vances in science and technology.’’ Experts in thyroid endocrinol- trophs, a specialized pituitary cell type, is under a multifactorial ogy, toxicologists with experience with endocrine disruption and control. A central regulator of thyrotroph activity is thyrotropin- neurotoxicology, computational experts, high-throughput screen- releasing hormone (TRH), synthesized by neurons residing in the ing (HTS) and regulatory experts reviewed the state of science paraventricular nucleus of the hypothalamus (Nillni, 2010). TRH for (1) known mechanisms for THD plus examples of THDC; (2) binding to its membrane receptor in thyrotrophs activates phos- in vitro THD tests currently available or under development related pholipase C signalling via Gq11 leading to increased gene expres- to these mechanisms; and (3) in silico methods for estimating the sion of TSH a and b subunits. In addition to stimulating TSH blood levels of THDC. production, TRH has also been shown to affect the glycosylation Based on this scientific review, the panel recommends a battery pattern of TSH thereby affecting its biological activity (Persani, of test methods to be able to classify chemicals as of less or high 1998). Importantly, as a target of several key pathways, these hyp- concern for further hazard and risk assessment for THD. In addi- ophysiotrophic TRH producing neurons integrate inputs from var- tion, research gaps and needs are identified in order to optimize ious neural circuitries to provide exact set points for the thyroid and validate the targeted THD in vitro test battery. This validation axis depending on negative feedback by circulating TH, nutritional can lead to a mechanism-based strategy used for deciding on status, illness and even circadian rhythms (Costa-e-Sousa and whether to opt out or to proceed with further THD testing. Hollenberg, 2012). To this aim, an inventory was taken of (1) the most relevant The neuroendocrine control of thyroidal TH synthesis and molecular targets and pathways that underlie thyroid functional secretion is very sensitive to negative feedback exerted by circulat- disturbances, which should be included in THD testing, (2) pres- ing TH, both at the level of the pituitary and the hypothalamus ently available or preliminary in vitro and in silico approaches that (Fliers et al., 2006), and involves various molecular mechanisms cover the most relevant mechanisms, and (3) areas in which assay such as specific expressions patterns of TR genes, TH transporters development is needed to fill data gaps in a comprehensive in vitro and deiodinases (Chiamolera and Wondisford, 2009; Fekete and THD testing strategy. Lechan, 2007; Fliers et al., 2006) that are discussed in the following boxes. 2. Thyroid hormone system and endpoints for THD Thus, TSH and TRH receptor binding is central to HPT control. Assays which may be used to detect chemicals that interfere with Current knowledge indicates that the majority of THD effects signalling through these receptors are described below. are mediated via influences on the HPTP axis rather on direct inter- ference with nuclear receptor function in the target tissues. For a 2.1.1. Endpoint: TRH receptor signalling conceptual framework, the workshop expanded earlier systems biology models of thyroid disruption (Capen, 1997) using an ap- Overall, very little is known to date about the potential of envi- proach recently developed( Keune et al., 2012; Ravnum et al., ronmental chemicals to specifically affect the neuroendocrine con- 2012; Smita et al., 2012; Zimmer et al., 2012 ) (Boxes 1–6; Fig. 1). trol mechanisms of thyroid gland function via alteration in TRH THDC can interfere with the central regulation and feedback mech- receptor signalling. Several drugs including somatostatin ana- anisms of the HPTP axis (Box 1); at the site of TH synthesis (Box 2, logues and synthetic rexinoids (retinoid X receptor (RXR)-selective e.g., by inhibition of iodide uptake or iodination of thyroglobulin); retinoids) have been shown to suppress TSH levels and cause cen- with the TH distribution via the blood (e.g., by competition for high tral hypothyroidism in humans as well as in experimental rodent affinity binding of TH to transthyretin (TTR) and TH binding glob- models (Haugen, 2009; Sharma et al., 2006; Zatelli et al., 2010). ulin (TBG)) and across placenta and blood brain barrier (Box 3); The rexinoid LG 268, for example, specifically suppressed expres- with central metabolism and excretion of TH (Box 4; e.g., by hepa- sion of TSH b and DIO2 genes in thyrotrophs without affecting tic deiodinases, and conjugating and deconjugating enzymes); hypothalamic TRH mRNA expression (Sharma et al., 2006). Such with cellular uptake by selective TH transporters of the cell mem- findings are particularly interesting in the light of recent observa- brane, and intracellular activation or inactivation of TH (Box 5); tions that certain environmental chemicals (e.g., bisphenol A (BPA), and finally some THDC interact with membrane, mitochondrial 2,4-dichlorophenol, p,p 0-dichlorodiphenyltrichlor oethane, tributyl- and nuclear TR receptors (Box 6). tin chloride) can also act as disrupters of retinoid X receptor (RXR) An in vitro test battery to identify THDC should include the most function (Grun and Blumberg, 2006; Li et al., 2008). RXR typically relevant targets known for THD. For each component of the model from heterodimers with TR on TR responsive elements of TH regu- in Fig. 1 the most relevant endpoints are briefly discussed. More lated genes and thus RXR ligands modulate TR action on gene detailed information can be found in the additional references gi- expression (Putcha et al., 2012). ven. For the selected endpoints, a panel of potential in vitro bioas- The deve lo pm ent of sens iti ve tools and assay s to characte ri ze says was identified (Table 2) that should cover the biologically or neuroend oc ri ne hypo thal am ic and pitu it ar y endoc ri ne cell re- toxicologically most relevant endpoints for THD. Table 2 includes spon se s to putat iv e THDCs clearl y repr ese nt s a majo r resea rc h need. existing HTS bioassays (96- or even higher density well formats) as well as assays for which a higher throughput format still needs 2.1.2. In vitro assays for TRH receptor signalling to be developed. While many more techniques and assays may One cell line that holds promise for in vitro toxicological studies provide valuable mechanistic information on pathways and mech- is the pituitary thyrotroph TaT1 cell line. This cell line has been anisms of TDHCs, only assays that can be performed in cell lines or proven a valid and physiologically relevant tool to dissect various with isolated proteins, and therefore amendable to medium to high responses of thyrotrophs to TH as well as TRH (Chiamolera et al., throughput screening (MTS–HTS) are included. 2012; Janssen et al., 2011). 1328 AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346

