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

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non-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, a

review 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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Contents lists available atScienceDirect

Molecular and Cellular Endocrinology

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

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

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 hormones Thyroid disrupting chemicals Biological assay

Endocrine disruption Alternative methods

A B S T R A C T

Thyroid hormones (THs) play critical roles in profound changes in many vertebrates, notably in mammalian neurodevelopment, although the precise molecular mechanisms of these fundamental biological processes are still being unravelled. Environmental and health concerns prompted the development of chemical safety testing and, 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 of TH signalling, screening tests for THADCs may not rely solely onin vitroligand/receptor binding to TH receptors.

Therefore, alternatives to mammalianin vivoassays featuring TH-related endpoints that are more sensitive than circulatory THs and more rapid than thyroid histopathology are needed to fulﬁl the ambition of higher throughput screening of the myriad of environmental chemicals. After a detailed introduction of the context, we have listed current assays and parameters to assess thyroid disruption following a literature search of recent publications referring to non-mammalian models. Potential THADCs were mostly investigated in zebraﬁsh and the frogXenopus laevis, an amphibian model extensively used to study TH signalling.

1. Background

A relatively modern threat to human health and ecosystems has emerged with the escalation in volume and diversity of substances produced by the chemical industry after World War II. Observations of adverse eﬀects in wildlife following widespread application of the pesticide DDT¹and, on human health after Diethylstilbestrol prescrip- tion (Carson, 1962; Herbst and Scully, 1970), served as a catalyst to promote chemical safety assessment programs promoted by the United States Environmental Protection Agency (US EPA) and the Food and Drug Administration (FDA) in the 1970s. In the 90's, global concern arose over the elusive impact of environmental endocrine disrupting chemicals (EDCs), capable of adversely aﬀecting normal hormone function in humans and in wildlife. Nowadays, global human activities release millions of tons of hazardous chemicals into the environment, exposing humans and other animals to a cocktail of chemicals through water, 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 their total volume (ICCA & UNEP, 2019^3,4). Recently over 600 compounds, some of which are produced in high volumes, have been included in a database of potential EDCs, with supporting evidence of adverse eﬀects in either humans or rodent models (Karthikeyan et al., 2019). Im- portantly, the vast majority of chemicals have yet to be assessed for their potential impact on human health or the environment (Applegate, 2008; Krimsky, 2017). Moreover, regulatory agencies are unable to keep pace with the rate of new compounds being introduced (600–1000 compounds per annum in the US alone) (Krimsky, 2017;Vandenberg, 2016).

1.1. Testing for endocrine disrupting chemicals

This context prompted the OECD⁵and the US EPA to develop new strategies to rapidly screen and characterize the endocrine activity of chemicals and environmental contaminants, speciﬁcally for estrogen, androgen and, more recently, for thyroid and metabolism-related

https://doi.org/10.1016/j.mce.2020.110779

Received 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).

1Dichlorodiphenyltrichloroethane.

2Center for Disease Control and Prevention.

3International Council of Chemical Associations.

4United Nations Environment Programme.

5Organization for Economic Co-operation and Development.

Available online 06 March 2020

T

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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 to identify and characterize a potential hazard. Initial tiers feature high- throughputin vitroor rapidin vivoassays in order to screen compounds via specific modes of action, while higher tiers of increased biological complexity consist of more expensive and relatively long-termin vivo tests (Manibusan and Touart, 2017). The latter may demonstrate biologically-relevant adverse effects on endocrine systems, and characterization of the dose-response functions may be extrapolated to human or wild-life populations (Pickford, 2010). Finally, the outcomes of the various tests and increasingly, data from open scientific literature, are compiled to determine the weight-of-evidence supporting potential endocrine activity of a chemical (Gross et al., 2017).

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

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

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

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

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

Therefore, assessment of endocrine disruption calls for speciﬁc endpoints and assays, to which the OECD and US EPA have responded by including speciﬁcin vitroscreening assays and adding endocrine- sensitive parameters to pre-existing mammalian and non-mammalianin vivoassays.

The current framework for next-generation assay development promotes the use of alternative approaches that rely on physiologically- based pharmacokinetic (PBPK) models and in vitro systems to re- capitulatein vivoresponses. This trend is coherent with the more ethical 3Rs principles advocating replacement, reduction and reﬁnement of animal testing protocols (Burden et al., 2015; Tannenbaum and Bennett, 2015). However, the complexity of endocrine regulation precludes the sole use ofin silicoandin vitrosystems. In particular, due to the many points of regulation of the hypothalamus-pituitary-thyroid (HPT) axis (OECD, 2018b;Zoeller et al., 2007), even a battery ofin vitro assays for thyroid disruption would not be as comprehensive as a thoroughin vivotest (Zoeller and Tan, 2007). Ultimately, several amphibian-based assays containing endpoints sensitive to thyroid hormone disruption were added to the list of standardizedin vivobioassays.

