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Testing for thyroid hormone disruptors, a review ofnon-mammalian in vivo models

Stephan Couderq, Michelle Leemans, Jean-Baptiste Fini

To cite this version:Stephan Couderq, Michelle Leemans, Jean-Baptiste Fini. Testing for thyroid hormone disruptors, areview of non-mammalian in vivo models. Molecular and Cellular Endocrinology, Elsevier, 2020, 508,pp.110779. �10.1016/j.mce.2020.110779�. �hal-02414719�

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Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Testing for thyroid hormone disruptors, a review of non-mammalian in vivomodels

Stephan Couderq, Michelle Leemans, Jean-Baptiste Fini∗

Unité PhyMA laboratory, Adaptation du Vivant, Muséum national d’Histoire naturelle, 7 rue Cuvier, 75005, Paris, France

A R T I C L E I N F O

Keywords:Thyroid hormonesThyroid disrupting chemicalsBiological assayEndocrine disruptionAlternative methods

A B S T R A C T

Thyroid hormones (THs) play critical roles in profound changes in many vertebrates, notably in mammalianneurodevelopment, although the precise molecular mechanisms of these fundamental biological processes arestill being unravelled. Environmental and health concerns prompted the development of chemical safety testingand, in the context of endocrine disruption, identification of thyroid hormone axis disrupting chemicals(THADCs) remains particularly challenging. As various molecules are known to interfere with different levels ofTH signalling, screening tests for THADCs may not rely solely on in vitro ligand/receptor binding to TH receptors.Therefore, alternatives to mammalian in vivo assays featuring TH-related endpoints that are more sensitive thancirculatory THs and more rapid than thyroid histopathology are needed to fulfil the ambition of higherthroughput screening of the myriad of environmental chemicals. After a detailed introduction of the context, wehave listed current assays and parameters to assess thyroid disruption following a literature search of recentpublications referring to non-mammalian models. Potential THADCs were mostly investigated in zebrafish andthe frog Xenopus laevis, an amphibian model extensively used to study TH signalling.

1. Background

A relatively modern threat to human health and ecosystems hasemerged with the escalation in volume and diversity of substancesproduced by the chemical industry after World War II. Observations ofadverse effects in wildlife following widespread application of thepesticide DDT1 and, on human health after Diethylstilbestrol prescrip-tion (Carson, 1962; Herbst and Scully, 1970), served as a catalyst topromote chemical safety assessment programs promoted by the UnitedStates Environmental Protection Agency (US EPA) and the Food andDrug Administration (FDA) in the 1970s. In the 90's, global concernarose over the elusive impact of environmental endocrine disruptingchemicals (EDCs), capable of adversely affecting normal hormonefunction in humans and in wildlife. Nowadays, global human activitiesrelease millions of tons of hazardous chemicals into the environment,exposing humans and other animals to a cocktail of chemicals throughwater, land, air and food (Bernanke and Köhler, 2009; CDC, 20192;Worldometers, 2019). Approximately 40,000–60,000 industrial

chemicals are sold worldwide, with 6000 representing 99% of theirtotal volume (ICCA & UNEP, 2019 3,4). Recently over 600 compounds,some of which are produced in high volumes, have been included in adatabase of potential EDCs, with supporting evidence of adverse effectsin either humans or rodent models (Karthikeyan et al., 2019). Im-portantly, the vast majority of chemicals have yet to be assessed fortheir potential impact on human health or the environment (Applegate,2008; Krimsky, 2017). Moreover, regulatory agencies are unable tokeep pace with the rate of new compounds being introduced (600–1000compounds per annum in the US alone) (Krimsky, 2017; Vandenberg,2016).

1.1. Testing for endocrine disrupting chemicals

This context prompted the OECD5 and the US EPA to develop newstrategies to rapidly screen and characterize the endocrine activity ofchemicals and environmental contaminants, specifically for estrogen,androgen and, more recently, for thyroid and metabolism-related

https://doi.org/10.1016/j.mce.2020.110779Received 16 September 2019; Received in revised form 26 February 2020; Accepted 27 February 2020

∗ Corresponding author.E-mail address: fini@mnhn.fr (J.-B. Fini).

1 Dichlorodiphenyltrichloroethane.2 Center for Disease Control and Prevention.3 International Council of Chemical Associations.4 United Nations Environment Programme.5 Organization for Economic Co-operation and Development.

Molecular and Cellular Endocrinology 508 (2020) 110779

Available online 06 March 20200303-7207/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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modalities of endocrine action (US EPA, 2014; OECD, 2018b).Current testing methods have yet to be harmonized worldwide but,

generally, involve a tiered-approach consisting of a series of assays toidentify and characterize a potential hazard. Initial tiers feature high-throughput in vitro or rapid in vivo assays in order to screen compoundsvia specific modes of action, while higher tiers of increased biologicalcomplexity consist of more expensive and relatively long-term in vivotests (Manibusan and Touart, 2017). The latter may demonstrate bio-logically-relevant adverse effects on endocrine systems, and char-acterization of the dose-response functions may be extrapolated tohuman or wild-life populations (Pickford, 2010). Finally, the outcomesof the various tests and increasingly, data from open scientific litera-ture, are compiled to determine the weight-of-evidence supportingpotential endocrine activity of a chemical (Gross et al., 2017).

In contrast to classical toxicology tests whereby “the dose makes thepoison” (Bus, 2017), assessing potential endocrine disruption posesfurther challenges (Fuhrman et al., 2015):

(i) The existence of non-monotonic responses to exposures calls intoquestion the determination of “safe” threshold doses (Vandenberget al., 2012).

(ii) The inherently integrative and complex endocrine system may leadto cross-talk between different hormonal pathways (Brüggemannet al., 2018; Kiyama, 2017; Sharma et al., 2017).

(iii) Critical windows of development (e.g., gestational or perinatalperiods) are particularly vulnerable to EDC exposure (Barrett,2009).

(iv) The effects of exposure may occur later in life or even extend tofuture generations (Xin et al., 2015).

Therefore, assessment of endocrine disruption calls for specificendpoints and assays, to which the OECD and US EPA have respondedby including specific in vitro screening assays and adding endocrine-sensitive parameters to pre-existing mammalian and non-mammalian invivo assays.

The current framework for next-generation assay developmentpromotes the use of alternative approaches that rely on physiologically-based pharmacokinetic (PBPK) models and in vitro systems to re-capitulate in vivo responses. This trend is coherent with the more ethical3Rs principles advocating replacement, reduction and refinement ofanimal testing protocols (Burden et al., 2015; Tannenbaum andBennett, 2015). However, the complexity of endocrine regulation pre-cludes the sole use of in silico and in vitro systems. In particular, due tothe many points of regulation of the hypothalamus-pituitary-thyroid(HPT) axis (OECD, 2018b; Zoeller et al., 2007), even a battery of in vitroassays for thyroid disruption would not be as comprehensive as athorough in vivo test (Zoeller and Tan, 2007). Ultimately, several am-phibian-based assays containing endpoints sensitive to thyroid hor-mone disruption were added to the list of standardized in vivo bioassays.

1.2. Thyroid hormones and the hypothalamus-pituitary-thyroid (HPT) axis

Concern over dysfunction of the HPT axis is spurred by the crucialrole that THs play in differentiation, growth, and metabolism, and theirrequirement for the proper functioning of virtually all tissues (Yen,2018). Notably, THs are essential for normal development and functionof the central nervous system due to their key roles in neuronal pro-liferation, migration, differentiation, synaptogenesis, synaptic plasticityand myelination (Bernal, 2005; Horn and Heuer, 2010; Howdeshell,2002). Deficiency in TH levels during neurological development caninduce severe manifestations in humans, as highlighted by congenitaliodine deficiency syndrome (Bernal, 2007). However even moderate ortransient TH insufficiency during the perinatal period may irreversiblyalter offspring neurodevelopmental outcomes (Bernal, 2005; Korevaaret al., 2016; Moog et al., 2017; Prezioso et al., 2018).

Fundamental research on TH signalling has elucidated the

importance of disturbances of the HPT axis beyond altered homeostasisevaluated by circulating levels of hormones. Indeed, TH axis disruptingchemicals (THADCs) are present in many classes of chemicals and maylead to a wide range of adverse effects in both wildlife and humansthrough disruption of the HPT axis at any of the multiple levels pre-sented hereafter (Calsolaro et al., 2017; Mughal et al., 2018; Oliveiraet al., 2018; Ghassabian and Trasande, 2018).

The HPT axis regulates TH synthesis through the hypothalamicthyrotropin-releasing hormone (TRH) which stimulates the pituitary tosecrete thyroid-stimulating hormone (TSH) necessary for the thyroidgland (or follicles in fishes) to produce THs. Homeostasis is regulated bynegative feedback on the release of both TRH and TSH. Synthesis of THsrequires the uptake of iodide mediated by the sodium/iodide symporter(NIS) in thyroid cells. Iodide ions are then oxidized by the enzymethyroid peroxidase (TPO) for incorporation into thyroglobulin to pro-duce precursors of the 3,3′,5-triiodothyronine (T3) (the active form ofTHs) and 3′,5′,3,5-tetraiodo-L-thyronine (thyroxine or T4) (Sellitti andSuzuki, 2013). The vast majority of T4 and T3 circulates in thebloodstream bound to thyroid distributor proteins (in humans: trans-thyretin (TTR), thyroid binding globulin (TBG), and albumin) and areconsidered biologically inactive (Bartalena and Robbins, 1993; Refetoff,2000). Free THs enter cells via the TH-specific transporter mono-carboxylate transporters (MCTs), several members of the organic anion-transporting polypeptide (OATP) family, and the heterodimeric L-typeamino acid transporters (LATs) (Hagenbuch, 2007; van der Deure et al.,2010). Intracellular TH availability is coordinated by specific deiodi-nation processes which either activate or deactivate THs (DIO1 maycontribute to both, while DIO2 and DIO3 exclusively activates and in-activates THs, respectively). Next, THs enter the nucleus where they caneither positively or negatively control transcription of target genes viaTH receptors (TRs) that bind to specific DNA segments containing THresponse elements (TREs) (Hönes et al., 2017). Finally, metabolicclearance of circulating THs is ensured by hepatic sulfotransferases oruridine disphosphate (UDP)-glucuronosyltransferases (Visser, 1988).

Some differences exist between vertebrates and should be borne inmind when evaluating thyroid disruption in different species. In parti-cular, in non-mammalian vertebrates, the release of TSH appears to beregulated by corticotrophin-releasing hormone (CRH) (De Groef et al.,2006), and the set of TH transport distributor proteins may differ aswell as their affinity, e.g., the main transport protein TTR has a higheraffinity for T3 instead of T4 compared to mammalian models (Zoelleret al., 2007). Interspecies differences are also found among mammalianspecies, namely between humans and rodent models - the gold standardof regulatory in vivo toxicology. These include differences in TH phar-macokinetics, basal levels of TSH, structure and expression patterns ofkey enzymes and/or transporters, and the timing of thyroid ontogenesisand neurodevelopmental events (Choksi et al., 2003; Fisher et al., 2012;Jomaa, 2015; Zoeller et al., 2007).

1.3. The challenge of assessing TH (dys)function

In environmental epidemiology, subtle variations of circulating THsand/or TSH may correlate with exposure to a chemical being scruti-nized but, without discernment of the mechanisms of action (MoA) orinsight into the potential adverse effect(s), these observations are in-sufficient to draw definitive conclusions on their endocrine activity (Leeand Jacobs, 2015; Slama et al., 2017).

Even though measuring circulating THs, and TSH has increased inspecificity, reproducibility and sensitivity (Kuyl, 2015; Spencer, 2000),inter-individual variations can mask discrete effects caused by THADCs(Boas et al., 2012), and discordant results in thyroid function tests maycomplicate interpretations, especially in cases of minor disturbances(Koulouri et al., 2013). In addition, effects in peripheral target tissues,such as alterations in activity of deiodinases, may not necessarily causedisruption of hormone homeostasis yet can alter TH availability at alocal level (Boas et al., 2012). These possibilities, coupled with the

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scarcity of information on the molecular mechanisms of THADCs, fur-ther complicate evaluation of thyroid disrupting effects.