TRH receptor is a G protein-coupled receptor. In pituitary cells, individual follicles providing intimate contact of thyroid follicular TRH binds to its receptor and activates phospholipase C leading to cells (thyrocytes) to the blood stream. Because TH are iodothyro- an increase of inositol 1,4,5-trisphosphates (IP3) that mobilizes nine derivatives, uptake of iodide from the blood stream represents intracellular calcium (Gershengorn, 1993). There are at least two a critical step in their biosynthesis. Iodine is concentrated in thyro- available assays for directly evaluating TRH receptor binding, bind- cytes by a factor of 20–40 over blood plasma iodide concentrations ing and activation of TRH, and intracellular calcium measurement. (Dohan et al., 2003) by the sodium–iodide (Na+/I) symporter (NIS) Both assays are commercially available (www.perkinelmer.com/ protein, a membrane glycoprotein located on the basolateral side GPCRcomplete, 2012). of thyroid follicular cells (Dohan et al., 2003). At the apical pole of thyrocytes the ion channel pendrin exports 2.1.3. Endpoint: TSH receptor signalling iodide into the follicular colloid lumen (Taylor et al., 2002; van den Hove et al., 2006). Organification of iodide takes place at the apical At the thyrocyte level, stimulation of the TSH receptor (TSHR) membrane of thyrocytes (Regard and Mauchamp, 1973). Localized by TSH activates several second messenger signalling cascades at the thyrocyte/colloid interface, the thyroid peroxidase (TPO) en- leading to increased iodide uptake, TH synthesis and secretion (Ro- zyme oxidises iodide for organification of iodine, it catalyzes the ger et al., 2010; Vassart and Costagliola, 2011). Among the various iodination of tyrosyl residues in the thyroglobulin polypeptide TSHR-regulated second messenger signalling cascades, the adenyl- chain to yield the TH precursors monoiodotyrosine (MIT) and diio- ate cyclase-cAMP-protein kinase A pathway has been most inten- dotyrosine (DIT). TPO also catalyzes the subsequent coupling of sively studied and the increase in cellular cAMP content is a MIT and DIT to produce T3 and T4 which remain covalently bound classical hallmark of TSHR stimulation in thyrocytes. A few studies to the thyroglobulin matrix (Taurog, 1970). In addition to thyro- are available that specifically analysed the interference of environ- globulin and iodide, TPO requires H2O2 as a third factor to carry mental chemicals with TSHR function (Picchietti et al., 2009; Rossi out the abovementioned reactions.H 2O2 production is likely a et al., 2007, 2009; Santini et al., 2003). Examples include the insec- rate-limiting step during TH generation (Song et al., 2007). Two ticide DDT and the PCB mixture Arochlor 1254 which were shown NADPH -d epe nde nt oxido re du ctas es , term ed dual oxidas es (DUOX1, to inhibit both basal and TSH-stimulated accumulation of cAMP in DUOX2 ), have been iden ti fied as impo rta nt comp onen ts of theH 2O2- TSHR-transfected cells (Rossi et al., 2007; Santini et al., 2003). Also genera ti ng syste m in thyro cyt es . several estrogen receptor agonists and antagonists such as diethyl- Iodinated thyroglobulin is stored extracellularly in the colloidal stilbestrol, quercetin, bisphenol A, and 17 b-estradiol have been compartment. Upon demand, thyroglobulin is endocytosed from shown to partially reduce TSH-induced cAMP accumulation (Rossi the colloid, enzymatically cleaved within the thyrocytes by cathep- et al., 2009). sin enzymes and finally TH is deliberated and actively secreted into the blood stream by monocarboxylate transporter 8 (MCT8) and 2.1.4. In vitro assays for TSH receptor signalling other TH transporters (Brix et al., 2011; Di Cosmo et al., 2010; In humans the TSHR activates both the cAMP and phospholi- Dunn and Dunn, 2001; Trajkovic-Arsic et al., 2010; Wirth et al., pase C-PIP2 cascades, which are both important for TH biosynthe- 2011). Several steps during TH synthesis are still not well under- sis (Song et al., 2010). There are several automated assays for TSHR stood such as the transport of the small charged iodine molecule binding by stimulating or blocking antibodies, there are small li- which cannot easily diffuse through plasma membranes and the gands activating and blocking TSH binding, and there are highly actual route of T4 and T3 release from the thyrocyte into the selective monoclonal antibodies to compete with TSH binding both bloodstream. blocking or stimulating the TSHR (Basaria and Cooper, 2005; The observation of hypothyroidism and goitre in patients carry- Kreuchwig et al., 2011; Nunez Miguel et al., 2012; Titus et al., ing mutations of genes coding for NIS, TPO, DUOX2, DEHAL and 2008). Measurements of cAMP accumulation in TSH-stimulated TSHR indicate that chemically-induced alterations in the activity thyroid cells (or TSHR-expressing non-thyroid cells) can be used of these molecules would also result in thyroid dysfunction. Fur- to evaluate chemicals for their potential to interfere with TSHR thermore, the thyroid gland is one of the most vascularized tissues function (Rossi et al., 2007, 2009 ). of our body, which allows for significant exposure of thyrocytes to A quantitative HTS assay has been developed for the identifica- THDC in the blood (Gerard et al., 2009). The effect of THDCs on TH tion of TSHR agonists (Neumann et al., 2008, 2009; Titus et al., synthesis and secretion is well established with experimental data 2008). This assay utilizes a cyclic nucleotide gated ion channel for inhibitors of NIS (perchlorate) and TPO (thiocarbamide drugs) (CNG)-coupled approach to measure changes in intracellular cAMP function. Also the unique continuous and life-long production of accumulation. In this cell system, TSHR stimulation by TSH leads to H2O2 and ROS (reactive oxygen species) for adequate TH biosyn- increased intracellular cAMP levels which activate a CNG cation thesis (Poncin et al., 2008) renders the thyroid gland highly vulner- channel leading to the influx of cations such as sodium and calcium able for low molecular weight agents, which may interfere with for and subsequently to a membrane depolarization. It is this mem- instance H2O2-catalyzed oxidation and activation (Divi and Doerge, brane depolarization that is detected by means of a membrane po- 1994; Jeong et al., 2005; Köhrle, 2008; Schmutzler et al., 2007a,b). tential dye in live cells. Changes in indirect measurements such as A central problem of in vitro detection of chemicals altering TH cAMP must be interpreted with caution. Xenobiotics may alter biosynthesis is the fact that the most important processes occur cAMP concentrations via a variety of mechanism, one of which extracellularly in the colloidal compartment of thyroid follicles may be TSH receptor activity. The same holds true for the recently and therefore require that thyrocytes are organized in a follicular developed TSH mediated cell proliferation assays in FRTL-5 rat thy- structure. Thus, typical monolayer cell culture techniques are roid cells (Jomaa et al., 2013). rarely useful for examining TH synthesis. Recently, however, mouse embryonic stem-cells were stimulated to differentiate into 2.2. Box 2. TH synthesis and secretion by the thyroid gland thyroid follicular cells in vitro. These cells formed functional folli- cles, which accumulate iodide, produce thyroglobulin and even In response to TSH stimulation, the thyroid tissue produces and organify iodide on thyroglobulin, hence mimicking functional thy- releases the T4 and to a lesser extent T3. Thyroid follicles represent roid tissue (Antonica et al., 2012). The application of this system in the functional subunit of thyroid tissue. Each follicle is lined in vitro assays needs to be further evaluated, but clearly represents by a single epithelial cell layer and is filled with a thyroglobulin- a breakthrough in in vitro methods that may lead to improved containing colloidal mass. A dense capillary network surrounds analysis of THDCs in the near future. Thus far the alternatives, AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346 1329 dispersed thyroid cell preparations from primary thyrocyte cul- port by the NIS. Perchlorate itself is also known to be transported tures as well immortalized thyroid cell lines had lacked the ability by NIS into thyroid cells and is concentrated in the thyroid gland of efficient iodide organification and TH synthesis. Although proto- (Clewell et al., 2004; Paroder-Belenitsky et al., 2011). This is likely cols exist to obtain follicle preparations from freshly dissected thy- true for other competitive inhibitors of NIS, as well. Recent work roid tissue or to reconstitute thyroid follicles under in vitro using an HTS NIS uptake inhibition assay revealed a novel class conditions, these techniques are not readily applicable to a HTS ap- of NIS inhibitors, imidazothiazoles (Lecat-Guillet and Ambroise, proach and have only recently been used to detect chemical inter- 2009). ference with TH biosynthesis (Hornung et al., 2010). Until an integrated in vitro testing model for HTS of chemicals interfering 2.2.3. Endpoint: TPO inhibition with TH synthesis, such as the functional follicles described above, is available, we recommend testing the two main aspects of TH To date, inhibitors of TPO activity make a large group of THDC biosynthesis, NIS and TPO activities, in specifically tailored affecting the thyroidal TH synthesis. TPO-inhibiting chemicals are in vitro assays that are currently available (Table 2, Box 2). a structurally diverse group of chemicals including thiourea deriv- atives (e.g., propylthiouracil (PTU)), flavonoids (e.g., genistein), 2.2.1. Endpoint: NIS-mediated iodide uptake m-dihydroxybenzene derivatives (e.g., resorcinol), tetracycline drugs (e.g., minocycline) and other structurally unrelated chemicals (e.g., Active uptake of iodide by thyroid follicular cell is indispensable amitrole, kojic acid). Potent TPO inhibitors such as the clinically for TH synthesis. The pivotal role of NIS in mediating thyroidal io- used thyrostatic drugs PTU and MMI can cause an almost complete dide uptake and thus facilitating TH synthesis is highlighted by inhibition of TH synthesis in vivo reflecting the crucial role of TPO several key findings. Competitive inhibition of NIS-mediated iodide activity for normal TH synthesis to occur. uptake by specific anions, for example, blocks not only thyroidal iodide uptake but impairs TH synthesis (Alexander and Wolff, 1966; De Groef et al., 2006; Tonacchera et al., 2004; Wolff, 2.2.4. In vitro assays for TPO inhibition 1998). In the human, mutations in the NIS protein are associated TPO-inhibiting chemicals could interfere with any of the dis- with congenital iodide transport defect, a condition characterized tinct steps of the TPO-catalyzed iodide organification process and by low iodide uptake, hypothyroidism and goitre (Bizhanova and a variety of enzyme assays have been historically used to evaluate Kopp, 2009; De La Vieja et al., 2000; Pohlenz and Refetoff, 1999). effects on iodide oxidation, tyrosine iodination and iodotyrosine coupling. Classical assays to evaluate the effects of chemicals on io- 2.2.2. In vitro assays for NIS-mediated iodide uptake dide oxidation use relatively simple spectrophotometric detection The classical assay to test the ability of a chemical to interfere systems to monitor the TPO-catalyzed oxidation of iodide or alter- with NIS-mediated iodide uptake is based on the measurement native substrates (e.g., guaiacol, azino-bis-(3-ethylbenzothiazo- 125 of radioiodine ( I ) uptake in NIS-expressing cells (Atterwill line-6-sulfonic acid)) in the presence of H2O2. Iodination assays, and Fowler, 1990; Schmutzler et al., 2007b). This type of assay is in turn, measure the TPO-catalyzed iodination of tyrosine to yield usually performed with a thyroid cell line (e.g., FRTL5 cells) but MIT and DIT which are usually determined spectrophotometrically non-thyroid cell lines transfected with NIS have also been success- or by chromatographic techniques (Divi et al., 1997; Freyberger fully used (Lecat-Guillet et al., 2007). Once a chemical shows inhib- and Ahr, 2006). The least straightforward type of assays are those itory effects on radioiodine uptake, further analyses are necessary that aim to specifically assess chemical interference with the to confirm a specific effect on NIS function and to identify false coupling reaction (Divi et al., 1997). Notably, the existence of dif- positives. Some chemicals might even increase iodide uptake be- ferent molecular mechanisms of TPO inhibition bears important cause of increased iodide retention resulting from inhibitory ef- implications regarding the suitability of different assays to sensi- fects on iodide efflux rates or interference with pendrin (Elisei tively detect TPO inhibitors. For example, many TPO inhibitors like et al., 2006; Lecat-Guillet and Ambroise, 2009). propylthiouracil (PTU), methimazole (MMI) and resorcinol deriva- A fully automated radioiodine uptake assay was developed for tives can be readily detected in assays measuring the TPO- rapid and quantitative screening of test chemicals in a 96-well for- mediated oxidation of guaiacol. Still other potent TPO inhibitors mat using HEK293 cells transfected with human NIS (Lecat-Guillet such as ethylenethiourea show no effects in the classical guaiacol et al., 2007, 2008; Lindenthal et al., 2009). This method has been oxidation assay but their inhibitory activity can be detected using used to screen a chemical library of 17,020 structures (Lecat- iodide oxidation or tyrosine iodination assays as readout for TPO Guillet et al., 2008). A nonradioactive iodide uptake assay was re- interference (Freyberger and Ahr, 2006). cently developed using FRLT5 cells (Waltz et al., 2010). The assay Common to all currently available TPO inhibition assays is that was robust and had a similar sensitivity to the classical radioiodine they are enzymatic assays performed in a cell-free environment. uptake assay using FRTL5 cells (Waltz et al., 2010). Recently also a Although the enzymatic assays used to demonstrate TPO inhibition fluorescent assay for cellular iodide uptake was developed on a bear the potential for the development of HTS protocols, no such yellow fluorescent protein variant, YFP-H148Q/I152L, which is sen- efforts have yet been reported. A major difficulty with regard to sitive to halide and several other voluminous anions and thus can TPO inhibition assays is the identification of a stable and reliable be employed as a biosensor to monitor the cellular uptake of nat- source of TPO for systematic screening approaches. Historically, ural and EDC NIS substrates. FRTL-5 cells expressing high NIS levels most studies used partially purified TPO preparations from hog as well as NIS transfected other cells can be used to study transport and bovine thyroids or from human goitre samples. However, re- kinetic parameters such as maximal velocity of NIS-mediated up- cent studies started to validate the use of TPO preparations derived take as well as the rate constant for passive efflux (Cianchetta from thyroid cancer lines stably transfected with human TPO et al., 2010; Di Bernardo et al., 2011). expression vectors for use in TPO inhibition assays, the results of To date, several inhibitors of NIS-mediated iodide uptake have which appear very promising (Schmutzler et al., 2007b ). Currently, been identified, including inorganic monovalent anions such as the most important endpoints of TH production can only be inves- perchlorate, thiocyanate, fluoroborates or nitrate (Dohan et al., tigated in separate assays. However, the number of assays for ini- 2003; Van Sande et al., 2003). Due to their similarity to iodide in tial screening for THDCs could be reduced with the development of charge and size, these voluminous anions (perchlorate, etc.) inter- a TH producing in vitro follicular system. When an effect is ob- fere with iodide uptake by competitive inhibition of iodide trans- served in such an integrated system, underlying mechanisms could 1330 AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346 then be further investigated in a second tier of specific in vitro and in the blood brain barrier, two important transporter mole- assays. cules are MCT8 and OATP1C1. MCT8 is also expressed in heart, kid- ney, liver, and skeletal muscle (Roberts et al., 2008). 2.3. Box 3. Transport of thyroid hormones The relevance of cell membrane transporters and assays for this endpoint are further discussed in Box 5 below on local cellular After TPO catalyzed production, TH still remain integral parts of concentrations. thyroglobulin deposited in the lumen of the thyroid follicle. Next, thyroglobulin molecules are internalized into the thyrocytes by 2.3.2. In vitro assays for binding to transport proteins TTR and TBG pinocytosis and intracellularly degraded by cathepsins( Brix Several TH binding assays have been developed and published, et al., 2001; Friedrichs et al., 2003). The TH are subsequently ex- most of them applying radioactive T4 displacement from TTR and ported via TH transporters (e.g., MCT8) across the basolateral thy- to a lesser extent from TBG (Brouwer and van den Berg, 1986; rocyte membrane into the circulation (Di Cosmo et al., 2010; Cheek et al., 1999; Hallgren and Darnerud, 2002; Hamers et al., Trajkovic-Arsic et al., 2010; Wirth et al., 2011 ). In the blood, most 2006; Lans et al., 1994 ). An alternative method is the detection of the TH are bound to TH binding proteins, leaving approximately of the TH transport disruptors with a biosensor based on the sur-