1.2. Thyroid hormones and the hypothalamus-pituitary-thyroid (HPT) axis Concern over dysfunction of the HPT axis is spurred by the crucial role that THs play in differentiation, growth, and metabolism, and their requirement for the proper functioning of virtually all tissues (Yen, 2018). Notably, THs are essential for normal development and function of the central nervous system due to their key roles in neuronal pro- liferation, migration, differentiation, synaptogenesis, synaptic plasticity and myelination (Bernal, 2005; Horn and Heuer, 2010; Howdeshell, 2002). Deficiency in TH levels during neurological development can induce severe manifestations in humans, as highlighted by congenital iodine deficiency syndrome (Bernal, 2007). However even moderate or transient TH insufficiency during the perinatal period may irreversibly alter offspring neurodevelopmental outcomes (Bernal, 2005;Korevaar et 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 homeostasis evaluated by circulating levels of hormones. Indeed, TH axis disrupting chemicals (THADCs) are present in many classes of chemicals and may lead to a wide range of adverse eﬀects in both wildlife and humans through disruption of the HPT axis at any of the multiple levels presented hereafter (Calsolaro et al., 2017;Mughal et al., 2018;Oliveira et al., 2018;Ghassabian and Trasande, 2018).

The HPT axis regulates TH synthesis through the hypothalamic thyrotropin-releasing hormone (TRH) which stimulates the pituitary to secrete thyroid-stimulating hormone (TSH) necessary for the thyroid gland (or follicles infishes) to produce THs. Homeostasis is regulated by negative feedback on the release of both TRH and TSH. Synthesis of THs requires the uptake of iodide mediated by the sodium/iodide symporter (NIS) in thyroid cells. Iodide ions are then oxidized by the enzyme thyroid peroxidase (TPO) for incorporation into thyroglobulin to produce precursors of the 3,3′,5-triiodothyronine (T3) (the active form of THs) and 3′,5′,3,5-tetraiodo-L-thyronine (thyroxine or T4) (Sellitti and Suzuki, 2013). The vast majority of T4 and T3 circulates in the bloodstream bound to thyroid distributor proteins (in humans: trans- thyretin (TTR), thyroid binding globulin (TBG), and albumin) and are considered 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-type amino acid transporters (LATs) (Hagenbuch, 2007;van der Deure et al., 2010). Intracellular TH availability is coordinated by specific deiodination processes which either activate or deactivate THs (DIO1 may contribute to both, while DIO2 and DIO3 exclusively activates and in- activates THs, respectively). Next, THs enter the nucleus where they can either positively or negatively control transcription of target genes via TH receptors (TRs) that bind to specific DNA segments containing TH response elements (TREs) (Hönes et al., 2017). Finally, metabolic clearance of circulating THs is ensured by hepatic sulfotransferases or uridine disphosphate (UDP)-glucuronosyltransferases (Visser, 1988).

Some differences exist between vertebrates and should be borne in mind when evaluating thyroid disruption in different species. In particular, in non-mammalian vertebrates, the release of TSH appears to be regulated by corticotrophin-releasing hormone (CRH) (De Groef et al., 2006), and the set of TH transport distributor proteins may differ as well as their affinity, e.g., the main transport protein TTR has a higher affinity for T3 instead of T4 compared to mammalian models (Zoeller et al., 2007). Interspecies differences are also found among mammalian species, namely between humans and rodent models - the gold standard of regulatoryin vivotoxicology. These include differences in TH phar- macokinetics, basal levels of TSH, structure and expression patterns of key enzymes and/or transporters, and the timing of thyroid ontogenesis and 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 THs and/or TSH may correlate with exposure to a chemical being scruti- nized but, without discernment of the mechanisms of action (MoA) or insight into the potential adverse effect(s), these observations are in- sufficient to draw definitive conclusions on their endocrine activity (Lee and Jacobs, 2015;Slama et al., 2017).

Even though measuring circulating THs, and TSH has increased in specificity, 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 may complicate 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 cause disruption of hormone homeostasis yet can alter TH availability at a local level (Boas et al., 2012). These possibilities, coupled with the

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scarcity of information on the molecular mechanisms of THADCs, further complicate evaluation of thyroid disrupting eﬀects.

Detailed insight into the HPT axis has elucidated multiple MoA through which thyroid disruption can occur (Crofton, 2008;Friedman et al., 2016). Numerous in vitroassays are designed to examine key steps in the synthesis, regulation, and action of TH: i) TRH and TSH production, ii) TSH and TRH receptor activation, iii) TPO activity or iodide uptake inhibition, iv) binding to TTR or TBG, v) deiodination activity, 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 been validated by either the OECD or US EPA. For this reason, there is a need for developing higher throughput screening assays that can detect substances that interfere with the activity of the thyroid system (Kortenkamp et al., 2017).

Newly required thyroid endpoints used to update existingin vivo tests consist of determining circulating levels of THs and TSH, measuring thyroid weight to inform of its stimulation by TSH over time, and especially, examining histopathologic changes in the thyroid gland.