Detailed insight into the HPT axis has elucidated multiple MoAthrough which thyroid disruption can occur (Crofton, 2008; Friedmanet al., 2016). Numerous in vitro assays are designed to examine keysteps in the synthesis, regulation, and action of TH: i) TRH and TSHproduction, ii) TSH and TRH receptor activation, iii) TPO activity oriodide uptake inhibition, iv) binding to TTR or TBG, v) deiodinationactivity, vi) cellular membrane transport, vii) binding to TRs (OECD,2017). Importantly, while several assays show promise for im-plementation into regulatory toxicology, currently, none have beenvalidated by either the OECD or US EPA. For this reason, there is a needfor developing higher throughput screening assays that can detectsubstances that interfere with the activity of the thyroid system(Kortenkamp et al., 2017).

Newly required thyroid endpoints used to update existing in vivotests consist of determining circulating levels of THs and TSH, mea-suring thyroid weight to inform of its stimulation by TSH over time, andespecially, examining histopathologic changes in the thyroid gland.Thyroid histopathology is a particularly valuable and sensitive diag-nostic parameter to disruption of TH homeostasis (Dang, 2019; Grimet al., 2009; Pickford, 2010). Together, these parameters of thyroidfunction have been used to identify the majority of thyroid toxicants inmammals and can be sufficient to detect clear signs of thyroid disrup-tion. However, it should be noted, that severity grading in thyroidhistopathology is based on an idealized model which used the powerfulinhibitor of TH synthesis propylthiouracil (PTU) (Brucker-Davis, 1998;Zoeller and Tan, 2007), and may therefore overlook the effects of otherTHADCs with more subtle effects. By combining curated data fromscientific literature and results from validated assays in the US EPAEndocrine Disruptor Screening Program, i.e., the female and male ratpubertal assays and the Amphibian Metamorphosis Assay (AMA),Wegner et al. (2016) proposed an additional 28 reference THADCs withvarious MoA which could serve to further characterize the response ofdifferent assays and endpoints.

Although alternative models represented by amphibians, fish andavian species may appear too far removed from mammals to be relevantto human health, molecular components of the HPT axis are highlyconserved across vertebrate taxa and TH signalling involves the samecirculating hormones (Noyes et al., 2018; Taylor and Heyland, 2017).Markedly, the peak of THs observed around the perinatal period inmammals is also found during developmental transitions in other ver-tebrates (Holzer and Laudet, 2013) as demonstrated by amphibianmetamorphosis (Galton, 1992), subtle or spectacular post-hatchingmetamorphosis in teleost fish (McMenamin and Parichy, 2013), andhatching in precocial birds (De Groef et al., 2013).

Consequently, alternative models used in toxicology have been in-creasingly popular for the purpose of chemical safety assessment ofpotential THADCs. Therefore, we focused this review on knowledge ofTH-related endpoints developed or under development in non-mam-malian vertebrates.

2. Methods

In order to supplement our knowledge on the subject and recentOECD, US EPA, EFSA6 and ANSES7 reports; a combination of severalsearch strategies were used in a single query in PubMed to review re-cent literature either assessing thyroid disruption in non-mammalianmodels in vivo or discussing regulatory issues on the topic. Fine-tuningof the search was achieved using Yale's Mesh analyzer to limit non-relevant articles (i.e., mammalian studies, articles treating TH in

passing, thyroid disease, etc.) and retrieve a maximum of publicationscontaining thyroid assays or fundamental research on TH of potentialuse to (eco)toxicology. A PubMed filter was applied to remove humanstudies and articles older than 5 years. Fig. 1 provides an overview ofthe final articles retrieved using the following search query performedon the February 14, 2019 (full search query available in SupplementaryDocument). In addition, a supplementary table features the followinginformation for each ecotoxicological article retrieved from the searchquery: the model employed, the test chemical(s), the concentrationranges and exposure period, and the endpoints examined (Supple-mentary Table).

Discarded articles from the 256 articles initially retrieved did notinvolve THs in an (eco)toxicology or regulatory context. TH-relatedexperimental studies were mainly in vivo. Among these, several per-formed additional in vitro, in silico or ex vivo experiments, or even usedmultiple animal models and endpoints, leading to some overlap be-tween the number of articles (indicated in parenthesis) per type ofstudy, model utilized and endpoints examined. Amphibian and teleostfish models both had distinct endpoints, underlining the advantagesand disadvantages of each model.

In vivo studies pertaining to chemical assessment that were retrievedfrom our literature search originated from academic research, andwhile most are not based on standardized assays tailored for risk as-sessment, common endpoints frequently appear within these models(Fig. 1 & Supplementary table). Novel methods and endpoints arehighlighted in this review and could be considered for future integra-tion into regulatory risk assessment. Particularly, the anuran (tail-less)amphibian Xenopus laevis and the teleost fish Danio rerio were thepredominant models employed and share common advantages: theability to easily produce a large numbers of free-living embryos, theiraccessibility during critical stages of development, and the absence ofcontinuous maternal hormonal influence on embryo and larvae devel-opment. Although the latter excludes maternal compensatory me-chanisms that can mask the effects of THADCs, this powerful advantagefor screening purposes comes at the expense of added difficulty forhuman health risk assessment.

3. Amphibian models for detecting THADCs

Besides their particular susceptibility to environmental pollutantsdue to their exposure through dermal, respiratory and dietary routes,amphibians have been extensively used as compelling vertebratemodels of thyroid function by virtue of their remarkable TH-dependantmetamorphosis – a process mimicking perinatal development in mam-mals and hatching in birds (Brown and Cai, 2007; Fini et al., 2012;Mengeling et al., 2018). During frog metamorphosis initiated by T3,various developmental processes are orchestrated spatio-temporally byTHs, including tail and gill resorption, limb growth, remodelling of theintestines, central nervous system, respiratory system, cranial carti-lages, and skin (Dodd and Dodd, 1976). Consequently, observation ofpotential interferences with these developmental processes in exposedtadpoles or in ex vivo organ cultures can reflect disturbances in the HPTaxis (Fu et al., 2018; Miyata and Ose, 2012; Taft et al., 2018; Yao et al.,2017). Strikingly, halting endogenous T3 synthesis by surgical removalof the thyroid gland or by chemical blockage of thyroid synthesis (e.g.,TPO inhibitors PTU and methimazole (MMI), or the NIS inhibitor per-chlorate) will inhibit metamorphosis. Conversely, pre-metamorphictadpoles can be induced to undergo precocious metamorphosis by theintroduction of exogenous THs or compounds that mimic TH activity(Opitz et al., 2005). On top of slight or profound metamorphic changes,histological analysis of amphibian thyroid glands can reinforce the in-dication of thyroid disrupting activity. For these reasons, the well-known Xenopus model was selected by both the US EPA and the OECDas a valuable and robust tool to assess the impact of potential EDCs onthe thyroid axis (Fig. 2), and has been used to evaluate multiple che-micals in both validated and alternative assays.

6 European Food Safety Authority.7 The French Agency for Food, Environmental and Occupational Health &

Safety (ANSES).

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Exposure periods and endpoints covered by the three validated(eco)toxicological assays employing Xenopus laevis are indicated withcorresponding developmental stage and approximate number of dayspost-fertilization. Levels of TH gradually rise until reaching a peak atmetamorphic climax at Nieuwkoop and Faber (NF) stage NF 62 andsubside at completion of metamorphosis at NF 66 (green gradient).

3.1. The amphibian metamorphosis assay (AMA)

The AMA is a validated and standardized assay in both the OECD

Test Guidelines (TG) (OECD TG 231, 2009) and the US EPA (US EPA TG809.1100, 2009). The assay involves exposing 20 Xenopus laevisNieuwkoop and Faber (NF) stage NF 51 tadpoles (17 days post-fertili-zation (dpf)) for 21 days to a test chemical using a flow-through system.After 7 and 21 days of exposure, determination of developmental stageand measurements of hindlimb length (HLL) inform on the rate ofmetamorphic development, in additional to snout-vent length (SVL)and body weight used as growth and health indicators. Delayed, ac-celerated or asynchronous development of any of the key features atday 7 and/or 21 (relative to the control group) could suggest thyroid

Fig. 1. Overview of the articles retrieved from the search query.

Fig. 2. Timeline of validated amphibian assays for detection of THADCs.

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disruption. In the absence of accelerated or asynchronous metamorphicdevelopment which are indicators of thyroid activity (asynchronousmetamorphic development may arise from disruption of peripheral THaction and/or metabolism in developing tissues), or in the case of de-velopmental delay potentially caused by stress or non-specific systemictoxicity, the AMA requires the evaluation of thyroid gland histology(OECD TG 231, 2009).

Diagnostic criteria of thyroid histopathology originate from AMAvalidation studies that were conducted using a wide range of chemicalsthat interact with the HPT axis through different mechanisms. Similarto mammalian thyroid histopathology, a semi-quantitative severitygrading is applied to the relative size of the thyroid gland, the numberand size of thyroid follicular cells, together with additional criteria suchas follicular lumen area, colloid quality, and follicular cell height andshape (Coady et al., 2010; Grim et al., 2009; OECD TG 231, 2009). Anychemical capable of adjusting TSH secretion will result in histopatho-logical changes of the thyroid gland, however the effects of TH an-tagonists (i.e., glandular hypertrophy due to follicular hypertrophyand/or hyperplasia) are more evident than those of weak agonists thatmay result in confounding advanced developmental stages (Miyata andOse, 2012; OECD TG 231, 2009). By comparing results from validationstudies, Dang et al. (2019) confirmed that thyroid histology is the mostsensitive endpoint for thyroid active chemicals which werepredominantly known antagonists, and only few thyroid inactive che-micals would change thyroid histology, reinforcing the value of thyroidhistopathology in reducing the number of false positive identification ofTHADCs. As outlined in a review by Pickford (2010), in the case ofthyroid agonists, developmental stage and hindlimb length are moresensitive and reliable. Importantly, using numerous reference THADCswith known MoA, a strong concordance was found without major dif-ferences in sensitivity between mammalian assays and amphibian me-tamorphosis assays (Pickford, 2010; Wegner et al., 2016). Moreover,none of the chemicals active in mammalian assays were negative inamphibians, highlighting the possibility to detect thyroid activity acrossthe vertebrate spectrum (OECD, 2018b).

The AMA uses Stage NF 51 tadpoles because it was assumed thattadpoles were functionally athyroid prior to the endogenous secretionof TH detected at stage NF 54 (26 dpf) yet metabolically competent andsensitive to exogenous TH as well as thyroid toxicants (Degitz et al.,2005; Y.-F. Zhang et al., 2019b). Evidence supporting the convenienceof using younger tadpoles in other assays for increased sensitivity andhigher-throughput have been described in studies indicating that ma-ternal THs are present in the egg yolk and start to rise at around NF 45,and that TH signalling may be functional during early embryogenesis(Fini et al., 2012; Morvan-Dubois et al., 2008). In order to optimize theAMA, Y.-F. Zhang et al. (2019b) compared results of NF 48 tadpolesexposed for 7, 21 & 28 days to the original AMA protocol without theuse of histopathological examination. Tadpoles at this earlier stagepossess the added benefit of being obtainable 10 days earlier (7 dpf)and are easily identifiable due to the appearance of forelimb buds. Thetreatment protocol covering a relatively longer part of the life cycle(due to the extra 10 days required for NF 48 tadpoles to reach NF 51)was more sensitive than the original AMA to reference thyroid an-tagonists MMI and perchlorate, when considering developmentalstages. Of particular importance for higher-throughput screening, whenusing smaller NF 48 tadpoles, only 7 days of exposure were needed toobserve significant development inhibition and at lower concentrationsthan were used in the original AMA after 7 days of exposure.