0.03% of T4 and 0.3% T3 unbound, the ‘free’ hormone fraction (Yen, face plasmon resonance (SPR) technique (Marchesini et al., 2006). 2001). In humans, the protein with highest affinity to TH is TBG, SPR biosensors such as Biacore measures binding as a change in which carries the majority of the TH in the circulation. The role the refractive index at the surface of the sensor caused by accumu- of TBG in TH transport is not only species-, but also age specific, lation of mass within the surface layer which in this case is loaded since it is undetectable in adult, but present in serum of young, with T4 to which TTR or TBG can bind (Marchesini et al., 2006, pregnant and obese rats (Savu et al., 1987, 1989 ). The second 2008). This method is fast and can be adapted to analyse the inter- high-affinity TH binding protein is TTR (reviewed by Blake et al. action of THDC with multiple TH binding sites in parallel. Recently (1978), Robbins (2000), Schreiber (2002) and Schussler (2000)). a 96-well non-radioactive fluorescence displacement assay has

Albumin is the most abundant TH-binding protein, but the binding been developed based on competition of T4-like chemicals with is unspecific and with low affinity (Schussler, 2000 ). the fluorescent probe ANSA 8-anilino-1-naphtalenesulfon ic acid TTR is synthesized in liver, brain, pancreas, retina (Buxbaum ammonium salt) (Montano et al., 2012). An alternative new devel- and Reixach, 2009) and placenta (Mortimer et al., 2012), and is in- opment makes use of immunomagnetic microbeads followed by volved in the transport of T4 and retinol across the blood–brain screening with flow cytometry and identification of THDCs with barrier and to the fetus from the placenta. TTR may be an impor- nano-liquid chromatography mass spectrometry (Aqai et al., 2012). tant target for THDCs, as many hydroxylated metabolites of persis- tent organic pollutants show high binding affinity for TTR that can 2.4. Box 4. Metabolism and excretion be even higher than those of the endogenous ligand T4 (Ghosh et al., 2000; Hamers et al., 2006; Lans et al., 1993; Meerts et al., The major pathways of TH metabolism resulting in activation,

2000a). Inhibition of T4-binding to TTR results in disruption of inactivation or excretion from the body are (1) deiodination, (2) the TTR-retinol binding protein complex followed by reduced plas- alanine side chain modification, (3) sulfation and (4) glucuronida- ma levels of TH and reduced retinol levels. tion. Deiodination is the most important metabolic pathway in both quantitative and quantitative terms for normal physiological 2.3.1. Endpoint: Binding to transport proteins TTR and TBG control of TH homeostasis. THDCs could interfere with both the activity of enzymes as well as the production of the enzymes by

Given the relevance of TTR and TBG for transport of T4 in the inhibiting or inducing their expression. plasma, and the relevance of TTR for T4-transport over the placen- Inclusion of catabolic enzymes and cellular transporters, e.g., tal and blood–brain barriers, competition of THDC for binding to UGTs, SULTs, OATPs, MCT, and multi-drug efflux transporters, as these transport proteins should be included in a comprehensive molecular targets for endocrine-disrupting chemicals is clearly THDC test battery. In vitro evaluation of TBG binding should also warranted due to the critical role these proteins play in regulating be considered when in vivo rodent studies are used, as TBG-binding concentrations of circulating thyroid hormones. Indeed, there are is not assessed in rodent studies. Binding to albumin is unspecific many published reports that correlate upregulation of these pro- and no competition for TH binding by xenobiotics has been re- teins with increased biliary elimination of glucuronidated or sul- ported. Apart from TH binding proteins TTR, TBG and albumin, fated T4 or T3 following exposure to xenobiotics (see for example, TH transport over the placental and blood–brain barrier as well Christenson et al., 1995, 1996), and/or to systemic decreases in as across other membranes strongly depends on their expression thyroid hormones. Xenobiotic nuclear receptors including AhR, levels of various TH transporters (MCT8, MCT10, OATP1c, LAT1, CAR, PXR, and PPAR transcriptionally regulate the expression of LAT2, etc. for transport and export of TH. these proteins, and many endocrine-disrupting xenobiotics are Several halogenated phenolic mono- and polycyclic chemicals known to activate these receptors (Boas et al., 2006; Crofton, such as hydroxylated PCB or PBDE metabolites (Brouwer et al., 2008; Kretschmer and Baldwin, 2005; Miller et al., 2009; Timsit 1990; Gutleb et al., 2010; Hamers et al., 2006; Lans et al., 1993, and Negishi, 2007; Zhang et al., 2004). For example, activators of 1994; Marchesini et al., 2008; Meerts et al., 2000a) or natural CAR or PXR are known to upregulate transcription of a wide variety and synthetic (iso-)flavonoids (Lueprasitsakul et al., 1990; Radovic of Phase I–III enzymes, which increase hepatic catabolism and et al., 2006) have been shown to bind to TTR with high affinity sometimes bioactivation of not only pharmaceuticals( Kretschmer resulting in displacement of T4. In contrast to TTR, the interference and Baldwin, 2005; Sinz et al., 2006) and other chemicals, but also of xenobiotics with binding of TH to TBG has not been studied as endogenous thyroid hormones (Visser, 1996). Some of the relevant intensively (Cao et al., 2010; Marchesini et al., 2006, 2008; Meerts Phase II enzymes capable of thyroid hormone metabolism include et al., 2000a). UGT1A1, UGT1A6, and SULT2A1 (Kato et al., 2008). Phase III influx In addition to TTR, placental TH plasma membrane transporters and efflux transporters including, but not limited to, MCT8, OAT- play a role in the transplacental passage of TH from mother to fe- P1A4, and MRP2, may also be important for increased excretion tus, including the TH transporters MCT8, MCT10, L-type amino acid of thyroid hormones and its conjugates, thus enabling an overall transporter (LAT) LAT1, LAT2, organic anion transporting peptide increase in systemic clearance (Lecureux et al., 2009; Morimoto (OATP) OATP1A2 and OATP4A1 (Loubiere et al., 2010). In the brain et al., 2008; van der Deure et al., 2010; Visser et al., 2010; Wong AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346 1331 et al., 2005). Therefore, any in vitro screening assay battery for thy- of the non-genomic effects of TH exerted at the cell membrane roid-disrupting chemicals should include biomarkers of nuclear through binding to the avb3 integrin (Davis et al., 2011). There receptor activation, because they represent upstream biological is, however, little or no information that environmental chemicals targets that indicate potential for modulation of the regulation of induce or inhibit alanine side chain modification of iodothyronines, hepatic (and other tissues) proteins important for thyroid hormone therefore we do not suggest including this endpoint in an in vitro homeostasis( Sinz et al., 2006). THD test battery. There are already several HTS assays for nuclear receptors (Huang et al., 2011; Kavlock et al., 2012; Raucy and Lasker, 2010; 2.4.3. Endpoint: Sulfation Schoonen et al., 2009) that are relevant for other toxic pathways as well. Therefore they will not be reviewed herein. Importantly, Iodothyronines are phenolic chemicals and thus readily metab- data from these assays must be included in any systems ap- olized by conjugation of the phenolic hydroxyl group with sulfate proaches to determine the actions of chemicals on thyroid hor- or glucuronic acid. Sulfation appears to be a primary step leading mone pathways. Additional measurement of expression and/or to the irreversible inactivation of TH (Kester and Visser, 2005; 0 activity UGT, SULT, or transporters would serve as a downstream Wu et al., 2005). The IRD of T4S to rT 3S and of T3S to 3,3 -T2S are confirmation of one or more chemical-nuclear receptor interac- orders of magnitude faster than the IRD of non-sulfated T4 and tions, and also an important marker of potential increases in thy- T3, whereas ORD of T4S to T3S is completely blocked. Normally, roid hormone turnover. plasma levels of iodothyronine sulfates such as T4S and T3S are very low, as these conjugates are rapidly deiodinated by D1 by