Thyroid histopathology is a particularly valuable and sensitive diagnostic parameter to disruption of TH homeostasis (Dang, 2019;Grim et al., 2009; Pickford, 2010). Together, these parameters of thyroid function have been used to identify the majority of thyroid toxicants in mammals and can be suﬃcient to detect clear signs of thyroid disruption. However, it should be noted, that severity grading in thyroid histopathology is based on an idealized model which used the powerful inhibitor of TH synthesis propylthiouracil (PTU) (Brucker-Davis, 1998;

Zoeller and Tan, 2007), and may therefore overlook the effects of other THADCs with more subtle effects. By combining curated data from scientific literature and results from validated assays in the US EPA Endocrine Disruptor Screening Program, i.e., the female and male rat pubertal assays and the Amphibian Metamorphosis Assay (AMA), Wegner et al. (2016)proposed an additional 28 reference THADCs with various MoA which could serve to further characterize the response of different assays and endpoints.

Although alternative models represented by amphibians,ﬁsh and avian species may appear too far removed from mammals to be relevant to human health, molecular components of the HPT axis are highly conserved across vertebrate taxa and TH signalling involves the same circulating hormones (Noyes et al., 2018;Taylor and Heyland, 2017).

Markedly, the peak of THs observed around the perinatal period in mammals is also found during developmental transitions in other vertebrates (Holzer and Laudet, 2013) as demonstrated by amphibian metamorphosis (Galton, 1992), subtle or spectacular post-hatching metamorphosis in teleost ﬁsh (McMenamin and Parichy, 2013), and hatching in precocial birds (De Groef et al., 2013).

Consequently, alternative models used in toxicology have been increasingly popular for the purpose of chemical safety assessment of potential THADCs. Therefore, we focused this review on knowledge of TH-related endpoints developed or under development in non-mammalian vertebrates.

2. Methods

In order to supplement our knowledge on the subject and recent OECD, US EPA, EFSA⁶and ANSES⁷reports; a combination of several search strategies were used in a single query in PubMed to review recent literature either assessing thyroid disruption in non-mammalian modelsin vivoor discussing regulatory issues on the topic. Fine-tuning of 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 publications containing thyroid assays or fundamental research on TH of potential use to (eco)toxicology. A PubMedﬁlter was applied to remove human studies and articles older than 5 years.Fig. 1provides an overview of theﬁnal articles retrieved using the following search query performed on the February 14, 2019 (full search query available in Supplementary Document). In addition, a supplementary table features the following information for each ecotoxicological article retrieved from the search query: the model employed, the test chemical(s), the concentration ranges and exposure period, and the endpoints examined (Supple- mentary Table).

Discarded articles from the 256 articles initially retrieved did not involve THs in an (eco)toxicology or regulatory context. TH-related experimental studies were mainly in vivo. Among these, several performed additionalin vitro,in silicoorex vivoexperiments, or even used multiple animal models and endpoints, leading to some overlap between the number of articles (indicated in parenthesis) per type of study, model utilized and endpoints examined. Amphibian and teleost ﬁsh models both had distinct endpoints, underlining the advantages and disadvantages of each model.

In vivostudies pertaining to chemical assessment that were retrieved from our literature search originated from academic research, and while most are not based on standardized assays tailored for risk assessment, common endpoints frequently appear within these models (Fig. 1 & Supplementary table). Novel methods and endpoints are highlighted 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 the predominant models employed and share common advantages: the ability to easily produce a large numbers of free-living embryos, their accessibility during critical stages of development, and the absence of continuous maternal hormonal influence on embryo and larvae development. Although the latter excludes maternal compensatory mechanisms that can mask the effects of THADCs, this powerful advantage for screening purposes comes at the expense of added difficulty for human health risk assessment.

3. Amphibian models for detecting THADCs

Besides their particular susceptibility to environmental pollutants due to their exposure through dermal, respiratory and dietary routes, amphibians have been extensively used as compelling vertebrate models of thyroid function by virtue of their remarkable TH-dependant metamorphosis–a process mimicking perinatal development in mammals 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 by THs, including tail and gill resorption, limb growth, remodelling of the intestines, central nervous system, respiratory system, cranial carti- lages, and skin (Dodd and Dodd, 1976). Consequently, observation of potential interferences with these developmental processes in exposed tadpoles or inex vivoorgan cultures can reﬂect disturbances in the HPT axis (Fu et al., 2018;Miyata and Ose, 2012;Taft et al., 2018;Yao et al., 2017). Strikingly, halting endogenous T3 synthesis by surgical removal of the thyroid gland or by chemical blockage of thyroid synthesis (e.g., TPO inhibitors PTU and methimazole (MMI), or the NIS inhibitor perchlorate) will inhibit metamorphosis. Conversely, pre-metamorphic tadpoles can be induced to undergo precocious metamorphosis by the introduction 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- knownXenopusmodel was selected by both the US EPA and the OECD as a valuable and robust tool to assess the impact of potential EDCs on the thyroid axis (Fig. 2), and has been used to evaluate multiple chemicals in both validated and alternative assays.

6European Food Safety Authority.