3.2. The larval amphibian growth and development assay (LAGDA)

Following the AMA, the OECD and the US EPA recently validatedand harmonized the LAGDA (OECD TG 241, 2015; US EPA 890.2300,2015). The LAGDA is considered a more sensitive and higher tier assayfor suspected thyroid-active chemicals to assess population relevanteffects. Covering multiple life-stages, beginning with early embryos (NF

8–15) and ending two months after completion of metamorphosis (NF66), this relatively long-term assay (lasting approximately 16 weeks)has the ability to confirm disruption of both the HPT axis at meta-morphic climax (NF 62) and of the hypothalamic-pituitary-gonadal axisat test termination, when gonads are fully differentiated. In addition torecording the time needed to reach NF 62, the LAGDA includes mea-surements plasma vitellogenin, histological examination of the thyroidgland and gonads, as well as liver and kidneys for detection of meta-bolic or systemic toxicity. While the LAGDA is not a life-cycle test dueto exclusion of the reproductive phase, it has the advantage of beingable to pry into potential gender differences and overlaps betweenseveral endocrine modalities. Using a wider range of concentrations,the LAGDA also has utility for risk assessment by providing a dose-response that can be derived to determine a No Observed Effect Con-centration (NOEC) for the measured endpoint (OECD TG 241, 2015).However, it also has the disadvantage of being relatively costly, timeconsuming, and requires large numbers of animals, water volumes andtest chemicals.

As such, validation of the LAGDA was performed using a limited setof EDCs in order to test the responsiveness of the assay to differentendocrine modalities (US EPA, 2013), such as the ultraviolet filterbenzophenone-2 (BP-2) which, in addition to documented estrogenicand anti-androgenic activity, has been shown to inhibit TPO in vivo andin vitro (Wang et al., 2016). Chronic exposure to BP-2 considerablyincreased levels of vitellogenin, caused sex-reversal of genotypic males,delayed gonad development of genotypic females, and increased theprevalence and severity of thyroid histopathological observations in adose-dependent manner, in line with BP-2's ability to inhibit THsynthesis (Haselman et al., 2016a, b). Unfortunately, neither referenceTH agonists nor antagonists were included in the validation process andfurthermore, neither are currently required as positive or negativecontrols in the test guidelines.

3.3. The Xenopus Eleutheroembryonic thyroid assay (XETA)

The XETA is the latest validated assay by the OECD (OECD TG 248,2019). Compared to the aforementioned assays, the XETA serves as arelatively short-term (72 h of exposure) and miniaturized test, parti-cularly suited for preliminary screening of a large number of chemicalsat various concentrations, while retaining the full spectrum of physio-logical relevance provided by in vivo analysis. Furthermore, eleuther-oembryos employed in the XETA (NF 45 to NF 48) still feed on theiryolk reserves and are therefore not considered as laboratory animals(Directive, 2010/63/EU, 2010), in compliance with the 3Rs principle.

This next-generation assay features a transgenic line of X. laevis, Tg(thibz:eGFP), which expresses the Green Fluorescent Protein (GFP)under the control of a portion of the regulatory region of TH/bZIP, aputative leucine zipper transcription factor highly sensitive to TH reg-ulation (Fini et al., 2007; Turque et al., 2005; Furlow and Brown, 1999).Exposure begins at stage NF 45 and is performed in 6-well plates ap-propriate for hosting 10 tadpoles per well, each containing only 8 mL ofthe test chemical with or without a T3 spike equivalent to the plasmaT3 concentration during tadpole metamorphosis (Leloup and Buscaglia,1977; OECD, 2018b). Daily renewal of the test chemical bypasses theneed of sophisticated flow-through systems absent in most laboratories,but analytical determination of the test chemical concentration is re-commended before and after each renewal to ascertain stability of thetest chemical and/or possible experimental errors (OECD TG 248,2019). The final endpoint is the quantification of the fluorescenceemitted by individual tadpoles carefully placed in 96-well plates, usingeither a fluorescent microscope or a fluorescence plate-reader. Thyroidhormone agonists will cause an increase in fluorescence with or withoutthe T3 spike, while chemicals acting as antagonists on the HPT axis arebest detected with the T3 spike and revealed by a decrease in fluores-cence compared to T3 alone (OECD, 2018b).

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3.4. Additional amphibian assays

Close to a third of the amphibian (eco)toxicology studies retrievedfrom our search query used the aforementioned validated assays. Whilethe majority mainly differ in exposure period and sometimes in themodel employed, many of the same endpoints were investigated toidentify potential THADCs (i.e., hindlimb length, snout-to-vent length,developmental stage and thyroid histology; Supplementary Table). Intadpoles, as morphological changes of the head region and the brain areinduced by THs, measurements such as mouth width, head area, brainwidth, brain length, or olfactory-organ-to-brain length may furthercharacterize disruption of TH-induced metamorphosis (Yao et al., 2017;Zhu et al., 2018; Mengeling et al., 2016, 2017). Several morphologicalendpoints were examined in conjunction with the readout of transgenicX. laevis tadpoles harbouring a luciferase reporter driven by TH/BZIPTREs (Mengeling et al., 2016, 2017). After 5 days of exposure to eitherTHs or the TR antagonist NH-3 at NF 48, a strong correlation was ob-served between measurements of brain width at the optic tectum, ol-factory-organ-to-brain length and luciferase activity. In particular,brain width at the optic tectum may be less sensitive to the confoundingeffects of general toxicity than other morphological parameters(Mengeling et al., 2017).

3.5. Additional endpoints in amphibian assays: gene transcription andbehaviour

In contrast to in vitro assays, the XETA provides neither an un-equivocal identification of the precise MoA of a chemical nor an asso-ciated apical endpoint relative to adverse outcomes as present in theAMA or in the more thorough LAGDA. As emphasised by Haselmanet al. (2016a, b) during validation of the LAGDA, a lack of endpoints forneurodevelopment or behaviour is apparent in all of these validatedassays. The refinement of these assays by integrating behaviouralendpoints and expression of key genes related to the HPT axis and/orgenes predictive of late adverse outcomes in neurodevelopment couldbe very valuable.

Morphological endpoints only provide indirect evidence of dis-rupted signalling since a series of molecular cascade events involved indevelopmental processes are triggered by THs (Shi et al., 2001). Genetranscription has been utilized to optimize T3-induced Xenopus meta-morphosis assays. After first sampling tadpole intestines which undergosignificant morphological and transcriptional variations during meta-morphosis (Yao et al., 2017), Wang et al. (2017) successfully sampledthe tail (a significantly easier tissue to dissect) for detection of alteredTH-responsive gene transcription. For example, in addition to effects onmorphological endpoints after 96h of exposure, the brominated flameretardant tetrabromobisphenol A (TBBPA) inhibited T3-induced ex-pression of key genes (e.g., trβ, th/bzip, krüppel-like factor 9 (klf9), anddio3) in the tail of NF 52 tadpoles after just 24h of exposure comparedto the T3 group.

The brain shows transcriptomic responsiveness to THs earlier thanthe tail, and with greater sensitivity (Yost et al., 2016). Subtle effects onTH-related and TH-responsive genes in whole brains of NF 48 tadpolescan be revealed following the XETA as detailed in a protocol bySpirhanzlova et al. (2018). Using this approach more pertinent to TH-mediated neurodevelopmental effects, Fini et al. (2017) reported that amixture of chemicals present in human amniotic fluid could induce adose-dependent increase of fluorescence in the XETA and modulate THsignalling genes (encoding TH transporters, and dio1, dio2). The mix-ture also downregulated genes implicated in neural differentiation(markers of pluripotency (sox 2), neuronal (tubb2) and oligodendrocyte(mbp) differentiation) and synaptic plasticity (brain derived neurotrophicfactor (bdnf)), and altered tadpole behaviour (assessed by motilitytracking under light/dark stimuli). Behavioural modifications assessedby motility tracking have been described in tadpoles in conjunctionwith TH disruption in other amphibian studies (Spirhanzlova et al.,

2019; W. Zhang et al., 2019a). Experimental techniques to examineneurobehavioural effects are available in Xenopus tadpoles (as reviewedby Pratt and Khakhalin, 2013) however, due to gaps in knowledge ofthe mechanistic pathways leading to behavioural responses, linkingaltered neurobehaviour to HPT dysfunction still remains a challenge inchemical testing and assessment.

In particular, as tadpoles at the stages employed in the XETA arefunctionally athyroid, inhibitors of TH synthesis (i.e., NIS or TPO in-hibitors) are not expected to be detected (OECD TG 248, 2019). Inlarger animals such as those used in the AMA or LAGDA, gene ex-pression in thyroid and pituitary tissue may complement effects onmetamorphosis-related endpoints (Lorenz et al., 2018). Using stage NF54 tadpoles characterized by the onset of thyroid gland-function, the invitro TPO inhibitor 2-Mercaptobenzothiazole was shown to upregulateNIS expression in the thyroid gland in a robust and sensitive mannerafter 7 days of exposure, consistent with others signs of classical HPTcompensatory responses (i.e., decreased T4 and elevated TSH levels inserum, and increased follicular cell hypertrophy (size) and hyperplasia(number) of the thyroid gland) (Tietge et al., 2013). Iodotyrosinedeiodinase (IYD) is a lesser known yet important enzyme for THsynthesis which may provide a substantial amount of recycled iodide tothe thyroid gland through deiodination of monoiodotyrosine (MIT) anddiiodotyrosine (DIT) (Rousset et al., 2015). After exposing tadpolesfrom stage NF 50 to 62 to an IYD inhibitor (3-nitro-L-tyrosine), Olkeret al. (2018) described effects similar to those incurred by NIS or TPOinhibitors. However, notable differences and specificities were ob-served, such as increased IYD mRNA expression in the thyroid gland,detection of MIT and DIT in plasma, and arrested development at a laterstage. As these effects could be rescued by iodine supplementation, IYDinhibition is a potential novel MoA for THADCs, especially relevant toenvironments with limited availability of iodine such as freshwaterecosystems.

4. Teleost fish models for identification of thyroid disruption

Teleost fish models are highly popular in developmental toxicityscreening and have been increasingly investigated in the context ofendocrine disruption, particularly along the hypothalamic-pituitary-gonadal (HPG) axis (Ankley and Johnson, 2004), and more recently forthe HPT axis. Indeed, while the roles of THs have historically been morethoroughly investigated in amphibians and mammals, the zebrafish(Danio rerio) is the predominant model employed in the articles re-trieved from our search query pertaining to non-mammalian modelsspanning the last 5 years (cf. Fig. 1). Remarkably, specific biomarkersfor thyroid disruption in teleost fishes have yet to be accepted forregulatory purposes due to discrepancies resulting from inconsistentexperimental parameters across a limited amount of studies usingknown TH disruptors, as emphasised by Spaan et al. (2019). However,given the relatively recent characterization of the zebrafish HPT axisand intrinsic advantages, the zebrafish is becoming a promising alter-native vertebrate model for thyroid signalling, amenable to high-throughput screening owing to their small size, ease of culture andshort generation time (Haggard et al., 2018; Kollitz et al., 2018; Marelliand Persani, 2017; Noyes et al., 2018; Sipes et al., 2011; Vancamp et al.,2018; Walter et al., 2019).

Tight regulation of circulating hormones by the HPT axis is wellconserved across vertebrates, and the structure and function of keycomponents TH signalling in teleosts closely resemble those of highervertebrates. Using zebrafish exposed to exogenous THs or PTU (with orwithout an mct8 morpholino) at 24, 72 and 120 h post-fertilization(hpf), Walter et al. (2019) confirmed that expression of key orthologgenes which mediate TH signalling in mammals appear to be co-ordinated by THs in D. Rerio. However, in teleosts, thyroid follicles (thefunctional unit of the gland) are dispersed along the ventral midline ofthe pharynx instead of being encapsulated in an organized pair ofthyroid glands (Alt et al., 2006), which therefore precludes the use of

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histopathological assessment as is employed in Xenopus and mammals.It remains that the structure and function of these follicles is conservedin vertebrates (Carr and Patiño, 2011), and the onset of thyroid de-velopment in teleosts resemble that of higher vertebrates, with keytranscription factors for thyroid follicular precursor cell specificationand differentiation (i.e., nkx2.1, pax2a, and hhex) having orthologs thatact similarly in mammals and amphibians (Elsalini et al., 2003; Porazziet al., 2009). As these precursors appear as early as 24 hpf, and maturethyroid follicles are identifiable 48 h later, the zebrafish has presented arelatively simple model of great utility for studying thyroid dysgenesis,growth and differentiation (Opitz et al., 2013).