2.4.1. Endpoint: Deiodination IRD. Plasma T4S and T3S are increased if D1 activity is inhibited during fetal development, non-thyroidal illness and fasting. Phenol

In most species, T4 is the main TH secreted by the thyroid gland sulfotransferases (SULT) with significant activity towards iodothy- under normal conditions. The bioactive hormone T3 is largely pro- ronines include human SULT1A1, 1A2, 1A3, 1B1 and 1C2. They 0 duced by enzymatic outer ring deiodination (ORD) of T4 in periph- show substrate preference for 3,3 -T2 T3, rT 3 T4. Iodothyro- eral tissues. Inner ring deiodination (IRD) of T4 produces the nines are also sulfated by human estrogen sulfotransferase 0 inactive metabolite rT3 .T3 is degraded by IRD to 3,3 -T2, an inactive (SULT1E1), in particular T4 (Kester et al., 1999). Drugs such as TH metabolite which is also generated by ORD of rT3 . Three iodo- PTU can inhibit D1-activity, while in rodents it has been shown thyronine deiodinases (DIO1, DIO2, DIO3) are involved in the deio- that phenobarbital may accelerate TH metabolism by induction dination of TH. DIO1 is expressed predominantly in the liver, the of sulfotransferases through activation of the constitutive andro- kidneys and the thyroid (Gereben et al., 2008). It catalyzes the stane receptor. The importance of this mechanism in humans is ORD and/or IRD of a variety of iodothyronine derivatives, but it is still unknown (Qatanani et al., 2005). most effective in the ORD of rT3 . Hepatic DIO1 is a major site for the production of plasma T3. The expression of DIO1 which codes 2.4.4. Endpoint: Glucuronidation for DIO1 is positively regulated by T3 at the transcriptional level. DIO2 is expressed primarily in brain, anterior pituitary, brown adi- In addition to sulfation, iodothyronines are metabolized by con- pose tissue (BAT), thyroid and skeletal muscle (Gereben et al., jugation of the phenolic hydroxyl group with glucuronic acid. Iod- 2008; Larsen, 2009). In brain, DIO2 is expressed predominantly in othyronine glucuronides are rapidly excreted in the bile, astrocytes. DIO2 only catalyzes the ORD of iodothyronines, with constituting the first step in the enterohepatic cycle of TH (Visser, a preference for T4 > rT3 . DIO2 plays an important role in the local 2008; Wu et al., 2005). In humans, about 20% of daily T4 production production of T3 from T4 in brain, pituitary and BAT. DIO2 ex- appears in the faeces, probably through incomplete intestinal pressed in the anterior pituitary and hypothalamus is important hydrolysis and reabsorption of the biliary-excreted conjugates. for the negative feed-back of T4 on TSH and TRH secretion. In gen- Glucuronidation of T4 and T3 is catalyzed by different members eral, DIO2 activity is negatively regulated by TH at the posttransla- of the UDP-glucuronyltransferase 1A family, i.e. UGT1A1, 1A3, tional level (Gereben et al., 2008). DIO3 is expressed in different 1A7-10 (Kato et al., 2008; Tong et al., 2007). Usually, this involves human tissues, i.e. brain, skin, liver, and intestine; DIO3 activities the glucuronidation of the hydroxyl group, but human UGT1A3 are much higher in fetal than in adult tissues (Gereben et al., also catalyzes the glucuronidation of the carboxyl group of iodo- 2008). DIO3 is also abundantly expressed in the placenta and the thyronine derivatives. pregnant uterus. DIO3 has only IRD activity and catalyzes the inac- In rodents, T4 metabolism is accelerated by different classes of tivation of T3 and T4 and is thought to protect the developing fetal chemicals, including barbiturates, fibrates, PCBs and PBDEs, which tissues against undue exposure to T3. Hence, fetal serum T3 levels is associated with the induction of hepatic T4 UGT activities (Morse are low. DIO2 and DIO3 show complex spatio-temporal expression et al., 1996; Hallgren et al., 2001; Hood et al., 2003; Visser et al., patterns in fetal and neonatal brain, which are critical for optimal 1993; Zhou et al., 2001). In rodent studies, induction of UGT activ- brain development. In the brain, DIO3 expression is stimulated by ity is a commonly proposed mechanism of interference for a num-

T3, probably at the transcriptional level (Gereben et al., 2008). ber of environmental contaminants (Axelrad et al., 2005; Hurley, Thus, tissue specific expression of deiodinases and regulation of 1998). This may result in hypothyroxinemia or hypothyroidism if DIO expression through HPTP feedback mechanisms are important the thyroid gland is not able to compensate for the increased hor- regulators of TH activity in tissues. mone loss. In humans, hypothyroxinemia may occur via induction

of T4 glucuronidation by anti-epileptics, but overt hypothyroidism 2.4.2. Endpoint: Alanine side chain modification is rare (Benedetti et al., 2005).

Iodothyronines are also metabolized by decarboxylation of the 2.4.5. Inhibition of enzyme functioning alanine side-chain to iodothyronamines which could have acute and dramatic effects on heart rate, body temperature and physical The deiodination of iodothyronines by the different deiodinases activity (Scanlan, 2009). Presumably by further conversion of iod- is inhibited by a variety of structurally related chemicals, including othyronamines, the iodothyroacetic acid metabolites Tetrac (TA4) iodinated X-ray contrast agents, amiodarone and its metabolites, and Triac (TA3) are generated (Wood et al., 2009). TA3 has signifi- and OH-PBDEs (Butt et al., 2011; Köhrle, 2011). However, the iod- cant TH activity (Freitas et al., 2011), and TA 4 is a strong inhibitor othyronine deiodinases have markedly higher affinities for iodin- 1332 AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346

ated phenols than for brominated or chlorinated phenols, suggest- T4 and T3 are also transported by 2 members of the hetero- ing that environmental levels of OH-PBDEs and OH-PCBs will affect dimeric amino acid transporters: LAT1 and LAT2 (Taylor and TH deiodination less if at all (Butt et al., 2011) (T.J. Visser, unpub- Ritchie, 2007). Both LAT1 and LAT2 facilitate the bidirectional lished work). The thyrostatic drug PTU potently inhibits DIO1 by transport of a variety of aliphatic and aromatic amino acids as well reacting with a selenenyl iodide intermediate but it has no effect as iodothyronines over the plasma membrane. on DIO2 and DIO3 (Gereben et al., 2008). The sulfation of iodothy- The MCT family contains 14 members. MCT8 (SLC16A2) and ronines by different SULTs is inhibited by OH-PCBs, OH-PDBEs and MCT10 (SLC16A10) have recently been identified as important other hydroxylated metabolites of polyhalogenated aromatic TH transporters which can be affected by THDC (Braun et al., hydrocarbons (OH-PHAHs) (Kester et al., 2002; Schuur et al., 2012; Friesema et al., 2006b, 2008; Halestrap, 2012; Kinne et al., 1998b). This is especially the case for the estrogen sulfotransferase 2010). So far, only iodothyronines have been identified as trans- SULT1E1, which is potently inhibited by a variety of OH-PHAHs. ported substrates for MCT8 but MCT10 also transports aromatic amino acids (Trp, Tyr, Phe). They are non-glycosylated proteins showing high homology and wide but different tissue distribu- 2.4.6. In vitro assays for induction and inhibition of enzymes for TH tions. In brain, MCT8 is importantly expressed in choroid plexus, metabolism capillaries and neurons in different brain areas. MCT8 is thought Both enzyme induction and inhibition of enzyme activity can to be critical for T uptake in central neurons and, thus, for the ac- have important implications for the availability of TH. The induc- 3 tion of TH during brain development. Patients with mutations in tion of enzymes can only be tested in vitro in cell-based assays, MCT8 suffer severe psychomotor retardation in combination with the inhibition of enzyme activities in cell lysates containing these highly elevated serum T levels (Friesema et al., 2006a). enzyme activities or isolated liver enzymes (Schuur et al., 3 1998a,b,c; Hamers et al., 2008). Since Phase II and III metabolism are partially controlled by specific nuclear receptors, assays for 2.5.1. Endpoint: cellular TH uptake these receptors could be used as surrogates for induction of the en- zymes (Kavlock et al., 2012). An important endpoint to include in an HTS battery is inhibition

While there are many published methods that have been used of cellular uptake of iodothyronines, in particular of T4 and T3. TH to measure expression of UGT and SULT mRNA and activity (Biegel uptake experiments are usually done by incubation of radioiodine- et al., 1995; Brucker-Davis, 1998; Hood et al., 2003; Hood and labelled TH with cells transiently or stably transfected with a Klaassen, 2000a,b; Liu and Klaassen, 1996; Paul et al., 2012 ), there transporter such as MCT8, MCT10 or OATPC1. After incubation are only limited reports of the use of medium or HTS assays for and washing, cells are lysed and counted for radioactivity measuring upregulation of these enzymes. Rotroff and colleagues (Friesema et al., 2006a, 2008), The assay may be adapted to (Rotroff et al., 2010), using quantitative nuclease protection assays measure the facilitation of intracellular TH metabolism in cells (qNPA) for UGT1A1, SULT2A1 screened 309 chemicals as part of co-transfected with transporter and one of the deiodinases ToxCast Phase I (Sinz et al., 2006). (Friesema et al., 2006a, 2008 ). However, to our knowledge HTS There is a clear need to improve and develop high-throughput assays have not been developed for the identification of chemicals assays for inhibition of Phase II enzymes that are involved in TH that inhibit cellular TH transport. The development of transporter metabolism. assays is a recommended area for assay development.