7The 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 employingXenopus laevis are indicated with corresponding developmental stage and approximate number of days post-fertilization. Levels of TH gradually rise until reaching a peak at metamorphic climax at Nieuwkoop and Faber (NF) stage NF 62 and subside 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 TG 809.1100, 2009). The assay involves exposing 20 Xenopus laevis Nieuwkoop and Faber (NF) stage NF 51 tadpoles (17 days post-fertilization (dpf)) for 21 days to a test chemical using aﬂow-through system.

After 7 and 21 days of exposure, determination of developmental stage and measurements of hindlimb length (HLL) inform on the rate of metamorphic development, in additional to snout-vent length (SVL) and body weight used as growth and health indicators. Delayed, accelerated or asynchronous development of any of the key features at day 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 metamorphic development which are indicators of thyroid activity (asynchronous metamorphic development may arise from disruption of peripheral TH action and/or metabolism in developing tissues), or in the case of developmental delay potentially caused by stress or non-speciﬁc systemic toxicity, the AMA requires the evaluation of thyroid gland histology (OECD TG 231, 2009).

Diagnostic criteria of thyroid histopathology originate from AMA validation studies that were conducted using a wide range of chemicals that interact with the HPT axis through different mechanisms. Similar to mammalian thyroid histopathology, a semi-quantitative severity grading is applied to the relative size of the thyroid gland, the number and size of thyroid follicular cells, together with additional criteria such as follicular lumen area, colloid quality, and follicular cell height and shape (Coady et al., 2010;Grim et al., 2009;OECD TG 231, 2009). Any chemical capable of adjusting TSH secretion will result in histopathological changes of the thyroid gland, however the effects of TH antagonists (i.e., glandular hypertrophy due to follicular hypertrophy and/or hyperplasia) are more evident than those of weak agonists that may result in confounding advanced developmental stages (Miyata and Ose, 2012;OECD TG 231, 2009). By comparing results from validation studies,Dang et al. (2019)confirmed that thyroid histology is the most sensitive endpoint for thyroid active chemicals which were predominantlyknown antagonists, and only few thyroid inactive chemicals would change thyroid histology, reinforcing the value of thyroid histopathology in reducing the number of false positive identification of THADCs. As outlined in a review byPickford (2010), in the case of thyroid agonists, developmental stage and hindlimb length are more sensitive and reliable. Importantly, using numerous reference THADCs with known MoA, a strong concordance was found without major differences in sensitivity between mammalian assays and amphibian metamorphosis assays (Pickford, 2010; Wegner et al., 2016). Moreover, none of the chemicals active in mammalian assays were negative in amphibians, highlighting the possibility to detect thyroid activity across the vertebrate spectrum (OECD, 2018b).

The AMA uses Stage NF 51 tadpoles because it was assumed that tadpoles were functionally athyroid prior to the endogenous secretion of TH detected at stage NF 54 (26 dpf) yet metabolically competent and sensitive to exogenous TH as well as thyroid toxicants (Degitz et al., 2005;Y.-F. Zhang et al., 2019b). Evidence supporting the convenience of using younger tadpoles in other assays for increased sensitivity and higher-throughput have been described in studies indicating that maternal 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 the AMA,Y.-F. Zhang et al. (2019b) compared results of NF 48 tadpoles exposed for 7, 21 & 28 days to the original AMA protocol without the use of histopathological examination. Tadpoles at this earlier stage possess the added benefit of being obtainable 10 days earlier (7 dpf) and are easily identifiable due to the appearance of forelimb buds. The treatment 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 antagonists MMI and perchlorate, when considering developmental stages. Of particular importance for higher-throughput screening, when using smaller NF 48 tadpoles, only 7 days of exposure were needed to observe significant development inhibition and at lower concentrations than 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 validated and harmonized the LAGDA (OECD TG 241, 2015;US EPA 890.2300, 2015). The LAGDA is considered a more sensitive and higher tier assay for suspected thyroid-active chemicals to assess population relevant eﬀects. Covering multiple life-stages, beginning with early embryos (NF

8–15) and ending two months after completion of metamorphosis (NF 66), this relatively long-term assay (lasting approximately 16 weeks) has the ability to confirm disruption of both the HPT axis at metamorphic climax (NF 62) and of the hypothalamic-pituitary-gonadal axis at test termination, when gonads are fully differentiated. In addition to recording the time needed to reach NF 62, the LAGDA includes measurements plasma vitellogenin, histological examination of the thyroid gland and gonads, as well as liver and kidneys for detection of metabolic or systemic toxicity. While the LAGDA is not a life-cycle test due to exclusion of the reproductive phase, it has the advantage of being able to pry into potential gender differences and overlaps between several 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, time consuming, and requires large numbers of animals, water volumes and test chemicals.