Furthermore, due to the existence of a comprehensive genetictoolbox, characterization of knock-downs or knock-out of key TH sig-nalling genes in zebrafish have resulted in fruitful modelling and havecontributed to our understanding of thyroid-related human diseases,e.g., Allan-Herndon-Dudley syndrome and TH resistance syndrome dueto loss-of-function of the gene encoding Mct8 and TRs, respectively(Campinho et al., 2014; Heijlen et al., 2013; Marelli and Persani, 2017;Silva et al., 2017; Trubiroha et al., 2018; Vancamp et al., 2018; Zadaet al., 2017). Finally, expression profiles of genes involved in TH sig-nalling during multiple time points encompassed by a validated teleosttoxicity assay (Fish, Early-Life Stage Toxicity Test” (OECD TG210,2018a)) have recently been obtained and have shown to be relativelysimilar in both the zebrafish and the fathead minnow (Vergauwen et al.,2018). Together, these achievements may facilitate interpretations ofthe consequences of THADC exposure in teleosts and aid in paving theway towards the inclusion of thyroid-related endpoints in validatedassays.

4.1. Endpoints in fish models for identification of thyroid disruption

In contrast to amphibian assays, TH content in eggs, whole-bodylarvae or even circulating levels in adults (Chen et al., 2018; Wei et al.,2018; Yu et al., 2014) have routinely been measured in teleosts (50 outof 73) ecotoxicological studies retrieved). Additionally, a particularvulnerable time windows to THADCs such as the transition from theembryonic to larval stage occurs relatively rapidly, i.e. only 2–3 dpf inzebrafish (Walter et al., 2019), and metamorphosis (larva-to-juveniletransition) initiates after 2 weeks (Sharma et al., 2016). The subsequentgeneration may be obtained in 2–4 months to provide evidence oftransgenerational effects following THADC exposure, whilst also havingthe advantage of taking into account gender-specific effects (Chen et al.,2018; Cheng et al., 2017; Han et al., 2017; Jianjie et al., 2016; Wanget al., 2015; Yu et al., 2014; Zhao et al., 2016). These features representoutstanding advantages for addressing the long-term effects (intoadulthood and on offspring) of EDCs which is more pertinent to real lifeexposure scenarios consisting of chronic exposure to low doses of en-vironmental chemicals, and invaluable for potential studies on heritableEDC-induced epigenetic alterations of gene expression (Alavian-Ghavanini and Rüegg, 2018; Baker et al., 2014).

Expression levels of TH signalling genes are often analysed inwhole-body teleost larvae in conjunction with TH content followingexposure to potential THADCs. For instance, principle componentanalysis (PCA) demonstrated that transcription patterns of most THsignalling genes showed a strong correlation to altered TH levels fol-lowing exposure of zebrafish embryos to the synthetic pyrethroid pes-ticide, permethrin (Tu et al., 2016). Furthermore, mercury exposureresulted in elevated levels of whole-body TH, upregulation of genesinvolved in thyroid development (hhex, nkx2.1) and thyroid synthesis(nis and tg (thyroglobulin)) (Sun et al., 2018). Particularly, Walter et al.(2019) identified a paralog of dio3 (dio3-b) as being highly sensitiveduring early zebrafish development to both hyperthyroidism and hy-pothyroidism induced by exogenous THs or PTU, respectively. How-ever, TH-responsive genes important for neurodevelopment and fre-quently assessed in mammals and amphibian were found to berelatively unresponsive in whole larvae homogenates (e.g., bdnf, klf9),

while expression of myelin basic protein (mbp) was upregulated by TRagonists (i.e., exogenous T3 or T4) (Walter et al., 2019). Similarly,transcriptomic profiling of 25 EDCs highlighted a suite of transcriptsthat may serve to identify TR agonists, including the myelin-relatedgene plp1b (Haggard et al., 2018). Further studies are warranted asmyelin-related genes were also found to be downregulated followingtreatment of zebrafish to xenobiotics that reduced levels of THs (Miaoet al., 2015; Wang et al., 2015).

In zebrafish, although behavioural studies are frequent in tox-icological studies, and fundamental research has observed aberrantneurobehaviour in association with HPT dysfunction generated by loss-of-function of core components of the HPT axis (e.g., Dio2 (Houbrechtset al., 2016), and Mct8 (Zada et al., 2014, 2017), the underlying MoA ofaltered behaviour remain unclear, as in Xenopus. Interestingly, Mct8-deficient zebrafish display neurological impairment, in contrast to micewhich require the additional knock-out of OATP1C1 to replicate thebehavioural abnormalities of Allan-Herndon-Dudley syndrome (Mayerlet al., 2014; Zada et al., 2017, 2014). The reduced mobility followingtouch-stimuli that was observed in Mct8-deficient zebrafish may in-volve reduced synaptic density of motor neurons, the quantity of oli-godendrocytes as well as altered expression of myelin-related genes(Zada et al., 2014). In ecotoxicology, zebrafish embryos exposed to themetabolite of the pesticide pentachlorophenol had similar hyperthyroideffects to that of T3 treatment, with increased expression of synapsin I(Syn 1), implicated in synaptic plasticity and learning and memory,which may have repercussions on the timing and development of thebrain (Cheng et al., 2015). In an exhaustive study on the effects of theorganophosphate flame retardant TDCPP,8 3-month exposure of adultzebrafish was reported to decrease plasma THs in F0 females and THcontent in unexposed F1 eggs/larvae, consistent with the previouscorrelation between decreased T4 levels in humans and TDCPP con-centrations in household dust. Additional effects reported in F1 off-spring include: downregulated mRNA and protein expression related toneurodevelopment (i.e., mbp, α1-tubulin encoding a neuron specificmicrotubule protein involved in developing and regenerating brain, andsynapsin IIa, important for synaptogenesis and neurotransmitter re-lease), decreased levels of neurotransmitters (e.g. dopamine, serotonin,gamma amino butyric acid) and reduced larval mobility (Wang et al.,2015). However, a more thorough examination is needed to clarify towhat extent TH disruption contributes to these affected neurodevelop-mental endpoints.

Differentiating the neurodevelopmental effects mediated by endo-crine disruption from neurotoxicity remains challenging in behaviouraltests. Fraser et al. (2017) compared larval zebrafish behaviour afterembryonic exposure to a wide range of hormones and potential EDCsand concluded that, while T4 affected locomotion (i.e., increasedswimming speed and total distance travelled under light/dark stimuli),perchlorate had no effects (Fraser et al., 2017). Using multiple end-points, Fetter et al. (2015) reported that reduced mobility followingexposure to MMI could be restored by T4, however conclusions on theMoA requires complementary gene expression analysis due to con-founding craniofacial malformations capable of affecting mobility andmay arise from either altered TH signalling or unspecific toxicity. In-deed, the ontogenesis in fish with less pronounced transformations thanthe flatfish also involves TH (Rastorguev et al., 2016). Interestingly,pectoral fin length, which can also affect motility, has been identified asa highly TH-sensitive morphological marker in zebrafish (Sharma et al.,2016). Another endpoint related to swimming activity is eye develop-ment. When impaired by two THADCs acting via different MoA (PTUand a known TR antagonist, TBBPA), Baumann et al. (2016) observeddiminished visual performances as determined by optokinetic responseand reduced vision-based behaviours.

One physiological endpoint potentially associated to transcriptional

8 Tris (1, 3-dichloro-2-propyl) phosphate.

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alterations by THs that has been well studied is swim bladder inflation,which can be examined in the validated “Fish, Early-Life Stage ToxicityTest” (OECD TG210, 2018a). Inhibitors of TPO and DIO1/2 were shownto impair swim bladder inflation in zebrafish and the Fathead minnow(Nelson et al., 2016; Stinckens et al., 2016; Vergauwen et al., 2018), akey event which could be incorporated in an Adverse Outcome Pathway(AOP) framework in combination with first-tier in chemico assays fordetermination of molecular initiating events (Stinckens et al., 2018).Coiling behaviour, another neurological-based endpoint, has beenlinked to thyroid receptor-β deficiency (Xu et al., 2018). In addition,endpoints included in a General Developmental Score (GDS) have beenassociated with developmental toxicity caused by thyroid disruptors,including abnormal iridophore pigmentation, beat and glide swimmingand resorption of the yolk sac (Jomaa et al., 2014).

Finally, given the aforementioned conservation of thyroid devel-opment in teleosts, early detection of thyroid disruption is possibleusing whole-mount intrafollicular TH staining which could potentiallybe implemented into current OECD zebrafish assays (Rehberger et al.,2018). Interestingly, a novel fluorescent transgenic line expressionunder the control of the zebrafish thyroglobulin promoter allows for invivo monitoring of thyroid gland development. Thyroid follicular cellcounts either increase following TH treatment or decrease followingantagonists MMI and PTU (Fetter et al., 2015; Trubiroha et al., 2018),and at concentrations in line with those affecting gene expression,highlighting the potential use of a transgenic line for efficient screeningpurposes (Jarque et al., 2018).

5. Conclusion

Identification of TH disrupting compounds is challenging as theymay act at multiple levels of the thyroid axis. As highlighted in thisreview, disruption of this axis in non-mammalian models is over-whelmingly investigated in medium-throughput assays using D. rerio &X. laevis embryos and larvae. The prevalence of these models should notcome as a surprise in context of the increasing pressure for chemicalrisk assessment to incorporate sublethal effects such as endocrine dis-ruption for an ever-growing list of compounds. However, avian specieswere relatively under-represented in ecotoxicology research despite theexistence of specific social behaviour tests that may be applied tochicken hatchlings exposed as embryos (Haba et al., 2014).

The urgency to efficiently identify potential THADCs requires amore integrated approach and higher throughput, currently lacking inharmonized and validated tests. In conjunction with the development ofadditional endpoints such as those related to neurobehaviour in vivo,framing endpoints in AOPs using both in silico and in vitro assays mayincrease the rapidity and the efficiency of hazard identification andcharacterization. A recent article published after our literature searchprovides multiple AOPs in a network that connects disruption of theHPT axis in various validated vertebrates assays to several promising invitro high-throughput screening assays (Noyes et al., 2019).

Assessing the ecological impact of THADCs may only benefit fromadded taxonomic breadth. Besides X. laevis and D. rerio studies retrievedfrom our literature search, other amphibians (i.e., Xenopus tropicalis,Rana nigromaculata, Bufo gargarizans) and teleost fishes (i.e, goldfish,killifish, medaka, fathead minnow, Mozambique tilapia, flatfishes, coralfishes, Japanese flounder) have been used to evaluate the potentialeffects of chemicals on the HPT axis with many of the same endpointsdescribed previously (Supplementary Table). Another recent articlereviewed the effects of exposure to reference THADCs and a variety ofenvironmental contaminants on thyroid signalling in diverse anuranamphibians, including the effects of environmentally relevant complexmixture and abiotic stressors (Thambirajah et al., 2019).

Following a recent report on the impact of endocrine disruptors onhuman health published by Demeneix and SLAMA (2019), the Eur-opean Parliament urged the EU commission to ensure a higher level ofprotection for European citizens, and specifically, to consider potential

mixture effects more representative of actual exposure. Indeed, it islikely that disruption of the thyroid axis by environmental chemicals isunderestimated when combined exposure to multiple substances isneglected. Finally, as the prevalence of adverse neurological effects areexpected to increase globally (WHO, 2012), recognition of the healthhazards of thyroid disruption that may contribute to some of thesedisorders has recently been reiterated by the funding of three Europeanprojects (ATHENA, ERGO and SCREENED) that promote new teststrategies and develop specific endpoints in thyroid hormone signalling.

Acknowledgements

We thank Gérard Benisti for supplying the photos that were editedfor Fig. 2. We thank the MNHN and the CNRS for the annual fundingprovided to the unit. SC is a PHD student implicated in EU H2020HBM4EU (n° 733032). ML worked in EU H2020 project-EDC-MixRisk(n° 634880) and lately in ATHENA EU H20202 (n° 825161).

Appendix A. Supplementary data

Supplementary data related to this article can be found at https://doi.org/10.1016/j.mce.2020.110779.

References

Alavian-Ghavanini, A., Rüegg, J., 2018. Understanding epigenetic effects of endocrinedisrupting chemicals: from mechanisms to novel test methods. Basic Clin. Pharmacol.Toxicol. 122 (1), 38–45. https://doi.org/10.1111/bcpt.12878.

Alt, B., Reibe, S., Feitosa, N.M., Elsalini, O.A., Wendl, T., Rohr, K.B., 2006. Analysis oforigin and growth of the thyroid gland in zebrafish. Dev. Dynam. 235 (7),1872–1883. https://doi.org/10.1002/dvdy.20831.