2.5. Box 5. Cellular concentrations and transporters 2.6. Box 6. Cellular responses

Cellular TH uptake is mediated by specific plasma membrane The best understood cellular actions of TH are carried out via transporters which may differ between cells from different tissues receptors found constitutively in the cell nucleus. Ligand-activated such as liver, pituitary heart, placenta and blood brain barrier and receptors directly control the transcription of specific sets of target showing preferential transport of T4 or T3 or their metabolites genes that carry out cell and developmental stage specific re- (Hennemann et al., 2001). Using transporter-specific ligands and sponses. There are two well-conserved TH receptor gene loci in inhibitors, it has been demonstrated that TH uptake by different all vertebrates, designated TRa and TR b. Several TR isoforms gener- cell types is mediated at least in part by amino acid transporters ated by alternative splicing and use of different initiation sites are showing preference for large, neutral amino acids (L-type) or aro- known and expressed in a tissue specific manner (i.e. TR a: TRa 1 matic amino acids (T-type). A number of transporters capable of and the non-hormone binding TR a2; TRb : TRb 1 and TR b2). These transporting TH have been identified, including different members have been reported in various species and cell types with both of the OATP family, 2 L-type amino acid transporters (LAT1,2), and overlapping and specific actions reported (reviewed in Flamant 2 members of the monocarboxylate transporter (MCT8,10) family and Gauthier (2012)). The nuclear TRs control transcription of spe- (Butt et al., 2011; Friesema et al., 2005; Köhrle, 2011; Visser cific genes directly through binding to thyroid hormone response et al., 2008). elements (TREs). Often, the TRs are found in a complex with the The human OATP family consists of 11 members, many of related retinoid-X receptor; in the heterodimer complex with TRs which have been shown to transport iodothyronine derivatives the RXR is usually a ‘‘silent partner’’ but in some cell types and/ (Hagenbuch, 2007; Obaidat et al., 2012). OATPs are glycoproteins or TRE configurations it may be activated by various retinoids and the human OATP1 subfamily contains four members (OAT- (Castillo et al., 2004). In the absence of ligand, co-repressor com- P1A2, 1B1, 1B3, 1C1) that are capable of transporting a variety of plexes repress transcription and upon ligand binding, co-repressor anionic, neutral and even cationic ligands. OATP1A2, 1B1 and complexes are replaced by co-activator complexes that initiate 1B3 show preference for sulfated over non-sulfated iodothyronines transcription (Rosenfeld et al., 2006; Shi, 2009). Most of the studies as ligands (van der Deure et al., 2009). OATP1A1 is expressed in dif- of TR action have examined up-regulated genes, since far less is ferent tissues but OATP1B1 and 1B3 are expressed only in liver. understood about direct ligand induced down-regulation. How-

OATP1C1 shows a high preference for T4 as the ligand and is almost ever, understanding the mechanism of down-regulation is impor- exclusively expressed in brain capillaries and choroid plexus, sug- tant for understanding negative feedback on TRH and TSH gesting that it is very important for T4 transport into the brain expression, a common endpoint for TH disruption assays in intact (Hagenbuch, 2007; Obaidat et al., 2012). animals. AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346 1333

Recently, TH have been shown to also directly act both at the Ultimately, changes in gene expression mediated by the nuclear cell membrane, in the cytoplasm (Cheng et al., 2010) and in mito- TRs result in observable cellular phenotypes that are specific for a chondria as well (Chocron et al., 2012; Pessemesse et al., 2012 ), given target cell type. For example, the rat pituitary tumour GH3 which is of interest given the important influence of TH on mito- cell line proliferates in response to T3.T3 treated primary cultures chondrial function in warm-blooded vertebrates. Often, these of cerebellar Purkinje cells show increased dendritic branching effects are referred to as ‘‘nongenomic’’ vs. the nuclear receptor- (Heuer and Mason, 2003; Ibhazehiebo et al., 2011), cardiomyocytes mediated ‘‘genomic’’ effects described above, although nongenom- self-organized into three dimensional heart muscle alter their con- ic actions may ultimately influence the transcription of specific tractility properties (Khait and Birla, 2008), and primary liver cells genes, and interface with nuclear TR actions as well. The actions show altered expression of metabolic enzymes (Attia et al., 2010; of TH at the cell membrane, in the cytoplasm or in mitochondria, Hwang et al., 2011) as observed in vivo. Although primary cell as- may be mediated by a bona fide TR isoform acting outside the nu- says may have more predictive value for effects in vivo than any of cleus, an amino terminal truncated form of the TRa , or a distinct the other models discussed here, animals would still be used as receptor for TH altogether. For example T4 (and to some extent source material and non-TR mediated mechanisms make the mode T3) can stimulate angiogenesis in various models via a membrane of action less certain. Miniaturizing the primary cell assays, or receptor (integrin avb3) mediated signalling pathway. T4 has been expansion of the primary cell population prior to assay, would shown to bind directly to the avb3 integrin and activate down- greatly reduce the number of animals needed, and development stream signalling including induction of genes linked to blood ves- of sensitive and specific assays for sets of key, direct TR target sel formation (Cheng et al., 2010). In addition, TRb 1 has been genes in these cell lines may help with mode of action uncertainty. reported to bind to PI3 kinase in the cytoplasm, inducing down- For Tier 1 HTS, however, this is not ideal with current technology. stream responses including gene activation linked to responses Finally, as more is learned about ‘‘non-genomic’’ TH action at to hypoxia, such as HIF1a (Moeller et al., 2005), although a classical the membrane, in the cytoplasm, or in mitochondria, in vitro assays ‘‘genomic’’ pathway to HIF1a induction has also been proposed will need to be developed for these endpoints as well, but at the (Otto and Fandrey, 2008). Clarification of relative importance of moment the relevance of non-genomic pathways in THD remains these alternate modes of action in the homeostatic and develop- unclear. mental roles for TH will be important to determine whether these are relevant targets of concern for TH disrupting chemicals and as- 2.6.2. In vitro assays for detecting disruption of TR activity say development. Ligand binding assays. Several ligand binding assays have been re- ported to detect chemicals binding to the TRs in vitro (Kitamura 2.6.1. Endpoint: TR binding and transcriptional activity et al., 2002, 2005a; You et al., 2006). Typically, these assays involve over-expressed TR ligand binding domains or intact proteins in While most TH disruption has been shown to occur at pre- bacteria or are obtained from mammalian cell extracts, and detect receptor steps as discussed, the TRs are the point in the response the efficiency of displacement of radiolabeled T3. The advantages pathway closest to the endpoints that result in altered cellular re- are speed and relative simplicity, and the ability to demonstrate sponses: the proper transcriptional control of specific sets of genes. a direct interaction of a TH disrupting chemical and an important