As such, validation of the LAGDA was performed using a limited set of EDCs in order to test the responsiveness of the assay to diﬀerent endocrine modalities (US EPA, 2013), such as the ultraviolet ﬁlter benzophenone-2 (BP-2) which, in addition to documented estrogenic and anti-androgenic activity, has been shown to inhibit TPOin vivoand in vitro (Wang et al., 2016). Chronic exposure to BP-2 considerably increased levels of vitellogenin, caused sex-reversal of genotypic males, delayed gonad development of genotypic females, and increased the prevalence and severity of thyroid histopathological observations in a dose-dependent manner, in line with BP-2's ability to inhibit TH synthesis (Haselman et al., 2016a, b). Unfortunately, neither reference TH agonists nor antagonists were included in the validation process and furthermore, neither are currently required as positive or negative controls 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 a relatively short-term (72 h of exposure) and miniaturized test, particularly suited for preliminary screening of a large number of chemicals at various concentrations, while retaining the full spectrum of physiological relevance provided byin vivoanalysis. Furthermore, eleuther- oembryos employed in the XETA (NF 45 to NF 48) still feed on their yolk 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 ofX. laevis,Tg (thibz:eGFP), which expresses the Green Fluorescent Protein (GFP) under the control of a portion of the regulatory region of TH/bZIP, a putative leucine zipper transcription factor highly sensitive to TH regulation (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 of the test chemical with or without a T3 spike equivalent to the plasma T3 concentration during tadpole metamorphosis (Leloup and Buscaglia, 1977;OECD, 2018b). Daily renewal of the test chemical bypasses the need of sophisticatedflow-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 the test chemical and/or possible experimental errors (OECD TG 248, 2019). The final endpoint is the quantification of the fluorescence emitted by individual tadpoles carefully placed in 96-well plates, using either afluorescent microscope or afluorescence plate-reader. Thyroid hormone agonists will cause an increase influorescence with or without the T3 spike, while chemicals acting as antagonists on the HPT axis are best detected with the T3 spike and revealed by a decrease influorescence 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 retrieved from our search query used the aforementioned validated assays. While the majority mainly diﬀer in exposure period and sometimes in the model employed, many of the same endpoints were investigated to identify potential THADCs (i.e., hindlimb length, snout-to-vent length, developmental stage and thyroid histology; Supplementary Table). In tadpoles, as morphological changes of the head region and the brain are induced by THs, measurements such as mouth width, head area, brain width, brain length, or olfactory-organ-to-brain length may further characterize disruption of TH-induced metamorphosis (Yao et al., 2017;

Zhu et al., 2018;Mengeling et al., 2016,2017). Several morphological endpoints were examined in conjunction with the readout of transgenic X. laevistadpoles harbouring a luciferase reporter driven by TH/BZIP TREs (Mengeling et al., 2016,2017). After 5 days of exposure to either THs or the TR antagonist NH-3 at NF 48, a strong correlation was observed between measurements of brain width at the optic tectum, olfactory-organ-to-brain length and luciferase activity. In particular, brain width at the optic tectum may be less sensitive to the confounding eﬀects of general toxicity than other morphological parameters (Mengeling et al., 2017).

3.5. Additional endpoints in amphibian assays: gene transcription and behaviour

In contrast to in vitro assays, the XETA provides neither an un- equivocal identiﬁcation of the precise MoA of a chemical nor an associated apical endpoint relative to adverse outcomes as present in the AMA or in the more thorough LAGDA. As emphasised by Haselman et al. (2016a, b)during validation of the LAGDA, a lack of endpoints for neurodevelopment or behaviour is apparent in all of these validated assays. The reﬁnement of these assays by integrating behavioural endpoints and expression of key genes related to the HPT axis and/or genes predictive of late adverse outcomes in neurodevelopment could be very valuable.

Morphological endpoints only provide indirect evidence of dis- rupted signalling since a series of molecular cascade events involved in developmental processes are triggered by THs (Shi et al., 2001). Gene transcription has been utilized to optimize T3-inducedXenopusmeta- morphosis assays. Afterfirst sampling tadpole intestines which undergo significant morphological and transcriptional variations during metamorphosis (Yao et al., 2017),Wang et al. (2017)successfully sampled the tail (a significantly easier tissue to dissect) for detection of altered TH-responsive gene transcription. For example, in addition to effects on morphological endpoints after 96h of exposure, the brominatedflame retardant tetrabromobisphenol A (TBBPA) inhibited T3-induced expression of key genes (e.g., trβ,th/bzip,krüppel-like factor 9(klf9), and dio3) in the tail of NF 52 tadpoles after just 24h of exposure compared to the T3 group.

The brain shows transcriptomic responsiveness to THs earlier than the tail, and with greater sensitivity (Yost et al., 2016). Subtle effects on TH-related and TH-responsive genes in whole brains of NF 48 tadpoles can be revealed following the XETA as detailed in a protocol by Spirhanzlova et al. (2018). Using this approach more pertinent to TH- mediated neurodevelopmental effects,Fini et al. (2017)reported that a mixture of chemicals present in human amnioticfluid could induce a dose-dependent increase offluorescence in the XETA and modulate TH signalling genes (encoding TH transporters, anddio1,dio2). The mixture also downregulated genes implicated in neural differentiation (markers of pluripotency (sox 2),neuronal (tubb2) and oligodendrocyte (mbp) differentiation) and synaptic plasticity (brain derived neurotrophic factor (bdnf)), and altered tadpole behaviour (assessed by motility tracking under light/dark stimuli). Behavioural modifications assessed by motility tracking have been described in tadpoles in conjunction with TH disruption in other amphibian studies (Spirhanzlova et al.,

2019; W. Zhang et al., 2019a). Experimental techniques to examine neurobehavioural eﬀects are available inXenopustadpoles (as reviewed byPratt and Khakhalin, 2013) however, due to gaps in knowledge of the mechanistic pathways leading to behavioural responses, linking altered neurobehaviour to HPT dysfunction still remains a challenge in chemical testing and assessment.