Ankley, G.T., Johnson, R.D., 2004. Small fish models for identifying and assessing theeffects of endocrine-disrupting chemicals. ILAR J. 45 (4), 469–483. https://doi.org/10.1093/ilar.45.4.469.

Applegate, J.S., 2008. Synthesizing TSCA and REACH: practical principles for chemicalregulation reform. Maurer Faculty. https://doi.org/10.2139/ssrn.1183942. Paper438.

ATHENA - Assays for the Identification of Thyroid Hormone Axis-Disrupting Chemicals:Elaborating Novel Assessment Strategies | Projects | H2020 | CORDIS | EuropeanCommission. Retrieved February 10, 2020, from. https://cordis.europa.eu/project/rcn/219094/factsheet/en.

Baker, T.R., King-Heiden, T.C., Peterson, R.E., Heideman, W., 2014. Dioxin induction oftransgenerational inheritance of disease in zebrafish. Mol. Cell. Endocrinol. 398(1–2), 36–41. https://doi.org/10.1016/j.mce.2014.08.011.

Barrett, J.R., 2009. Endocrine disruption: developmental picture window. Environ.Health Perspect. 117 (3), A101. https://doi.org/10.1289/ehp.117-a101.

Bartalena, L., Robbins, J., 1993. Thyroid hormone transport proteins. Clin. Lab. Med. 13(3), 583–598. Retrieved from. http://www.ncbi.nlm.nih.gov/pubmed/8222576.

Baumann, L., Ros, A., Rehberger, K., Neuhauss, S.C.F., Segner, H., 2016. Thyroid dis-ruption in zebrafish (Danio rerio) larvae: different molecular response patterns leadto impaired eye development and visual functions. Aquat. Toxicol. 172, 44–55.https://doi.org/10.1016/j.aquatox.2015.12.015.

Bernal, J., 2005. Thyroid Hormones and Brain Development. https://doi.org/10.1016/S0083-6729(05)71004-9.

Bernal, J., 2007. Thyroid hormone receptors in brain development and function. Nat.Clin. Pract. Endocrinol. Metabol. 3 (3), 249–259. https://doi.org/10.1038/ncpendmet0424.

Bernanke, J., Köhler, H.R., 2009. The impact of environmental chemicals on wildlifevertebrates. Rev. Environ. Contam. Toxicol. 198, 1–47. https://doi.org/10.1007/978-0-387-09647-6_1.

Boas, M., Feldt-rasmussen, U., Main, K.M., 2012. Molecular and Cellular EndocrinologyThyroid effects of endocrine disrupting chemicals. Mol. Cell. Endocrinol. 355 (2),240–248. https://doi.org/10.1016/j.mce.2011.09.005.

Brown, D.D., Cai, L., 2007. Amphibian metamorphosis. Dev. Biol. 306 (1), 20–33. https://doi.org/10.1016/j.ydbio.2007.03.021.

Brucker-Davis, F., 1998. Effects of environmental synthetic chemicals on thyroid function.Thyroid: Official Journal of the American Thyroid Association 8 (9), 827–856.https://doi.org/10.1089/thy.1998.8.827.

Brüggemann, M., Licht, O., Fetter, É., Teigeler, M., Schäfers, C., Eilebrecht, E., 2018.Knotting nets: molecular junctions of interconnecting endocrine axes identified byapplication of the adverse outcome pathway concept. Environ. Toxicol. Chem. 37 (2),318–328. https://doi.org/10.1002/etc.3995.

Burden, N., Benstead, R., Clook, M., Doyle, I., Edwards, P., Maynard, S.K., et al., 2015.Advancing the 3Rs in Regulatory Ecotoxicology: A Pragmatic Cross-Sector Approach.https://doi.org/10.1002/ieam.1703.

Bus, J.S., 2017. “The dose makes the poison”: key implications for mode of action (me-chanistic) research in a 21st century toxicology paradigm. Current Opinion inToxicology 3, 87–91. https://doi.org/10.1016/j.cotox.2017.06.013.

S. Couderq, et al. Molecular and Cellular Endocrinology 508 (2020) 110779

8

Calsolaro, V., Pasqualetti, G., Niccolai, F., Caraccio, N., Monzani, F., 2017. Thyroid dis-rupting chemicals. Int. J. Mol. Sci. 18 (12), 2583. https://doi.org/10.3390/ijms18122583.

Campinho, M.A., Saraiva, J., Florindo, C., Power, D.M., 2014. Maternal thyroid hormonesare essential for neural development in zebrafish. Mol. Endocrinol. 28 (7),1136–1149. https://doi.org/10.1210/me.2014-1032.

Carr, J.A., Patiño, R., 2011. The hypothalamus-pituitary-thyroid axis in teleosts andamphibians: endocrine disruption and its consequences to natural populations. Gen.Comp. Endocrinol. 170 (2), 299–312. https://doi.org/10.1016/j.ygcen.2010.06.001.

Carson, R., 1962. Silent Spring. Houghton Mifflin.Centers for Disease Control and Prevention, 2019. Fourth Report on Human Exposure to

Environmental Chemicals, Updated Tables. Retrieved May 5, 2019, from. https://www.cdc.gov/exposurereport/.

Chen, L., Hu, C., Tsui, M.M.P., Wan, T., Peterson, D.R., Shi, Q., et al., 2018.Multigenerational disruption of the thyroid endocrine system in marine medaka aftera life-cycle exposure to perfluorobutanesulfonate. Environ. Sci. Technol. 52 (7),4432–4439. https://doi.org/10.1021/acs.est.8b00700.

Cheng, H., Yan, W., Wu, Q., Liu, C., Gong, X., Hung, T.C., Li, G., 2017. Parental exposureto microcystin-LR induced thyroid endocrine disruption in zebrafish offspring, atransgenerational toxicity. Environ. Pollut. 230, 981–988. https://doi.org/10.1016/j.envpol.2017.07.061.

Cheng, Y., Ekker, M., Chan, H.M., 2015. Relative developmental toxicities of penta-chloroanisole and pentachlorophenol in a zebrafish model (Danio rerio). Ecotoxicol.Environ. Saf. 112, 7–14. https://doi.org/10.1016/j.ecoenv.2014.10.004.

Choksi, N.Y., Jahnke, G.D., St Hilaire, C., Shelby, M., 2003. Role of thyroid hormones inhuman and laboratory animal reproductive health. Birth Defects Res B 68, 479–491.https://doi.org/10.1002/bdrb.10045.

Coady, K., Marino, T., Thomas, J., Currie, R., Hancock, G., Crofoot, J., et al., 2010.Evaluation of the amphibian metamorphosis assay: exposure to the goitrogen me-thimazole and the endogenous thyroid hormone L-thyroxine. Environ. Toxicol. Chem.29 (4), 869–880. https://doi.org/10.1002/etc.74.

Crofton, K.M., 2008. Thyroid disrupting chemicals: mechanisms and mixtures. Int. J.Androl. 31 (2), 209–223. https://doi.org/10.1111/j.1365-2605.2007.00857.x.

Dang, Z.C., 2019. Endpoint sensitivity in Amphibian metamorphosis assay. Ecotoxicol.Environ. Saf. 167, 513–519. https://doi.org/10.1016/j.ecoenv.2018.10.028.

De Groef, B., Grommen, S.V.H., Darras, V.M., 2013. Hatching the cleidoic egg: the role ofthyroid hormones. Front. Endocrinol. 4, 63. https://doi.org/10.3389/fendo.2013.00063.

De Groef, B., Van Der Geyten, S., Darras, V.M., Kühn, E.R., 2006. Role of corticotropin-releasing hormone as a thyrotropin-releasing factor in non-mammalian vertebrates.Gen. Comp. Endocrinol. 146, 62–68. https://doi.org/10.1016/j.ygcen.2005.10.014.

Degitz, S.J., Holcombe, G.W., Flynn, K.M., Kosian, P.A., Korte, J.J., Tietge, J.E., 2005.Progress towards development of an Amphibian-based thyroid screening assay usingXenopus laevis. Organismal and thyroidal responses to the model compounds 6-propylthiouracil, methimazole, and thyroxine. Toxicol. Sci. 87 (2), 353–364. https://doi.org/10.1093/toxsci/kfi246.

Demeneix, B., SLAMA, R., 2019. Endocrine Disruptors: from Scientific Evidence to HumanHealth. PETI Committee of the European Parliament Retrieved from. http://www.europarl.europa.eu/RegData/etudes/STUD/2019/608866/IPOL_STU(2019)608866_EN.pdf.

Dodd, M., Dodd, J., 1976. The Biology of Metamorphosis. Retrieved from. https://www.researchgate.net/publication/309456044_The_biology_of_metamorphosis.

Elsalini, O.A., Von Gartzen, J., Cramer, M., Rohr, K.B., 2003. Zebrafish hhex, nk2.1a, andpax 2.1 regulate thyroid growth and differentiation downstream of Nodal-dependenttranscription factors. Dev. Biol. 263 (1), 67–80. https://doi.org/10.1016/S0012-1606(03)00436-6.

Ergo - EndocRine Guideline Optimisation, 2019. Retrieved February 10, 2020, from.https://ergo-project.eu/.

Fetter, E., Baldauf, L., Da Fonte, D.F., Ortmann, J., Scholz, S., 2015. Comparative analysisof goitrogenic effects of phenylthiourea and methimazole in zebrafish embryos.Reprod. Toxicol. 57, 10–20. https://doi.org/10.1016/j.reprotox.2015.04.012.

Fini, J.-B., Le Mevel, S., Turque, N., Palmier, K., Zalko, D., Cravedi, J.-P., Demeneix, B.A.,2007. An in vivo multiwell-based fluorescent screen for monitoring vertebratethyroid hormone disruption. Environ. Sci. Technol. 41 (16), 5908–5914. Retrievedfrom. http://www.ncbi.nlm.nih.gov/pubmed/17874805.

Fini, J.B., Le Mével, S., Palmier, K., Darras, V.M., Punzon, I., Richardson, S.J., et al., 2012.Thyroid hormone signaling in the Xenopus laevis embryo is functional and suscep-tible to endocrine disruption. Endocrinology 153 (10), 5068–5081. https://doi.org/10.1210/en.2012-1463.

Fini, J., Mughal, B.B., Mével, S. Le, Leemans, M., Lettmann, M., Spirhanzlova, P., et al.,2017. Human Amniotic Fluid Contaminants Alter Thyroid Hormone Signalling andEarly Brain Development in Xenopus Embryos. Nature Publishing Group, pp. 1–12.https://doi.org/10.1038/srep43786. October 2016.

Fisher, J., Lumen, A., Latendresse, J., Mattie, D., 2012. Extrapolation of hypothalamic-pituitary-thyroid axis perturbations and associated toxicity in rodents to humans:case study with perchlorate. J. Environ. Sci. Health C Environ. Carcinog. Ecotoxicol.Rev. 30 (1), 81–105. https://doi.org/10.1080/10590501.2012.653889.

Fraser, T.W.K., Khezri, A., Lewandowska-Sabat, A.M., Henry, T., Ropstad, E., 2017.Endocrine disruptors affect larval zebrafish behavior: testing potential mechanismsand comparisons of behavioral sensitivity to alternative biomarkers. Aquat. Toxicol.193, 128–135. https://doi.org/10.1016/j.aquatox.2017.10.002.

Friedman, K.P., Watt, E.D., Hornung, M.W., Hedge, J.M., Judson, R.S., Crofton, K.M.,et al., 2016. Tiered high-throughput screening approach to identify thyroperoxidaseinhibitors within the toxcast phase I and II chemical libraries. Toxicol. Sci. 151 (1),160–180. https://doi.org/10.1093/toxsci/kfw034.

Fu, L., Wen, L., Shi, Y.-B., 2018. Role of Thyroid Hormone Receptor in Amphibian

Development. https://doi.org/10.1007/978-1-4939-7902-8_20.Fuhrman, V.F., Tal, A., Arnon, S., 2015. Why endocrine disrupting chemicals (EDCs)

challenge traditional risk assessment and how to respond. J. Hazard Mater. 286,589–611. https://doi.org/10.1016/j.jhazmat.2014.12.012.