The relatively snug fit of T3 in the TR ligand binding domain only defined target. Further, since these are typically cell free assays, translates to those chemicals with a structure closely resembling there are no confounding issues with cytotoxicity of the chemical that of the hormone itself and which specifically interact with in question. Beyond the less desirable use of radioiodine-labelled the TR ligand binding domains with reasonable affinities (DeVito ligands, the primary disadvantages are that binding does not pre- et al., 1999; Zoeller, 2005). Examples are T3-like OH-PCBs and dict in vivo agonism or antagonism per se , and isolated receptors OH-PBDEs (Freitas et al., 2011). A functional consequence of ago- or receptor fragments are not in their native environment where nist binding to the TR ligand binding domain is co-activators proper folding or stability may be quite different. For example, re- recruitment, and the few antagonists that have been described cent studies with the glucocorticoid receptor have demonstrated for TRs act at least in part via disruption of this interaction (Webb an important allosteric effect of various response element se- et al., 2002). In principle, assays that measure coactivator vs. core- quences on ligand binding that has not been widely appreciated pressor interactions may have more predictive value than simple for other receptors (Meijsing et al., 2009). Improvements in in silico ligand binding. Both cell free and cell based assays are amenable methods to predict ligand binding of the TRs are an important to HTS of TR-coregulator interactions (Table 2). For the nuclear complementary approach to the direct binding assays (Park et al., TRs, however, measurement of their transcriptional regulatory 2010). activity in their native environment at target genes, including in native chromatin context, should be most relevant for predicting Coregulator interaction assays. Another related approach uses the THD acting via this pathway. recruitment of interaction domain peptides derived from nuclear It should also be emphasized that multiple means for disrupting receptor coactivators or corepressors to the TR ligand binding do- TR function are possible beyond direct binding to the receptors and main. These peptides are labelled and recruitment or displacement altering coactivator/corepressor interactions, given the complexi- can be followed by fluorescence polarization (Levy-Bimbot et al., ties of transcriptional control by these receptors. Interactions 2012); furthermore, such assays have been adapted for HTS with RXRs may affect a subset of important TH target tissues or (Johnson et al., 2011). specific genes in specific cells. Further, the TRs themselves and various coactivator and corepressor proteins are subject to post- Reporter gene assays. To be of highest concern, THDCs should alter translational modifications such as phosphorylation that may be TR transcriptional activity in a cellular context. To address this a target for endocrine disruption. TR and coactivator stability, question transient and stable transfection assays are deployed to nuclear localization, and interactions with other cofactors may be develop TRE driven reporter gene assays. Reporter gene assays additional targets of THD chemicals. Arsenic trioxide exposure, for TR activity have taken advantage of cells with endogenous for example, activates a MAP kinase cascade that leads to phos- receptor expression (Freitas et al., 2011; Sugiyama et al., 2005b), phorylation of the corepressor SMRT and its dissociation from co-transfected specific TR expression vectors with the TRE based its nuclear receptor partners including the TRs, leading to de- reporters (Jugan et al., 2007; Freitas et al., 2012b), or co-transfected repression of target genes (Hong et al., 2001). gal4 TR expression vectors and UAS (upstream activating 1334 AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346 sequence) based reporters (Huang et al., 2011; Invitrogen, 2010; tivation assays (Freitas et al., 2011; Gauger et al., 2007; Li et al., Tiefenbach et al., 2010) of which the response is relatively more 2010; You et al., 2006). A summary of suggested chemicals is found specific for TRs so for example RXR activation would be less likely in Table 3. to contribute to a Gal4 based assay. T3 induces reporter gene activ- ity from a low baseline and the assay can be fairly sensitive and 3. Chemical bio-activation and availability in vitro reproducible. Multiplexing responses with a reporter for non- receptor specific cytotoxicity can enhance the assay efficiency In addition to chemicals directly interfering with endpoints of (commercial assays from Promega, and Indigo, no scientific publi- the TH system, other potential THDCs such as PCBs and PBDEs have cations yet), especially when confirming antagonistic action. Tran- to undergo metabolic activation to become a THD hazard. This bio- sient transfection allows flexible analysis of multiple reporter activation into hydroxylated chemicals occurs in vivo by Phase I configurations, multiple receptor isoforms and in various cell lines metabolic enzymes (Marsh et al., 2006; Morse and Brouwer, and has been used to demonstrate the effect of chemicals like BPA, 1995). These OH-metabolites can show remarkable structural sim- TBBPA, and various PCB and PBDE variants and their metabolites ilarities with TH thereby causing direct effects on the TH homeo- on TR transcriptional activity (reviewed by Zoeller (2005)), and stasis by mimicking T and to a lesser extent T (Freitas et al., some other examples are listed here (Hofmann et al., 2009; 4 3 2011; Meerts et al., 2002). As was explained above, this can inter- Ibhazehiebo et al., 2011; You et al., 2006). A limitation of transient fere with T and T transport, action, feedback mechanisms and transfection assays for the use in HTS is that the transfection of 4 3 metabolism. In mammals including humans, OH-metabolites, DNA has to be performed again for each experiments which is bound with high affinity to specific transport proteins like TTR time-consuming and results in relatively great variations in re- and TGB (Gutleb et al., 2010; Lans et al., 1994; Marchesini et al., sponses. Another disadvantage is that the reporter gene in a tran- 2006; Meerts et al., 2000a), can accumulate in blood plasma reach- siently transfected cell is not fully wrapped in chromatin like ing levels similar or even much higher concentrations than of the endogenous genes are; this may be an important distinction with respective parent chemicals (Berg et al., 2010; Bergman et al., stably transfected cells given the TR’s role in modifying chromatin 1994; Gebbink et al., 2008a,b; Sjodin et al., 2000) Montano et al., as part of its transcriptional repression/activation properties. submitted for publication. In addition, the TH-like OH-metabolites Therefore stably transfected reporter gene assays are much can be selectively transported over barriers such as the placenta more suited for HTS applications. Full-length endogenous or co- and blood brain barrier (Meerts et al., 2002; Morse et al., 1993). transfected receptors have the advantage of the full range of Given the high plasma levels and the toxicological relevance of the interactions with a native response element configuration and metabolite effects, it is very important to include quantification of the co-regulatory proteins, while the gal4 fusion system may be some- toxic potencies of the OH-chemicals for toxicological hazard assess- what more specific and show a wider dynamic range of regulation. ment. However, cell-based in vitro test systems almost all lack Phase Yeast based reporter assays have also been reported for full length I metabolism. Therefore, this bio-activation step needs to be added TRs or to measure TR ligand binding domain-coactivator interac- for example by using in vitro biotransformation techniques with tions (Arulmozhiraja et al., 2005; Shiizaki et al., 2010), but there microsomal fractions, although biotransformation efficiencies, can have been concerns raised about ligand availability in those sys- be low and matrix components co-extracted from the metabolizing tems and the absence of a full complement of the TH signalling system may interfere with the assay (Hamers et al., 2008; Meerts pathway and the typically minimal nature of the reporter gene reg- et al., 2000a; Schriks et al., 2006). Recently a new method has been ulatory elements. developed to separate the OH-metabolites from the parent followed by a non-destructive clean-up to remove potentially interfering ma- Induced cellular phenotypes. While further development of primary trix chemicals such as microsomal fatty acids from the extract (Mont- cell based assays is desirable, to date few continuous cell-line- anoetal.,2012). Alternatively, an in vitro system can be exposed based assays for THD have been developed. One is the so-called directlytothemetaboliteofinterest, if known and available. ‘‘T screen’’ that relies on a T induced proliferative response in 3 Another important issue to take into account is the availability rat somatotrophic tumour cell line GH (Ghisari and Bonefeld-Jor- 3 of the chemicals in the test conditions. In cell-based assays with at gensen, 2009; Gutleb et al., 2005; Schriks et al., 2006). The assay least 5% serum lipophilic chemicals will remain available for the has shown the ability to detect agonistic and antagonistic chemi- cell (Li et al., 2010; Weiss et al., 2009), although it also is argued cals presumably acting via a TR mediated mechanism (Table 2). that when they bind to serum proteins their bioavailability for cells In terms of assay development and validation for TR binding might decrease. In tests without serum or other carrier proteins or and transcriptional activation endpoints, beyond using the native lipids relatively lipophilic chemicals may be lost to the plastic of ligands T and T , several synthetic thyromimetics have been de- 3 4 the test plates. This can also be an issue when chemicals are added scribed that are quite useful for this purpose. For example, the to aqueous test media before proteins such as enzymes or recep- T -analogue GC-1 is a TR b selective iodine free thyromimetic with 3 tors are present. Depending on their chemical nature, chemicals high affinity and works well across species (Chiellini et al., 1998; could also be lost during exposure due to oxidation or light- Furlow et al., 2004). Tetrac is another interesting chemical for con- induced degradation. These issues should get ample attention in sideration since it induced as a weak agonist the nuclear TR (Freitas the validation phase of every in vitro bioassay to prevent overesti- et al., 2011), possibly after it is deiodinated to Triac in the cell, but mation of the exposure concentration. Tetrac itself acts as antagonist at the avb3 integrin membrane Finally it is important to characterize the phase 2 and cellular receptor (Davis et al., 2009). Unfortunately, few antagonists have efflux mechanisms in cell based systems, as hydroxylated chemi- been developed for the TRs; nevertheless, NH-3 is an interesting cals may undergo rapid phase 2 metabolism and excretion from chemical that induces release of corepressor but blocks coactivator the cells resulting in an overestimation of the potency (Vandenberg recruitment to the TR ligand binding domain (Nguyen et al., 2002). et al., 2010 ). However, in these in vitro assays (and also in vivo) the endogenous level of T3 available for TR binding (i.e. hypo-, eu-, or hyperthyroid condition) will influence whether some of the T3 analogues act as 4. Suggested testing strategy agonists or antagonists as illustrated by the case of NH 3 (Figueira et al., 2011; Grover et al., 2007). The hydroxylated versions of PCBs The goal of this review was to identify a mechanism-based and PBDEs are most active in TR binding and in cell based transac- battery of non-animal tests that can be applied for THD hazard AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346 1335

Table 3 Examples of chemicals reported to alter critical elements of thyroid hormone regulation or action. This list is not exhaustive, nor all the results are critically checked in detail.

Thyroid regulation target Effect on thyroid regulation Examples of active chemicals References or action TRH receptor signalling Interfere with TSH Somatostatin analogues and synthetic rexinoids Haugen (2009), Sharma et al. (2006) and Zatelli et al. production by thyrotrophic (2010) cells TSH receptor signalling Decreased stimulation of DDT, Aroclor 1254; plant extracts (e.g. Auf’mkolk et al. (1985) and Santini et al. (2003) thyrocytes 3,4-dihydroxycinnamic acid derivatives) NIS-mediated iodide Decreased thyroidal Perchlorate, nitrate, thiocyanate, Lecat-Guillet and Ambroise (2009), Schmutzler et al.

uptake synthesis of T3 and T4 4-methylbenzylidene-camphor, imidazothiazoles (2007b), Van Sande et al. (2003) and Wolff (1998) TPO inhibition Decreased thyroidal Methimazole, propylthiourea, amitrole, mancozeb, Biegel et al. (1995), Capen (1997), Doerge and

synthesis of T3 and T4 soy isoflavones, benzophenone 2, 1-methyl- Sheehan (2002), Higa et al. (2002), Jarry et al. (2004) 3-propyl-imidazole-2-thionem, kojic acid, and Schmutzler et al. (2007b) 4-nonophenol Binding to serum Decreased serum levels Hydroxy-PCBs and -PBDEs, EMD 49209; Lans et al. (1993), Marchesini et al. (2008), Meerts transport proteins TTR pentachlorophenol, HCB, phenols et al. (2000b), Montano et al. (2012), Schröder-van and TBG der Elst et al. (1997) and van den Berg (1990) Cellular TH transporters Altered cellular availability Antidepressants, amino acids, aromatic dyes, Kinne et al. (2010) and Roth et al. (2010) of TH for target cells, flavonoids metabolism, action and elimination Inhibition of deiodination Decreased peripheral FD&C Red Dye #3, PTU, PCBs, octyl- Capen (1998), Klammer et al. (2007), Köhrle et al.