In particular, as tadpoles at the stages employed in the XETA are functionally athyroid, inhibitors of TH synthesis (i.e., NIS or TPO inhibitors) are not expected to be detected (OECD TG 248, 2019). In larger animals such as those used in the AMA or LAGDA, gene expression in thyroid and pituitary tissue may complement effects on metamorphosis-related endpoints (Lorenz et al., 2018). Using stage NF 54 tadpoles characterized by the onset of thyroid gland-function, thein vitroTPO inhibitor 2-Mercaptobenzothiazole was shown to upregulate NIS expression in the thyroid gland in a robust and sensitive manner after 7 days of exposure, consistent with others signs of classical HPT compensatory responses (i.e., decreased T4 and elevated TSH levels in serum, and increased follicular cell hypertrophy (size) and hyperplasia (number) of the thyroid gland) (Tietge et al., 2013). Iodotyrosine deiodinase (IYD) is a lesser known yet important enzyme for TH synthesis which may provide a substantial amount of recycled iodide to the thyroid gland through deiodination of monoiodotyrosine (MIT) and diiodotyrosine (DIT) (Rousset et al., 2015). After exposing tadpoles from stage NF 50 to 62 to an IYD inhibitor (3-nitro-L-tyrosine),Olker et al. (2018)described effects similar to those incurred by NIS or TPO inhibitors. However, notable differences and specificities were observed, such as increased IYD mRNA expression in the thyroid gland, detection of MIT and DIT in plasma, and arrested development at a later stage. As these effects could be rescued by iodine supplementation, IYD inhibition is a potential novel MoA for THADCs, especially relevant to environments with limited availability of iodine such as freshwater ecosystems.

4. Teleostfish models for identification of thyroid disruption Teleostfish models are highly popular in developmental toxicity screening and have been increasingly investigated in the context of endocrine disruption, particularly along the hypothalamic-pituitary- gonadal (HPG) axis (Ankley and Johnson, 2004), and more recently for the HPT axis. Indeed, while the roles of THs have historically been more thoroughly investigated in amphibians and mammals, the zebrafish (Danio rerio) is the predominant model employed in the articles retrieved from our search query pertaining to non-mammalian models spanning the last 5 years (cf.Fig. 1). Remarkably, specific biomarkers for thyroid disruption in teleost fishes have yet to be accepted for regulatory purposes due to discrepancies resulting from inconsistent experimental parameters across a limited amount of studies using known TH disruptors, as emphasised bySpaan et al. (2019). However, given the relatively recent characterization of the zebrafish HPT axis and intrinsic advantages, the zebrafish is becoming a promising alternative vertebrate model for thyroid signalling, amenable to high- throughput screening owing to their small size, ease of culture and short generation time (Haggard et al., 2018;Kollitz et al., 2018;Marelli and 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 well conserved across vertebrates, and the structure and function of key components TH signalling in teleosts closely resemble those of higher vertebrates. Using zebraﬁsh exposed to exogenous THs or PTU (with or without anmct8 morpholino) at 24, 72 and 120 h post-fertilization (hpf),Walter et al. (2019)conﬁrmed that expression of key ortholog genes which mediate TH signalling in mammals appear to be coordinated by THs inD. Rerio. However, in teleosts, thyroid follicles (the functional unit of the gland) are dispersed along the ventral midline of the pharynx instead of being encapsulated in an organized pair of thyroid glands (Alt et al., 2006), which therefore precludes the use of

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histopathological assessment as is employed inXenopusand mammals.

It remains that the structure and function of these follicles is conserved in vertebrates (Carr and Patiño, 2011), and the onset of thyroid development in teleosts resemble that of higher vertebrates, with key transcription factors for thyroid follicular precursor cell specification and differentiation (i.e.,nkx2.1,pax2a, andhhex) having orthologs that act similarly in mammals and amphibians (Elsalini et al., 2003;Porazzi et al., 2009). As these precursors appear as early as 24 hpf, and mature thyroid follicles are identifiable 48 h later, the zebrafish has presented a relatively simple model of great utility for studying thyroid dysgenesis, growth and differentiation (Opitz et al., 2013).