Furlow, J.D., Brown, D.D., 1999. In vitro and in vivo analysis of the regulation of atranscription factor gene by thyroid hormone during Xenopus laevis metamorphosis.Mol. Endocrinol. 13 (12), 2076–2089. https://doi.org/10.1210/mend.13.12.0383.

Galton, V.A., 1992. The role of thyroid hormone in amphibian metamorphosis. TrendsEndocrinol. Metabol.: TEM (Trends Endocrinol. Metab.) 3 (3), 96–100. Retrievedfrom. http://www.ncbi.nlm.nih.gov/pubmed/18407086.

Ghassabian, A., Trasande, L., 2018. Disruption in thyroid signaling pathway: a me-chanism for the effect of endocrine-disrupting chemicals on child neurodevelopment.Front. Endocrinol. 9. https://doi.org/10.3389/fendo.2018.00204.

Grim, K.C., Wolfe, M., Braunbeck, T., Iguchi, T., Ohta, Y., Tooi, O., et al., 2009. Thyroidhistopathology assessments for the Amphibian metamorphosis assay to detectthyroid-active substances. Toxicol. Pathol. 37 (4), 415–424. https://doi.org/10.1177/0192623309335063.

Gross, M., Green, R.M., Weltje, L., Wheeler, J.R., 2017. Weight of evidence approaches forthe identification of endocrine disrupting properties of chemicals: review and re-commendations for EU regulatory application. Regul. Toxicol. Pharmacol. 91, 20–28.https://doi.org/10.1016/j.yrtph.2017.10.004.

Haba, G., Nishigori, H., Sasaki, M., Tohyama, K., Kudo, K., Matsumura, Y., et al., 2014.Altered magnetic resonance images of brain and social behaviors of hatchling, andexpression of thyroid hormone receptor βmRNA in cerebellum of embryos afterMethimazole administration. Psychopharmacology 231 (1), 221–230. https://doi.org/10.1007/s00213-013-3229-z.

Hagenbuch, B., 2007. Cellular entry of thyroid hormones by organic anion transportingpolypeptides. Best Pract. Res. Clin. Endocrinol. Metabol. 21 (2), 209–221. https://doi.org/10.1016/j.beem.2007.03.004.

Haggard, D.E., Noyes, P.D., Waters, K.M., Tanguay, R.L., 2018. Transcriptomic andphenotypic profiling in developing zebrafish exposed to thyroid hormone receptoragonists. Reprod. Toxicol. 77, 80–93. https://doi.org/10.1016/j.reprotox.2018.02.006.

Han, Z., Li, Y., Zhang, S., Song, N., Xu, H., Dang, Y., et al., 2017. Prenatal transfer ofdecabromodiphenyl ether (BDE-209) results in disruption of the thyroid system anddevelopmental toxicity in zebrafish offspring. Aquat. Toxicol. 190, 46–52. https://doi.org/10.1016/j.aquatox.2017.06.020.

Haselman, J.T., Kosian, P.A., Korte, J.J., Olmstead, A.W., Iguchi, T., Johnson, R.D.,Degitz, S.J., 2016a. Development of the Larval Amphibian Growth and DevelopmentAssay: effects of chronic 4- tert -octylphenol or 17β-trenbolone exposure in Xenopuslaevis from embryo to juvenile. J. Appl. Toxicol. 36 (12), 1639–1650. https://doi.org/10.1002/jat.3330.

Haselman, J.T., Sakurai, M., Watanabe, N., Goto, Y., Onishi, Y., Ito, Y., et al., 2016b.Development of the larval Amphibian growth and development assay: effects ofbenzophenone-2 exposure in Xenopus laevis from embryo to juvenile. J. Appl. Toxicol.36 (12), 1651–1661. https://doi.org/10.1002/jat.3336.

Heijlen, M., Houbrechts, A.M., Darras, V.M., 2013. Zebrafish as a model to study per-ipheral thyroid hormone metabolism in vertebrate development. Gen. Comp.Endocrinol. 188, 289–296. https://doi.org/10.1016/j.ygcen.2013.04.004.

Herbst, A.L., Scully, R.E., 1970. Adenocarcinoma of the vagina in adolescence.A report of7 cases including 6 clear-cell carcinomas (so-called mesonephromas). Cancer 25 (4),745–757. https://doi.org/10.1002/1097-0142(197004)25:4<745::AID-CNCR2820250402>3.0.CO;2-2.

Holzer, G., Laudet, V., 2013. Thyroid hormones and postembryonic development inamniotes. In: Current Topics in Developmental Biology, vol. 103. pp. 397–425.https://doi.org/10.1016/B978-0-12-385979-2.00014-9.

Hönes, G.S., Rakov, H., Logan, J., Liao, X.-H., Werbenko, E., Pollard, A.S., et al., 2017.Noncanonical thyroid hormone signaling mediates cardiometabolic effects in vivo.Proc. Natl. Acad. Sci. Unit. States Am. 114 (52), E11323–E11332. https://doi.org/10.1073/pnas.1706801115.

Horn, S., Heuer, H., 2010. Thyroid hormone action during brain development: morequestions than answers. Mol. Cell. Endocrinol. 315 (1–2), 19–26. https://doi.org/10.1016/j.mce.2009.09.008.

Houbrechts, A.M., Delarue, J., Gabriëls, I.J., Sourbron, J., Darras, V.M., 2016. Permanentdeiodinase type 2 deficiency strongly perturbs zebrafish development, growth, andfertility. Endocrinology 157 (9), 3668–3681. https://doi.org/10.1210/en.2016-1077.

Howdeshell, K.L., 2002. A model of the development of the brain as a construct of thethyroid system developing a timeline model of thyroid system and brain. Environ.Health Perspect. 110 (Suppl. 3), 337–348.

International Council of Chemical Associations (Icca) and the United NationsEnvironment Programme (Unep), 2019. Submission of the International Council ofChemical Associations (ICCA) and the United Nations Environment Programme(UNEP): Knowledge Management and Information Sharing for the SoundManagement of Industrial Chemicals. Retrieved from. http://www.saicm.org/Portals/12/Documents/meetings/OEWG3/inf/OEWG3-INF-28-KMI.pdf.

Jarque, S., Fetter, E., Veneman, W.J., Spaink, H.P., Peravali, R., Strähle, U., Scholz, S.,2018. An automated screening method for detecting compounds with goitrogenicactivity using transgenic zebrafish embryos. PloS One 13 (8), e0203087. https://doi.org/10.1371/journal.pone.0203087.

Jianjie, C., Wenjuan, X., Jinling, C., Jie, S., Ruhui, J., Meiyan, L., 2016. Fluoride causedthyroid endocrine disruption in male zebrafish (Danio rerio). Aquat. Toxicol. 171,48–58. https://doi.org/10.1016/j.aquatox.2015.12.010.

Jomaa, B., 2015. Thyroid in a Jar: towards an Integrated in Vitro Testing Strategy forThyroid-Active Compounds. Wageningen University, Wageningen, pp. 187 2015.

Jomaa, B., Hermsen, S.A.B., Kessels, M.Y., Van Den Berg, J.H.J., Peijnenburg, A.A.C.M.,Aarts, J.M.M.J.G., et al., 2014. Developmental toxicity of thyroid-active compounds

S. Couderq, et al. Molecular and Cellular Endocrinology 508 (2020) 110779

9

in a zebrafish embryotoxicity test. ALTEX 31 (3), 303–317. https://doi.org/10.14573/altex.1402011.

Karthikeyan, B.S., Ravichandran, J., Mohanraj, K., Vivek-Ananth, R.P., Samal, A., 2019. Acurated knowledgebase on endocrine disrupting chemicals and their biological sys-tems-level perturbations. Sci. Total Environ. 692, 281–296. https://doi.org/10.1016/j.scitotenv.2019.07.225.

Kiyama, R., 2017. Endocrine disruptor actions through receptor crosstalk. EnvironmentalBiotechnology 12 (1), 1–16. https://doi.org/10.14799/ebms262.

Kollitz, E.M., De Carbonnel, L., Stapleton, H.M., Lee Ferguson, P., 2018. The affinity ofbrominated phenolic compounds for human and zebrafish thyroid receptor β: influ-ence of chemical structure. Toxicol. Sci. 163 (1), 226–239. https://doi.org/10.1093/toxsci/kfy028.

Korevaar, T.I.M., Muetzel, R., Medici, M., Chaker, L., Jaddoe, V.W.V., de Rijke, Y.B.,et al., 2016. Association of maternal thyroid function during early pregnancy withoffspring IQ and brain morphology in childhood: a population-based prospectivecohort study. The Lancet Diabetes and Endocrinology 4 (1), 35–43. https://doi.org/10.1016/S2213-8587(15)00327-7.

Kortenkamp, A., Martin, O., Baynes, A., Silva, E., Axelstad, M., Hass, U., 2017. Supportingthe Organisation of a Workshop on Thyroid Disruption - Final Report. https://doi.org/10.2779/921523.

Koulouri, O., Moran, C., Halsall, D., Chatterjee, K., Gurnell, M., 2013. Pitfalls in themeasurement and interpretation of thyroid function tests. Best Pract. Res. Clin.Endocrinol. Metabol. 27 (6), 745–762. https://doi.org/10.1016/j.beem.2013.10.003.

Krimsky, S., 2017. The unsteady state and inertia of chemical regulation under the USToxic Substances Control Act. PLoS Biol. 15 (12), e2002404. https://doi.org/10.1371/journal.pbio.2002404.

Kuyl, J.M., 2015. The evolution of thyroid function tests. J. Endocrinol. Metabol. DiabetesS. Afr. 20 (2), 11–16. https://doi.org/10.1080/16089677.2015.1056468. Retrievedfrom. http://search.ebscohost.com/login.aspx?direct=true&db=a9h&AN=109154233&site=ehost-live.

Lee, D.H., Jacobs, D.R., 2015. Methodological issues in human studies of endocrine dis-rupting chemicals. Rev. Endocr. Metab. Disord. 16, 289–297. https://doi.org/10.1007/s11154-016-9340-9.

Leloup, J., Buscaglia, M., 1977. La triiodothyronine, hormone de la metamorphose desAmphibiens. C. R. Acad. Sci. Paris, D 284, 2261–2263. Retrieved from. https://ci.nii.ac.jp/naid/10027773429/.

Lorenz, C., Krüger, A., Schöning, V., Lutz, I., 2018. The progestin norethisterone affectsthyroid hormone-dependent metamorphosis of Xenopus laevis tadpoles at en-vironmentally relevant concentrations. Ecotoxicol. Environ. Saf. 150, 86–95. https://doi.org/10.1016/j.ecoenv.2017.12.022.

Manibusan, M.K., Touart, L.W., 2017. A comprehensive review of regulatory test methodsfor endocrine adverse health effects. Crit. Rev. Toxicol. 47 (6), 440–488. https://doi.org/10.1080/10408444.2016.1272095.

Marelli, F., Persani, L., 2017. How zebrafish research has helped in understanding thyroiddiseases. F1000Research 6, 2137. https://doi.org/10.12688/f1000research.12142.1.

Mayerl, S., Müller, J., Bauer, R., Richert, S., Kassmann, C.M., Darras, V.M., et al., 2014.Transporters MCT8 and OATP1C1 maintain murine brain thyroid hormone home-ostasis. J. Clin. Invest. 124 (5), 1987–1999. https://doi.org/10.1172/JCI70324.

McMenamin, S.K., Parichy, D.M., 2013. Metamorphosis in Teleosts. https://doi.org/10.1016/B978-0-12-385979-2.00005-8.

Mengeling, B.J., Goodson, M.L., Furlow, J.D., 2018. RXR ligands modulate thyroid hor-mone signaling competence in young xenopus laevis tadpoles. Endocrinology 159 (7),2576–2595. https://doi.org/10.1210/en.2018-00172.

Mengeling, B.J., Murk, A.J., Furlow, J.D., 2016. Trialkyltin rexinoid-X receptor agonistsselectively potentiate thyroid hormone induced programs of Xenopus laevis meta-morphosis. Endocrinology 157 (7), 2712–2723. https://doi.org/10.1210/en.2016-1062.

Mengeling, B.J., Wei, Y., Dobrawa, L.N., Streekstra, M., Louisse, J., Singh, V., et al., 2017.A multi-tiered, in vivo, quantitative assay suite for environmental disruptors ofthyroid hormone signaling. Aquat. Toxicol. 190, 1–10. https://doi.org/10.1016/j.aquatox.2017.06.019.