enzyme activity synthesis of T3 methoxycinnamate, 4-methylbenzylidene- (1986), Morse et al. (1993), Renko et al. (2012) and camphor, iodinated X-ray contrast agents, Visser et al. (1979) halogenated aromatic chemicals Induction of sulfation and Increased biliary Acetochlor, phenobarbital, 3-methylcolanthrene, Crofton (2008) and Vansell and Klaassen (2002)

glucuronidation elimination of T3 and T4 PCBs, 1-methyl-3-propyl-imidazole-2-thione enzyme activity Inhibition of sulfation and Decreased biliary Hydroxy PCBs and –PBDEs Hamers et al. (2008) and Schuur et al. (1999)

glucuronidation elimination of T3 and T4 enzyme activity Hepatic Cellular Altered biliary elimination TCPOBOP, pregnenolone-16- a-carbonitrile, TCDD, Guo et al. (2002), Jigorel et al. (2006), Petrick and

Transporters of T3 and T4 rifampicin, phenobarbital, oltipraz, di-n-butyl Klaassen (2007), Shimada and Yamauchi (2004) and phthalate, n-butylbenzyl phthalate, dicofol Staudinger et al. (2001)

TRbinding Altered activation of TH T3-like hydroxy PCBs and -PBDEs, linuron, Davey et al. (2008), Freitas et al. (2011), Gauger et al. dependent gene Tetrabromobisphenol A, arsenic (2004), Hong et al. (2001), Kitamura et al. (2005b), transcription Moriyama et al. (2002) and Schmutzler et al. (2007b) assessment. To minimize the risk of false negatives as well as of Importantly, any in vitro approach should account for possible false positives, the in vitro assays should cover all relevant end- bio-activation of the test chemical to prevent false negatives, as points of THD to distinguish between chemicals of less concern several chemicals only become hormone disrupting after meta- and chemicals that need further study. Prioritization of chemicals bolic activation. based on a well-chosen in vitro assay battery, possibly extended Once the test battery would be validated and accepted at with in silico approaches, will be much more specific compared OECD level, a Tier 1 in vitro battery screening outcome can be 1 to in vivo tests, will improve the efficiency of testing methods by of 3 classifications. (1) No indication for THD effects even at high providing empirical data on still untested chemicals and will in- concentrations. The chemical can then be classified as of less con- crease the ability of risk managers to protect human health. Testing cern and does not need to be selected for further testing. (2) of chemicals for THD activity will likely need to be done in a tiered Strong indications for THD in the in vitro test battery. Further fashion, and in contrast to most proposed tiered approaches (Dix testing is needed at exposure concentrations that approach ex- et al., 2007; Willett et al., 2011 ), we suggest that the first 2 tiers pected in vivo concentrations (based on exposure assessment can be done fully in vitro. After every tier there is then an option and toxicokinetic modelling) unless hazard-based decisions are not to proceed with further testing of a chemical. taken. (3) The responses in the Tier 1 in vitro test battery may not be conclusive or only weak and these chemicals should go to Tier 2 screening. 4.1. Tier 1 During optimization and in vivo validation of the in vitro test battery, clearly defined triggers need to be developed, in order to A major goal of the pathway-based chemical hazard assessment be able to classify the chemicals as of less concern, high concern is to be able to focus resources, both laboratory and regulatory, on or inconclusive. While the classification ‘of less concern’ does not those chemicals of highest concern. Thus, the first function of a mean ‘‘no need for evaluation’’ category, the reality is that chemi- comprehensive battery of in vitro tests is to prioritize the thou- cals with low priority may never see testing because of limited sands of poorly characterized chemicals. Table 2 presents the range resources and test capacity. Therefore, it is important to minimize of endpoints covering the most important mechanisms of TH sys- the number of false negatives, while not allowing the numbers of tem regulation (Fig. 1) that currently are known to be sensitive false positives to increase too much. for THDC and are now or in the near future, applicable to an in vitro HTS test battery. As current Tier 1 testing is aimed at pro- tection of humans from THDC, in vitro human cell-based assays are 4.2. Tier 2 preferentially applied. Nevertheless the high evolutionary preser- vation of THD targets clearly allows inclusion of other cell types, The purpose of this Tier 2 testing is to further identify and char- while on the other hand results obtained with human cells are acterize the interactions of putative THDCs with the TH system. As usable for ecotoxicological purposes. an intermediate step between the in vitro Tier 1 assay battery and 1336 AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346

Table 4 In vitro bioassay battery recommended for Tier 1 screenin gof chemicals for THD. Summary of MTS and HTS assay usage and recomm endations for assay development by target. The last three assays are not specific for THD, but can indirectly induce a THD effect. Bioactivation of the test chemicals is needed to prevent false negative responses.

Target Relevance Status State of science References TRH receptor Feedback control of TH Technology available and 1. Commercially available CHO-K1 cells with Boutin et al. (2012) signalling synthesis research gap stable rhTRH1 (PerkinElmer). No literature reports on use 2. LTS assay using HEK-EM293 cells stably expressing mouse TRH-R1 or TRH-R2 that uses TSH receptor Feedback control of TH In use and research gap 1. HTS assay available using HEK293 cell line Jomaa et al. (2013) and Titus signalling synthesis stably transfected with the TSHR coupled to a et al. (2008) cyclic nucleotide gated ion channel as a biosensor. Potential confounds by chemicals that interfere with cAMP pathways 2. MTS assay available for TSH mediated FRTL-5 cells rat thyroid cell proliferation NIS-mediated iodide TH synthesis In use 1. HTS assays using HEK293 cells stably Cianchetta et al. (2010), Lecat- uptake transfected with hNIS. Requires use of Guillet et al. (2007) and Waltz radiolabeled chemical et al. (2010) 2. MTS assays available include FRTL-5 cells with yellow cerium indicator or with yellow fluorescent protein (YFP) variant YFP-H148Q/ I152L. Could be adapted for HTS TPO inhibition TH synthesis Potential for adaptation for 1. LTS assays published that may be amendable Schmutzler et al. (2007a,b), MTS or HTS, research gap: to MTS or HTS. Both employ FTC-238 cells Song et al. (2011) and complex Fe-dependent transfected with hrTPO Verhaeghe et al. (2008) mechanisms of action 2. HTS assay available that uses vanadium haloperoxidases. Will require comparison to vertebrate TPO Binding to serum TH availability in tissues, In use, needs optimization 1. HTS assay available that uses Surface plasmon Marchesini et al. (2008), transport proteins THDC transport, serum and validation resonance based biosensor Montano et al. (2012) and TTR and TBG hormone levels Yamauchi et al. (2003) 2. MTS available based on non-radioactive fluorescence displacement assay

Inhibition of T3/T4 ratio Potential for adaptation for 1. MTS assay available for D1 based on tissue or Kunisue et al. (2011), Piehl deiodination MTS or HTS and Research cell lysates, also applicable to intact cells et al. (2008) and Renko et al. enzyme activity Gap (2012) Tissue hormone levels 2. Mass spectrometry of tissue thyroid hormone and metabolite profile Inhibition of sulfation Hormone turnover Potential for adaptation for 1. MTS assays currently being used. Very limited Hamers et al. (2008), Kavlock and MTS or HTS coverage of the biology and requires primary et al. (2012) and Paul et al. glucuronidation cells or enzyme preparations. (2010) enzyme activity 2. Commercial assays from CellzDirect (UGT1A1, SULT2A1) Tissue flux of THs via Tissue hormone levels Research gap Low-throughput assays available that use Freitas et al. (2011), Morimoto membrane radiolabeled transporter substrates et al. (2008), van der Deure transporters et al. (2010) and Visser et al. (2010)