Furthermore, due to the existence of a comprehensive genetic toolbox, characterization of knock-downs or knock-out of key TH signalling genes in zebraﬁsh have resulted in fruitful modelling and have contributed to our understanding of thyroid-related human diseases, e.g., Allan-Herndon-Dudley syndrome and TH resistance syndrome due to 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;Zada et al., 2017). Finally, expression proﬁles of genes involved in TH signalling during multiple time points encompassed by a validated teleost toxicity assay (Fish, Early-Life Stage Toxicity Test” (OECD TG210, 2018a)) have recently been obtained and have shown to be relatively similar in both the zebraﬁsh and the fathead minnow (Vergauwen et al., 2018). Together, these achievements may facilitate interpretations of the consequences of THADC exposure in teleosts and aid in paving the way towards the inclusion of thyroid-related endpoints in validated assays.

4.1. Endpoints infish models for identification of thyroid disruption In contrast to amphibian assays, TH content in eggs, whole-body larvae 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 out of 73) ecotoxicological studies retrieved). Additionally, a particular vulnerable time windows to THADCs such as the transition from the embryonic to larval stage occurs relatively rapidly, i.e. only 2–3 dpf in zebrafish (Walter et al., 2019), and metamorphosis (larva-to-juvenile transition) initiates after 2 weeks (Sharma et al., 2016). The subsequent generation may be obtained in 2–4 months to provide evidence of transgenerational effects following THADC exposure, whilst also having the advantage of taking into account gender-specific effects (Chen et al., 2018;Cheng et al., 2017;Han et al., 2017;Jianjie et al., 2016;Wang et al., 2015;Yu et al., 2014;Zhao et al., 2016). These features represent outstanding advantages for addressing the long-term effects (into adulthood and on offspring) of EDCs which is more pertinent to real life exposure scenarios consisting of chronic exposure to low doses of environmental chemicals, and invaluable for potential studies on heritable EDC-induced epigenetic alterations of gene expression (Alavian- Ghavanini and Rüegg, 2018;Baker et al., 2014).

Expression levels of TH signalling genes are often analysed in whole-body teleost larvae in conjunction with TH content following exposure to potential THADCs. For instance, principle component analysis (PCA) demonstrated that transcription patterns of most TH signalling genes showed a strong correlation to altered TH levels following exposure of zebraﬁsh embryos to the synthetic pyrethroid pesticide, permethrin (Tu et al., 2016). Furthermore, mercury exposure resulted in elevated levels of whole-body TH, upregulation of genes involved in thyroid development (hhex, nkx2.1) and thyroid synthesis (nisand tg (thyroglobulin)) (Sun et al., 2018). Particularly,Walter et al.

(2019)identiﬁed a paralog ofdio3(dio3-b) as being highly sensitive during 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 frequently assessed in mammals and amphibian were found to be relatively unresponsive in whole larvae homogenates (e.g.,bdnf, klf9),

while expression ofmyelin basic protein(mbp)was upregulated by TR agonists (i.e., exogenous T3 or T4) (Walter et al., 2019). Similarly, transcriptomic proﬁling of 25 EDCs highlighted a suite of transcripts that may serve to identify TR agonists, including the myelin-related geneplp1b(Haggard et al., 2018). Further studies are warranted as myelin-related genes were also found to be downregulated following treatment of zebraﬁsh to xenobiotics that reduced levels of THs (Miao et al., 2015;Wang et al., 2015).

In zebrafish, although behavioural studies are frequent in toxicological studies, and fundamental research has observed aberrant neurobehaviour in association with HPT dysfunction generated by loss- of-function of core components of the HPT axis (e.g., Dio2 (Houbrechts et al., 2016), and Mct8 (Zada et al., 2014,2017), the underlying MoA of altered behaviour remain unclear, as inXenopus.Interestingly, Mct8- deficient zebrafish display neurological impairment, in contrast to mice which require the additional knock-out of OATP1C1 to replicate the behavioural abnormalities of Allan-Herndon-Dudley syndrome (Mayerl et al., 2014;Zada et al., 2017,2014). The reduced mobility following touch-stimuli that was observed in Mct8-deficient zebrafish may involve 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 the metabolite of the pesticide pentachlorophenol had similar hyperthyroid effects to that of T3 treatment, with increased expression ofsynapsin I (Syn 1), implicated in synaptic plasticity and learning and memory, which may have repercussions on the timing and development of the brain (Cheng et al., 2015). In an exhaustive study on the effects of the organophosphateflame retardant TDCPP,⁸3-month exposure of adult zebrafish was reported to decrease plasma THs in F0 females and TH content in unexposed F1 eggs/larvae, consistent with the previous correlation between decreased T4 levels in humans and TDCPP concentrations in household dust. Additional effects reported in F1 offspring include: downregulated mRNA and protein expression related to neurodevelopment (i.e., mbp, α1-tubulin encoding a neuron specific microtubule protein involved in developing and regenerating brain, and synapsin IIa, important for synaptogenesis and neurotransmitter release), 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 to what extent TH disruption contributes to these affected neurodevelopmental endpoints.