Miao, W., Zhu, B., Xiao, X., Li, Y., Dirbaba, N.B., Zhou, B., Wu, H., 2015. Effects of ti-tanium dioxide nanoparticles on lead bioconcentration and toxicity on thyroid en-docrine system and neuronal development in zebrafish larvae. Aquat. Toxicol. 161,117–126. https://doi.org/10.1016/j.aquatox.2015.02.002.

Miyata, K., Ose, K., 2012. Thyroid hormone-disrupting effects and the Amphibian me-tamorphosis assay. J. Toxicol. Pathol. 25 (1), 1–9. https://doi.org/10.1293/tox.25.1.

Moog, N.K., Entringer, S., Heim, C., Wadhwa, P.D., Kathmann, N., Buss, C., 2017.Influence of maternal thyroid hormones during gestation on fetal brain development.Neuroscience 342, 68–100. https://doi.org/10.1016/j.neuroscience.2015.09.070.

Morvan-Dubois, G., Demeneix, B.A., Sachs, L.M., 2008. Xenopus laevis as a model forstudying thyroid hormone signalling: from development to metamorphosis. Mol. Cell.Endocrinol. 293 (1–2), 71–79. https://doi.org/10.1016/j.mce.2008.06.012.

Mughal, B.B., Fini, J.-B., Demeneix, B.A., 2018. Thyroid-disrupting chemicals and braindevelopment: an update. Endocrine Connections 7 (4), R160–R186. https://doi.org/10.1530/ec-18-0029.

Nelson, K.R., Schroeder, A.L., Ankley, G.T., Blackwell, B.R., Blanksma, C., Degitz, S.J.,et al., 2016. Impaired anterior swim bladder inflation following exposure to thethyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: fathead minnow. Aquat.Toxicol. 173, 192–203. https://doi.org/10.1016/j.aquatox.2015.12.024.

Noyes, P.D., Friedman, K.P., Browne, P., Haselman, J.T., Gilbert, M.E., Hornung, M.W.,et al., 2019. Evaluating chemicals for thyroid disruption: Opportunities and chal-lenges with in Vitro testing and adverse outcome pathway approaches. Environ.Health Perspect. 127. https://doi.org/10.1289/EHP5297.

Noyes, P.D., Garcia, G.R., Tanguay, R.L., 2018. Advances in the use of zebrafish in de-velopmental toxicology: linking genetics, behavior, and high-throughput testing

strategies. In: Comprehensive Toxicology, vols. 5–15. Third Edit, pp. 298–326.https://doi.org/10.1016/B978-0-12-801238-3.64294-0.

Organisation for Economic Co-operation and Development, 2009. TG 231: AmphibianMetamorphosis Assay. AMA)https://doi.org/10.1787/9789264076242-en.

Organisation for Economic Co-operation and Development, 2015. TG 241: the LarvalAmphibian Growth and Development Assay (LAGDA). https://doi.org/10.1787/9789264242340-en.

Organisation for Economic Co-operation and Development, 2017. New ScopingDocument on in Vitro and Ex Vivo Assays for the Identification of Modulators ofThyroid Hormone Signalling. https://doi.org/10.1787/9789264274716-en.

Organisation for Economic Co-operation and Development, 2018a. TG 210: Fish, Early-Life Stage (FELS) Toxicity Test. https://doi.org/10.1787/9789264304741-13-en.

Organisation for Economic Co-operation and Development, 2018b. Revised GuidanceDocument 150 on Standardised Test Guidelines for Evaluating Chemicals forEndocrine Disruption. https://doi.org/10.1787/9789264304741-en.

Organisation for Economic Co-operation and Development, 2019. TG 248: XenopusEleutheroembryonic Thyroid Assay (XETA). https://doi.org/10.1787/a13f80ee-en.

Oliveira, K.J., Chiamolera, M.I., Giannocco, G., Pazos-Moura, C.C., Ortiga-Carvalho, T.M.,2018. Thyroid function disruptors: from nature to chemicals. J. Mol. Endocrinol. 62(1). https://doi.org/10.1530/JME-18-0081.

Olker, J.H., Haselman, J.T., Kosian, P.A., Donnay, K.G., Korte, J.J., Blanksma, C., et al.,2018. Evaluating iodide recycling inhibition as a novel molecular initiating event forthyroid Axis disruption in Amphibians. Toxicol. Sci.: An Official Journal of theSociety of Toxicology 166 (2), 318–331. https://doi.org/10.1093/toxsci/kfy203.

Opitz, R., Antonica, F., Costagliola, S., 2013. New model systems to illuminate thyroidorganogenesis. Part I: an update on the zebrafish toolbox. European Thyroid Journal2 (4), 229–242. https://doi.org/10.1159/000357079.

Opitz, R., Braunbeck, T., Bögi, C., Pickford, D.B., Nentwig, G., Oehlmann, J., et al., 2005.Description and initial evaluation of a Xenopus metamorphosis assay for detection ofthyroid system-disrupting activities of environmental compounds. Environ. Toxicol.Chem. 24 (3), 653–664. Retrieved from. http://www.ncbi.nlm.nih.gov/pubmed/15779766.

Pickford, D.B., 2010. Screening chemicals for thyroid-disrupting activity: a critical com-parison of mammalian and amphibian models. Crit. Rev. Toxicol. 40 (10), 845–892.https://doi.org/10.3109/10408444.2010.494250.

Porazzi, P., Calebiro, D., Benato, F., Tiso, N., Persani, L., 2009. Thyroid gland develop-ment and function in the zebrafish model. Mol. Cell. Endocrinol. 312, 14–23. https://doi.org/10.1016/j.mce.2009.05.011.

Pratt, K.G., Khakhalin, A.S., 2013. Modeling human neurodevelopmental disorders in theXenopus tadpole: from mechanisms to therapeutic targets. DMM Disease Models andMechanisms 6, 1057–1065. https://doi.org/10.1242/dmm.012138.

Prezioso, G., Giannini, C., Chiarelli, F., 2018. Effect of thyroid hormones on neurons andneurodevelopment. Hormone Research in Paediatrics 90 (2), 73–81. https://doi.org/10.1159/000492129.

Rastorguev, S.M., Nedoluzhko, A.V., Levina, M.A., Prokhorchuk, E.B., Skryabin, K.G.,Levin, B.A., 2016. Pleiotropic effect of thyroid hormones on gene expression in fish asexemplified from the blue bream Ballerus ballerus (Cyprinidae): results of tran-scriptomic analysis. Dokl. Biochem. Biophys. 467 (1), 124–127. https://doi.org/10.1134/S1607672916020137.

Refetoff, S., 2000. Thyroid hormone serum transport proteins. In: Endotext, Retrievedfrom. http://www.ncbi.nlm.nih.gov/pubmed/25905421.

Rehberger, K., Baumann, L., Hecker, M., Braunbeck, T., 2018. Intrafollicular thyroidhormone staining in whole-mount zebrafish (Danio rerio) embryos for the detectionof thyroid hormone synthesis disruption. Fish Physiol. Biochem. 44 (3), 997–1010.https://doi.org/10.1007/s10695-018-0488-y.

Rousset, B., Dupuy, C., Miot, F., Dumont, J., 2015. Chapter 2 thyroid hormone synthesisand secretion. In: Endotext, Retrieved from. http://www.ncbi.nlm.nih.gov/pubmed/25905405.

SCREENED - A Multistage Model of Thyroid Gland Function for Screening Endocrine-Disrupting Chemicals in a Biologically Sex-specific Manner | Projects | H2020 |CORDIS | European Commission. Retrieved February 10, 2020, from. https://cordis.europa.eu/project/rcn/219710/factsheet/en.

Sellitti, D.F., Suzuki, K., 2013. Intrinsic regulation of thyroid function by thyroglobulin.Thyroid 24 (4), 625–638. https://doi.org/10.1089/thy.2013.0344.

Sharma, P., Grabowski, T.B., Patiño, R., 2016. Thyroid endocrine disruption and externalbody morphology of Zebrafish. Gen. Comp. Endocrinol. 226, 42–49. https://doi.org/10.1016/J.YGCEN.2015.12.023.

Sharma, R.P., Schuhmacher, M., Kumar, V., 2017. Review on crosstalk and commonmechanisms of endocrine disruptors: Scaffolding to improve PBPK/PD model of EDCmixture. Environ. Int. 99, 1–14. https://doi.org/10.1016/j.envint.2016.09.016.

Shi, Y.B., Fu, L., Hsia, S.C.V., Tomita, A., Buchholz, D., 2001. Thyroid hormone regulationof apoptotic tissue remodeling during anuran metamorphosis. Cell Res. 11, 245–252.https://doi.org/10.1038/sj.cr.7290093.

Silva, N., Louro, B., Trindade, M., Power, D.M., Campinho, M.A., 2017. Transcriptomicsreveal an integrative role for maternal thyroid hormones during zebrafish embry-ogenesis. Sci. Rep. 7 (1). https://doi.org/10.1038/s41598-017-16951-9.

Sipes, N.S., Padilla, S., Knudsen, T.B., 2011. Zebrafish-As an integrative model for twenty-first century toxicity testing. Birth Defects Res. Part C Embryo Today - Rev. 93 (3),256–267. https://doi.org/10.1002/bdrc.20214.

Slama, R., Vernet, C., Nassan, F.L., Hauser, R., Philippat, C., 2017. Characterizing theeffect of endocrine disruptors on human health: the role of epidemiological cohorts.Comptes Rendus Biol. 340 (9–10), 421–431. https://doi.org/10.1016/j.crvi.2017.07.008.

Spaan, K., Haigis, A.-C., Weiss, J., Legradi, J., 2019. Effects of 25 thyroid hormone dis-ruptors on zebrafish embryos: a literature review of potential biomarkers. Sci. TotalEnviron. 656, 1238–1249. https://doi.org/10.1016/j.scitotenv.2018.11.071.

S. Couderq, et al. Molecular and Cellular Endocrinology 508 (2020) 110779

10

Spencer, C.A., 2000. Assay of thyroid hormones and related substances. In: Endotext,Retrieved from. http://www.ncbi.nlm.nih.gov/pubmed/25905337.

Spirhanzlova, P., Fini, J.-B., Demeneix, B., Lardy-Fontan, S., Vaslin-Reimann, S., Lalere,B., Krief, S., 2019. Composition and endocrine effects of water collected in the Kibalenational park in Uganda. Environ. Pollut. 251, 460–468. https://doi.org/10.1016/j.envpol.2019.05.0 06.

Spirhanzlova, P., Leemans, M., Demeneix, B.A., Fini, J.-B., 2018. Following endocrine-disrupting effects on gene expression in Xenopus laevis. Cold Spring Harb. Protoc.(7), 526–537. https://doi.org/10.1101/pdb.prot098301.

Stinckens, E., Vergauwen, L., Ankley, G.T., Blust, R., Darras, V.M., Villeneuve, D.L., et al.,2018. An AOP-based alternative testing strategy to predict the impact of thyroidhormone disruption on swim bladder inflation in zebrafish. Aquat. Toxicol. 200,1–12. https://doi.org/10.1016/j.aquatox.2018.04.009.

Stinckens, E., Vergauwen, L., Schroeder, A.L., Maho, W., Blackwell, B.R., Witters, H.,et al., 2016. Impaired anterior swim bladder inflation following exposure to thethyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish. Aquat.Toxicol. 173, 204–217. https://doi.org/10.1016/j.aquatox.2015.12.023.

Sun, Y., Li, Y., Liu, Z., Chen, Q., 2018. Environmentally relevant concentrations of mer-cury exposure alter thyroid hormone levels and gene expression in the hypothala-mic–pituitary–thyroid axis of zebrafish larvae. Fish Physiol. Biochem. 44 (4),1175–1183. https://doi.org/10.1007/s10695-018-0504-2.

Taft, J.D., Colonnetta, M.M., Schafer, R.E., Plick, N., Powell, W.H., 2018. Dioxin exposurealters molecular and morphological responses to thyroid hormone in Xenopus laeviscultured cells and prometamorphic tadpoles. Toxicol. Sci. 161 (1), 196. https://doi.org/10.1093/TOXSCI/KFX213.