TR binding and Receptor activation/ In use 1. MTS assay for T3 induced proliferation of GH3 Freitas et al. (2011), Gutleb transcription inhibition cells. Potential confounds by chemicals that et al. (2005), Johnson et al. interfere with non-T3 mediated proliferation (2011), Rotroff et al. (2012) pathways. and Schriks et al. (2006) 2. HTS assays currently use a variety of assay technologies for a TR a and TR b as well as a TR b- SCR2 coactivator assay 3. HTS assay for TR activation (TR-GH3.Luc) Activation of other Influences TRE-mediated In use HTS assays currently use a variety of assay Huang et al. (2011), Knudsen nuclear receptors gene transcription technologies for a number of nuclear receptors et al. (2011) and Shah et al. that (e.g., CAR, PXR, PPAR) known to interfere with (2011) heterodimerize TR-activation with TRs Upregulation of Influences hormone In use HTS assays currently use a variety of assay Huang et al. (2011), Knudsen enzymes involved turnover technologies for a number of nuclear receptors et al. (2011) and Shah et al. in TH conjugation (e.g., CAR, PXR, PPAR) known to be involved in (2011) or deiodination the regulation of hepatic Phase 2 catabolic enzymes enzymes and Phase 3 cellular transporters In vitro bioactivation Bioactivation of THDC Potential for adaptation for In vitro metabolism followed by selective Montano et al. (2012) of parent crucial in vivo but absent HTS and research gap extraction of metabolites with limited co- chemicals to test in or not potent enough in extraction of disturbing matrix chemicals. This the other in vitro in vitro assays method needs further optimization and bioassays validation for HTS application. in vivo bioassays, dedicated and modern tissue engineering tech- more focusing and targeting on more time-consuming but also niques may be used to further select and refine the choice of chem- more physiologically based assays e.g., with primary tissue culture, icals that need full Tier 3 testing in vivo. Tier 2 testing should allow functional thyroid follicular cells differentiated from mouse AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346 1337 embryonic stem-cells( Antonica et al., 2012), embryonic zebrafish mine the blood plasma levels of the chemicals and/or its bioactive eleuthero-embryos tissue to identify chemicals impairing thyroid metabolites in the in vivo experiments. gland morphogenesis (Raldua et al., 2008), or with fish- or amphib- ian embryos, targeting endpoints suggested by the results in Tier 1. 6. Further outlook: using computational modelling to interpret Once Tier 2 assays qualify the chemical as a potential THD haz- in vitro assays for in vivo effects ard, evaluation could proceed to Tier 3 in vivo testing. However, be- fore this takes place, the toxicokinetics of the chemicals could be Although it was not the ambition of this review to present an determined based on in vitro metabolism studies combined with in vitro approach to be able to predict in vivo effects, this could at in silico modelling, as this has been shown to allow translation of a later stage be further elaborated. Despite the many advantages in vitro results to in vivo effects (Punt et al., 2013). From the of an in vitro testing battery, estimating in vivo toxicity from in vitro EC50 (which statistically is most robust) it is then possible in vitro results can be a daunting task, as it is not possible to fully to predict not only the in vivo EC50, but also the in vivo EC10 or reproduce the in vivo situation in vitro. There are two major uncer- BMD10 (benchmark dose at which a benchmark response equiva- tainties in extrapolation from in vitro to in vivo. One is the need to lent to a 10% effect is induced) which is more relevant for the hu- determine the ability of simple, in vitro cell culture or isolated pro- man situation (Louisse et al., 2010). tein preparations to fully reflect the complex biology found in Tier 3 in vivo experiments should preferably make use of assays whole organisms. The second is the complexity in extrapolating that need to be performed anyhow for other ED endpoints, and in vitro concentrations to in vivo pharmacokinetics (Blaauboer, THD-relevant endpoints indicated by the in vitro assays should 2010). The absence of whole-body pharmacokinetic processes be added to the standard in vivo tests. (absorption, distribution, metabolism and excretion) in the With both Tier 1 and Tier 2 testing, the challenge is to have a in vitro assay system is a challenge in implementing in vitro based battery of assays that comprehensively evaluate the pathways re- risk assessments for chemicals. Integration of data on the toxic lated to phenotype of interest together with a weight of evidence mode of action of a chemical with data on its pharmacokinetic approach to ranking chemicals. The ToxCast program has devel- behaviour is essential for the interpretation of in vitro studies on oped one such approach, termed the ‘‘ToxPi’’ that takes large the toxicity of a chemical in the whole animal. With physiologi- amounts of information about chemical interactions with particu- cally based biokinetic (PBBK) models, a better interpretation of lar biological pathways and displays them in a visual format for in vivo relevance of in vitro results can be made by linking an decision makers to use (Reif et al., 2010). in vitro concentration–response curve to an in vivo dose–response curve. These approaches are also known as IVIVE or reverse dosim- etry (Forsby and Blaauboer, 2007; Judson et al., 2011; Louisse et al., 5. Advantages of in vitro testing 2010; Punt et al., 2013; Verwei et al., 2006; Wetmore et al., 2012 ). IVIVE is a process for estimating the environmental exposures to a Although in vivo bioassays are generally perceived as being the chemical that could produce tissue exposures at the site of toxicity most reliable tests for assessing the hazard of chemicals, including in humans equivalent to those associated with effects in an in vitro THDC, there also are several limitations for this approach. The toxicity test (e.g., an EC , a Benchmark concentration, etc.) (Louis- functioning of the TH system depends on life-stage, species and 50 se et al., 2010). IVIVE can provide an estimate of the likelihood of health condition. Healthy animals may cope with certain harmful effects from expected environmental exposures to chemi- exposures by compensation via HPT feedback and effects may be cals by integrating diverse information from targeted in vitro toxic- unnoticed, while people with specific health conditions, such as ity and kinetic assays using a computational modelling approach. hypo- or hyperthyroidism or malnutrition, may be more vulnera- Pharmacokinetic modelling plays a pivotal role in this quantitative ble. Some animals may have targets for THD that are not relevant extrapolation, by incorporating the in vivo bioavailability, distribu- for humans and vice versa (e.g., Capen, 1997). For example TBG is tion, and clearance of the chemical into the process. an important human TH plasma transport protein while this is not Computational systems biology pathway (CSBP) models are also the case for adult male rats. Life-stage specific differences may also a crucial component in the transition away from animal testing in result in under-estimating hazards for humans. For example, most toxicology( Krewski et al., 2009). CSBP models provide quantitative in vivo animal studies are conducted using adult euthyroid ani- descriptions of molecular circuits responsible for the basal opera- mals, and the developing, or hypo- or hyperthyroid organisms tion of toxicity pathways (i.e., thyroid disruption) and characterize may be more susceptible to THDC. A well-chosen in vitro test bat- their alteration with chemical exposure (Zhang et al., 2010b ). Using tery should cover mechanisms and targets that are relevant for quantitative descriptions of chemical interactions with specific multiple life stages and conditions. This way a much broader range receptors or other molecular targets, these models describe the of chemicals can be assessed for potential perturbations of THD dose–response behaviour observed in the various assays. These pathways of relevance for human biology in a shorter time, with dose response models provide points of departure concentrations virtually no animals and at lower costs. To be able to make the step to serve as inputs for IVIV extrapolation and prediction of human from hazard to risk assessment based on in vitro test batteries risk from environmental exposures. An especially attractive oppor- more fundamental knowledge is needed to reveal the relationship tunity exists in linking these cellular models across multiple cell between the extent and timing of exposure, with critical pathway types, representative of different tissues within the body (e.g., liver, perturbations, physiological effects, and disease outcomes. With hypothalamus, pituitary, gonads, etc.). CSBP models can also help insight in new mechanisms, in vitro test battery results may offer explaining non-monotonic dose–response relationships that are greater ability to predict which chemicals are more relevant for sometimes observed in in vivo experiments, as these might be specifically sensitive groups or conditions. In vitro test results also the result of the combined result of multiple pathways with differ- offer more possibilities to predict THD effects for other species ent dose–response relationships in different tissues and over time. than humans, once more is known about possible species specific differences in cellular and molecular targets, and physiological sys- tems. Comparing in vitro and in vivo responses will help to deter- 7. Recommendations for assay use and development mine specific mechanisms of THD chemicals and further develop better predictive research models for use in regulatory toxicology. The overall goal of this paper is to summarize the potential thy- To allow in vitro-in vivo comparisons, it is very important to deter- roid targets of environmental chemicals and in vitro assays that 1338 AlberTinka J. Murk et al. /Toxicology in Vitro 27 (2013) 1320–1346 cover the important mechanisms for TH disruption by environ- mechanisms for TH disruption by chemicals. For these endpoints, mental chemicals. From the above review it is clear that the state currently available or desired in vitro assays are discussed that of the science for these assays ranges from those already being are or could be suitable for medium to high throughput testing. used in medium-throughput screening (MTS) or HTS, to potential Bio-activation of the tested chemicals is a critical consideration targets that lack cost efficient testing methods. To help focus in the in vitro testing, as several cases are known when testing of research and development efforts we provide a list of recommen- the parent chemical alone results in false negative responses. The dations for assay use and development( Table 4). These recommen- application of an in vitro test battery in a tiered testing approach dations fall into three basic categories; assays already in use for for THDC is discussed as well as the outlook for combination with MTS and HTS that are assays not currently used but show promise in silico alternatives that eventually may further reduce the use of for adaption to MTS or HTS, and endpoints for important mecha- animals for testing chemicals for THD while enhancing the knowl- nisms that either lack in vitro assays or available assays are not edge of the mechanisms of action of the chemicals. Technical and easily amenable to adaptation for MTS or HTS. in vivo validation of the in vitro test battery will be necessary to in- The first category contains assays that target known molecular crease confidence for use of its results in regulatory decisions. events important for thyroid processes and are already employed in MTS or HTS of environmental chemicals. These include the Tox- Conflict of interest statement Cast and Tox21 assays (Kavlock et al., 2012; Freitas et al., 2012a). Some other assays are currently in use (Lecat-Guillet et al., 2008 ) There are no conflicts of interest issues. or could be used (Marchesini et al., 2008; Montano et al., 2012 ). Data from some of these assays has begun to be analysed (Johnson et al., 2011; Rotroff et al., 2012) and will provide important infor- Disclaimer mation on assay performance and utility in screening environmen- tal chemicals for potential interactions with receptors and receptor The views expressed in this paper are those of the authors and signalling systems. Incorporation of existing HTS assays for NIS and do not necessarily reflect the views or policies of the U.S. Environ- TTR would expand coverage of screening systems to include mental Protection Agency. Mention of trade names or commercial important targets for THD chemicals. While there is little evidence products does not constitute endorsement or recommendation for that many xenobiotics target TRH and TSH, there has also been use. very little testing. Therefore, existing HTS assays could be used to determine whether these are important targets for inclusion in Acknowledgements HTS screening batteries. The second group consists of assays that may be suitable for This paper is based on the results of a workshop organized by MTS or HTS. These assays require research to determine whether the Institute for Risk Assessment Sciences, Utrecht University, for they could be adapted from current low-through put technology which financial support was kindly provided by ‘‘Proefdiervrij’’ to allow for efficient testing of larger number of chemicals. For (the Dutch Society for the Replacement of Animal Testing (dsRAT) example, stable reporter cell lines with human recombinant TPO www.proefdiervrij.nl/engl ish ) and the ASAT Foundation (www. have been developed, but require adaptation testing. There are asat-initiative.eu/ ). We want to thank Maurico Montaño for the guidelines available that list requirements for use in HTS screening nicer version he made of Fig. 1. programs (e.g., NCATS, 2012). Due to the large number and variety of chemicals known to inhibit TPO, a high priority should be given References to development of MTS or HTS TPO assays. The third group includes endpoints for which no screening as- Aamir, I.S., Tauheed, S., Majid, F., Atif, A., 2010. Frequency of autoimmune thyroid says currently exist that could be easily modified for MTS or HTS. disease in chronic urticaria. JCPSP – J. Coll. Phys. Surg. Pakistan 20, 158–161. These will require assay development research to devise basic Alexander, W.D., Wolff, J., 1966. Thyroidal iodide transport. VIII. Relation between transport, goitrogenic and Antigoitrogenic properties of certain anions. in vitro methods that could then be adapted to MTS or HTS. This in- Endocrinology 78, 581–590. cludes a number of the THDC targets reviewed above. For example, Andersson, M., Karumbunathan, V., Zimmermann, M.B., 2012. 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