Differentiating the neurodevelopmental effects mediated by endocrine disruption from neurotoxicity remains challenging in behavioural tests.Fraser et al. (2017) compared larval zebrafish behaviour after embryonic exposure to a wide range of hormones and potential EDCs and concluded that, while T4 affected locomotion (i.e., increased swimming speed and total distance travelled under light/dark stimuli), perchlorate had no effects (Fraser et al., 2017). Using multiple endpoints,Fetter et al. (2015)reported that reduced mobility following exposure to MMI could be restored by T4, however conclusions on the MoA requires complementary gene expression analysis due to confounding craniofacial malformations capable of affecting mobility and may arise from either altered TH signalling or unspecific toxicity. In- deed, the ontogenesis infish with less pronounced transformations than theflatfish also involves TH (Rastorguev et al., 2016). Interestingly, pectoralfin length, which can also affect motility, has been identified as a highly TH-sensitive morphological marker in zebrafish (Sharma et al., 2016). Another endpoint related to swimming activity is eye development. When impaired by two THADCs acting via different MoA (PTU and a known TR antagonist, TBBPA),Baumann et al. (2016)observed diminished visual performances as determined by optokinetic response and reduced vision-based behaviours.

One physiological endpoint potentially associated to transcriptional

8Tris (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 Toxicity Test”(OECD TG210, 2018a). Inhibitors of TPO and DIO1/2 were shown to impair swim bladder inflation in zebrafish and the Fathead minnow (Nelson et al., 2016;Stinckens et al., 2016;Vergauwen et al., 2018), a key event which could be incorporated in an Adverse Outcome Pathway (AOP) framework in combination withfirst-tierin chemicoassays for determination of molecular initiating events (Stinckens et al., 2018).

Coiling behaviour, another neurological-based endpoint, has been linked to thyroid receptor-βdeﬁciency (Xu et al., 2018). In addition, endpoints included in a General Developmental Score (GDS) have been associated with developmental toxicity caused by thyroid disruptors, including abnormal iridophore pigmentation, beat and glide swimming and resorption of the yolk sac (Jomaa et al., 2014).

Finally, given the aforementioned conservation of thyroid development in teleosts, early detection of thyroid disruption is possible using whole-mount intrafollicular TH staining which could potentially be implemented into current OECD zebrafish assays (Rehberger et al., 2018). Interestingly, a novel fluorescent transgenic line expression under the control of the zebrafishthyroglobulinpromoter allows forin vivo monitoring of thyroid gland development. Thyroid follicular cell counts either increase following TH treatment or decrease following antagonists 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 screening purposes (Jarque et al., 2018).

5. Conclusion

Identiﬁcation of TH disrupting compounds is challenging as they may act at multiple levels of the thyroid axis. As highlighted in this review, disruption of this axis in non-mammalian models is over- whelmingly investigated in medium-throughput assays usingD. rerio&

X. laevisembryos and larvae. The prevalence of these models should not come as a surprise in context of the increasing pressure for chemical risk assessment to incorporate sublethal eﬀects such as endocrine disruption for an ever-growing list of compounds. However, avian species were relatively under-represented in ecotoxicology research despite the existence of speciﬁc social behaviour tests that may be applied to chicken hatchlings exposed as embryos (Haba et al., 2014).

The urgency to efficiently identify potential THADCs requires a more integrated approach and higher throughput, currently lacking in harmonized and validated tests. In conjunction with the development of additional endpoints such as those related to neurobehaviourin vivo, framing endpoints in AOPs using bothin silicoandin vitroassays may increase the rapidity and the efficiency of hazard identification and characterization. A recent article published after our literature search provides multiple AOPs in a network that connects disruption of the HPT axis in various validated vertebrates assays to several promisingin vitrohigh-throughput screening assays (Noyes et al., 2019).

Assessing the ecological impact of THADCs may only benefit from added taxonomic breadth. BesidesX. laevisandD. reriostudies retrieved from our literature search, other amphibians (i.e., Xenopus tropicalis, Rana nigromaculata, Bufo gargarizans)and teleostfishes (i.e, goldfish, killifish, medaka, fathead minnow, Mozambique tilapia,flatfishes, coral fishes, Japanese flounder) have been used to evaluate the potential effects of chemicals on the HPT axis with many of the same endpoints described previously (Supplementary Table). Another recent article reviewed the effects of exposure to reference THADCs and a variety of environmental contaminants on thyroid signalling in diverse anuran amphibians, including the effects of environmentally relevant complex mixture and abiotic stressors (Thambirajah et al., 2019).

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

mixture effects more representative of actual exposure. Indeed, it is likely that disruption of the thyroid axis by environmental chemicals is underestimated when combined exposure to multiple substances is neglected. Finally, as the prevalence of adverse neurological effects are expected to increase globally (WHO, 2012), recognition of the health hazards of thyroid disruption that may contribute to some of these disorders has recently been reiterated by the funding of three European projects (ATHENA, ERGO and SCREENED) that promote new test strategies and develop specific endpoints in thyroid hormone signalling.

Acknowledgements

We thank Gérard Benisti for supplying the photos that were edited forFig. 2.We thank the MNHN and the CNRS for the annual funding provided to the unit. SC is a PHD student implicated in EU H2020 HBM4EU (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 athttps://

doi.org/10.1016/j.mce.2020.110779.

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