Tannenbaum, J., Bennett, B.T., 2015. Russell and Burch's 3Rs then and now: the need forclarity in definition and purpose. JAALAS: JAALAS 54 (2), 120–132. Retrieved from.http://www.ncbi.nlm.nih.gov/pubmed/25836957.

Taylor, E., Heyland, A., 2017. Evolution of thyroid hormone signaling in animals: non-genomic and genomic modes of action. Mol. Cell. Endocrinol. 459, 14–20. https://doi.org/10.1016/j.mce.2017.05.019.

Thambirajah, A.A., Koide, E.M., Imbery, J.J., Helbing, C.C., 2019. Contaminant and en-vironmental influences on thyroid hormone action in amphibian metamorphosis.Front. Endocrinol. 10. https://doi.org/10.3389/fendo.2019.00276.

The European parliament and the Council of the European Union, 2010. Directive 2010/63/on the Protection of Animals Used for Scientific Purposes. Retrieved from.http://ec.europa.eu/environment/chemicals/lab_animals/pdf/2010_63.pdf.

Tietge, J.E., Degitz, S.J., Haselman, J.T., Butterworth, B.C., Korte, J.J., Kosian, P.A., et al.,2013. Inhibition of the thyroid hormone pathway in Xenopus laevis by 2-mercapto-benzothiazole. Aquat. Toxicol. 126, 128–136. https://doi.org/10.1016/j.aquatox.2012.10.013.

Trubiroha, A., Gillotay, P., Giusti, N., Gacquer, D., Libert, F., Lefort, A., et al., 2018. Arapid CRISPR/Cas-based mutagenesis assay in zebrafish for identification of genesinvolved in thyroid morphogenesis and function. Sci. Rep. 8 (1), 5647. https://doi.org/10.1038/s41598-018-24036-4.

Tu, W., Xu, C., Jin, Y., Lu, B., Lin, C., Wu, Y., Liu, W., 2016. Permethrin is a potentialthyroid-disrupting chemical: in vivo and in silico evidence. Aquat. Toxicol. 175,39–46. https://doi.org/10.1016/j.aquatox.2016.03.006. Amsterdam, Netherlands.

Turque, N., Palmier, K., Le Mével, S., Alliot, C., Demeneix, B.A., 2005. A rapid, physio-logic protocol for testing transcriptional effects of thyroid-disrupting agents in pre-metamorphic Xenopus tadpoles. Environ. Health Perspect. 113 (11), 1588–1593.https://doi.org/10.1289/ehp.7992.

U.S. Environmental Protection Agency, 2013. Validation of the Larval Amphibian Growthand Development Assay: Integrated Summary Report.

U.S. Environmental Protection Agency, 2009. Endocrine Disruptor Screening ProgramTest Guidelines - OPPTS 890.1100: Amphibian Metamorphosis. (Frog) [EPA 740-C-09-002]. Retrieved from. https://www.regulations.gov/document?D=EPA-HQ-OPPT-2009-0576-0002.

U.S. Environmental Protection Agency, 2014. Endocrine Disruptor Screening ProgramComprehensive Management Plan. Retrieved from. https://www.epa.gov/sites/production/files/2015-08/documents/edsp_comprehesive_management_plan_021414_f.pdf.

U.S. Environmental Protection Agency, 2015. Endocrine Disruptor Screening ProgramTest Guidelines 890.2300: Larval Amphibian Growth and Development Assay(LAGDA) [EPA Pub No. 740-C-15-001]. Retrieved from. https://www.regulations.gov/document?D=EPA-HQ-OPPT-2009-0576-0018.

van der Deure, W.M., Peeters, R.P., Visser, T.J., 2010. Molecular aspects of thyroidhormone transporters, including MCT8, MCT10, and OATPs, and the effects of ge-netic variation in these transporters. J. Mol. Endocrinol. 44 (1), 1–11. https://doi.org/10.1677/JME-09-0042.

Vancamp, P., Houbrechts, A.M., Darras, V.M., 2018. Insights from zebrafish deficiencymodels to understand the impact of local thyroid hormone regulator action on earlydevelopment. Gen. Comp. Endocrinol. 279, 45–52. https://doi.org/10.1016/j.ygcen.2018.09.011.

Vandenberg, L.N., Colborn, T., Hayes, T.B., Heindel, J.J., Jacobs Jr., D.R., Lee, D.H., et al.,2012. Hormones and endocrine-disrupting chemicals: low-dose effects and non-monotonic dose responses. Endocr. Rev. 33 (3), 378–455. https://doi.org/10.1210/er.2011-1050.

Vandenberg, L.N., 2016. Reform of the toxic substances control act (TSCA): an endocrinesociety policy perspective. Endocrinology 157 (12), 4514–4515. https://doi.org/10.1210/en.2016-1712.

Vergauwen, L., Cavallin, J.E., Ankley, G.T., Bars, C., Gabriëls, I.J., Michiels, E.D.G., et al.,2018. Gene transcription ontogeny of hypothalamic-pituitary-thyroid axis develop-ment in early-life stage fathead minnow and zebrafish. Gen. Comp. Endocrinol. 266,

87–100. https://doi.org/10.1016/j.ygcen.2018.05.001.Visser, T.J., 1988. Chapter 6 Metabolism of thyroid hormone. In: New Comprehensive

Biochemistry, vol. 18. pp. 81–103. https://doi.org/10.1016/S0167-7306(08)60641-9.

Walter, K.M., Miller, G.W., Chen, X., Yaghoobi, B., Puschner, B., Lein, P.J., 2019. Effectsof thyroid hormone disruption on the ontogenetic expression of thyroid hormonesignaling genes in developing zebrafish (Danio rerio). Gen. Comp. Endocrinol. 272(November), 20–32. https://doi.org/10.1016/j.ygcen.2018.11.007.

Wang, J., Pan, L., Wu, S., Lu, L., Xu, Y., Zhu, Y., et al., 2016. Recent advances on en-docrine disrupting effects of UV filters. Int. J. Environ. Res. Publ. Health 13. https://doi.org/10.3390/ijerph13080782.

Wang, Q., Lai, N.L.S., Wang, X., Guo, Y., Lam, P.K.S., Lam, J.C.W., Zhou, B., 2015.Bioconcentration and transfer of the organophorous flame retardant 1,3-dichloro-2-propyl phosphate causes thyroid endocrine disruption and developmental neuro-toxicity in zebrafish larvae. Environ. Sci. Technol. 49 (8), 5123–5132. https://doi.org/10.1021/acs.est.5b00558.

Wang, Y., Li, Y., Qin, Z., Wei, W., 2017. Re-evaluation of thyroid hormone signalingantagonism of tetrabromobisphenol A for validating the T3-induced Xenopus meta-morphosis assay. J. Environ. Sci. (China) 52, 325–332. https://doi.org/10.1016/j.jes.2016.09.021.

Wegner, S., Browne, P., Dix, D., 2016. Identifying reference chemicals for thyroidbioactivity screening. Reprod. Toxicol. 65, 402–413. https://doi.org/10.1016/j.reprotox.2016.08.016.

Wei, P., Zhao, F., Zhang, X., Liu, W., Jiang, G., Wang, H., Ru, S., 2018. Transgenerationalthyroid endocrine disruption induced by bisphenol S affects the early development ofzebrafish offspring. Environ. Pollut. 243, 800–808. https://doi.org/10.1016/j.envpol.2018.09.042.

World Health Organization, 2012. World Health Organization - Neurological Disorders:Public Health Challenges. Retrieved from. https://www.who.int/mental_health/neurology/neurodiso/en/.

Worldometers, 2019. Toxic Chemicals Released by Industries. Retrieved May 5, 2019,from. https://www.worldometers.info/view/toxchem/.

Xin, F., Susiarjo, M., Bartolomei, M.S., 2015. Multigenerational and transgenerationaleffects of endocrine disrupting chemicals: a role for altered epigenetic regulation?Semin. Cell Dev. Biol. 43, 66–75. https://doi.org/10.1016/j.semcdb.2015.05.008.

Xu, C., Li, X., Jin, M., Sun, X., Niu, L., Lin, C., Liu, W., 2018. Early life exposure ofzebrafish (Danio rerio) to synthetic pyrethroids and their metabolites: a comparisonof phenotypic and behavioral indicators and gene expression involved in the HPT axisand innate immune system. Environ. Sci. Pollut. Res. Int. 25 (13), 12992–13003.https://doi.org/10.1007/s11356-018-1542-0.

Yao, X., Chen, X., Zhang, Y., Li, Y., Wang, Y., Zheng, Z., et al., 2017. Optimization of theT3-induced Xenopus metamorphosis assay for detecting thyroid hormone signalingdisruption of chemicals. J. Environ. Sci. 52, 314–324. https://doi.org/10.1016/j.jes.2016.09.020.

Yen, M., 2018. Physiological and Molecular Basis of Thyroid Hormone Action 81 (3),1097–1142.

Yost, A.T., Thornton, L.M., Venables, B.J., Sellin Jeffries, M.K., 2016. Dietary exposure topolybrominated diphenyl ether 47 (BDE-47) inhibits development and alters thyroidhormone-related gene expression in the brain of Xenopus laevis tadpoles. Environ.Toxicol. Pharmacol. 48, 237–244. https://doi.org/10.1016/j.etap.2016.11.002.

Yu, L.-Q., Zhao, G.-F., Feng, M., Wen, W., Li, K., Zhang, P.-W., et al., 2014. Chronicexposure to pentachlorophenol alters thyroid hormones and thyroid hormonepathway mRNAs in zebrafish. Environ. Toxicol. Chem. 33 (1), 170–176. https://doi.org/10.1002/etc.2408.

Zada, D., Blitz, E., Appelbaum, L., 2017. Zebrafish – an emerging model to explore thyroidhormone transporters and psychomotor retardation. Mol. Cell. Endocrinol. 459,53–58. https://doi.org/10.1016/j.mce.2017.03.004.

Zada, D., Tovin, A., Lerer-Goldshtein, T., Vatine, G.D., Appelbaum, L., 2014. Alteredbehavioral performance and live imaging of circuit-specific neural deficiencies in azebrafish model for psychomotor retardation. PLoS Genet. 10 (9), e1004615. https://doi.org/10.1371/journal.pgen.1004615.

Zhang, W., Chen, L., Xu, Y., Deng, Y., Zhang, L., Qin, Y., et al., 2019a. Amphibian (Rananigromaculata) exposed to cyproconazole: changes in growth index, behavioralendpoints, antioxidant biomarkers, thyroid and gonad development. Aquat. Toxicol.208, 62–70. https://doi.org/10.1016/J.AQUATOX.2018.12.015.

Zhang, Y.-F., Xu, H.-M., Yu, F., Yang, H.-Y., Jia, D.-D., Li, P.-F., 2019b. Comparison thesensitivity of amphibian metamorphosis assays with NF 48 stage and NF 51 stageXenopus laevis tadpoles. Toxicol. Mech. Methods 1–7. https://doi.org/10.1080/15376516.2019.1579291. 0(0).

Zhao, X., Ren, X., Ren, B., Luo, Z., Zhu, R., 2016. Life-cycle exposure to BDE-47 results inthyroid endocrine disruption to adults and offsprings of zebrafish (Danio rerio).Environ. Toxicol. Pharmacol. 48, 157–167. https://doi.org/10.1016/j.etap.2016.10.004.

Zhu, M., Chen, X.Y., Li, Y.Y., Yin, N.Y., Faiola, F., Qin, Z.F., Wei, W.J., 2018. Bisphenol Fdisrupts thyroid hormone signaling and postembryonic development in Xenopuslaevis. Environ. Sci. Technol. 52 (3), 1602–1611. https://doi.org/10.1021/acs.est.7b06270.

Zoeller, R.T., Tan, S.W., 2007. Implications of research on assays to characterize thyroidtoxicants. Crit. Rev. Toxicol. 37 (1–2), 195–210. https://doi.org/10.1080/10408440601123578.

Zoeller, R.T., Tan, S.W., Tyl, R.W., 2007. General background on the hypothalamic-pi-tuitary-thyroid (HPT) Axis. Crit. Rev. Toxicol. 37 (1–2), 11–53. https://doi.org/10.1080/10408440601123446.

S. Couderq, et al. Molecular and Cellular Endocrinology 508 (2020) 110779

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