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

Stephan Couderq, Michelle Leemans, Jean-Baptiste Fini

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Stephan Couderq, Michelle Leemans, Jean-Baptiste Fini. Testing for thyroid hormone disruptors, a review of non-mammalian in vivo models. Molecular and Cellular Endocrinology, 2020, 508, pp.110779. 10.1016/j.mce.2020.110779. hal-02414719

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

<figure><img src="image_1.png" /></figure>

Stephan Couderq, Michelle Leemans, Jean-Baptiste Fini*

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

## ARTICLE INFO

**Keywords:**

Thyroid hormones

Thyroid disrupting chemicals

Biological assay

Endocrine disruption

Alternative methods

## ABSTRACT

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 on *in vitro* ligand/receptor binding to TH receptors. Therefore, alternatives to mammalian *in vivo* assays 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 frog *Xenopus 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 DDT<fnref n="1" /> and, on human health after Diethylstilbestrol prescription (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 <fnref n="3" />,<fnref n="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). Importantly, 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 OECD<fnref n="5" /> and 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

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: fini@mnhn.fr (J.-B. Fini).</footnote>

<footnote n="1">¹ Dichlorodiphenyltrichloroethane.</footnote>

<footnote n="2">² Center for Disease Control and Prevention.</footnote>

<footnote n="3">³ International Council of Chemical Associations.</footnote>

<footnote n="4">⁴ United Nations Environment Programme.</footnote>

<footnote n="5">⁵ Organization for Economic Co-operation and Development.</footnote>
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-throughput *in vitro* or rapid *in vivo* assays in order to screen compounds via specific modes of action, while higher tiers of increased biological complexity consist of more expensive and relatively long-term *in vivo* tests (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 to human or wild-life populations (Pickford, 2010). Finally, the outcomes of the various tests and increasingly, data from open scientific litera- ture, 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 specific *in vitro* screening assays and adding endocrine- sensitive parameters to pre-existing mammalian and non-mammalian *in vivo* assays.

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- capitulate *in vivo* responses. 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 pre- cludes the sole use of *in silico* and *in vitro* systems. 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 of *in vitro* assays for thyroid disruption would not be as comprehensive as a thorough *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 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 pre- sented 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 in fishes) 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 pro-
duce 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 deiodi-
nation 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 partic-
ular, 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 regulatory in vivo toxicology. 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 scrutinized but, without discernment of the mechanisms of action (MoA) or insight into the potential adverse effect(s), these observations are insufficient 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
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 MoA
through which thyroid disruption can occur (Crofton, 2008; Friedman
et al., 2016). Numerous *in vitro* assays 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 existing *in vivo*
tests consist of determining circulating levels of THs and TSH, mea-
suring 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 diag-
nostic 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 disrup-
tion. 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 ver-
tebrates (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 in-
creasingly 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-mam-
malian vertebrates.

## 2. Methods

In order to supplement our knowledge on the subject and recent
OECD, US EPA, EFSA<fnref n="6" /> and ANSES<fnref n="7" /> reports; a combination of several
search strategies were used in a single query in PubMed to review re-
cent literature either assessing thyroid disruption in non-mammalian
models *in vivo* or 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 PubMed filter was applied to remove human
studies and articles older than 5 years. Fig. 1 provides an overview of
the final 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 additional *in vitro*, *in silico* or *ex vivo* experiments, 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 vivo* studies 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 integration 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 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 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 in *ex vivo* organ 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 per-
chlorate) 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-
known *Xenopus* model 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 che-
micals in both validated and alternative assays.

<footnote n="6">⁶European Food Safety Authority.</footnote>

<footnote n="7">⁷The French Agency for Food, Environmental and Occupational Health & Safety (ANSES).</footnote>
<figure><img src="image_2.png" /><figcaption>Fig. 1. Overview of the articles retrieved from the search query.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>Fig. 2. Timeline of validated amphibian assays for detection of THADCs.</figcaption></figure>

Exposure periods and endpoints covered by the three validated (eco)toxicological assays employing *Xenopus 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 a flow-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 addition 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
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 de-
velopmental 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 histopatho-
logical changes of the thyroid gland, however the effects of TH an-
tagonists (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
predominantly known antagonists, and only few thyroid inactive che-
micals 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 by Pickford (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 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 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 Concentration (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 TPO *in vivo* and *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 by *in vivo* analysis. Furthermore, eleutheroembryos 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 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, a
putative 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 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 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 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 a fluorescent microscope or a fluorescence plate-reader. Thyroid
hormone agonists will cause an increase in fluorescence 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 in fluores-
cence compared to T3 alone (OECD, 2018b).
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. laevis tadpoles 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 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 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 asso-
ciated 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-induced *Xenopus* meta-
morphosis assays. After first sampling tadpole intestines which undergo
significant morphological and transcriptional variations during meta-
morphosis (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 brominated flame
retardant tetrabromobisphenol A (TBBPA) inhibited T3-induced ex-
pression 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 amniotic fluid could induce a
dose-dependent increase of fluorescence in the XETA and modulate TH
signalling genes (encoding TH transporters, and *dio1*, *dio2*). The mix-
ture also downregulated genes implicated in neural differentiation
(marker 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 in *Xenopus* tadpoles (as reviewed
by Pratt 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 in-
hibitors) are not expected to be detected (OECD TG 248, 2019). In
larger animals such as those used in the AMA or LAGDA, gene ex-
pression 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, the in
vitro TPO 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 ob-
served, 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. Teleost fish models for identification of thyroid disruption

Teleost fish 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 re-
trieved 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 by Spaan et al. (2019). However,
given the relatively recent characterization of the zebrafish HPT axis
and 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 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 an *mct8* 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 co-
ordinated by THs in *D. 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
histopathological assessment as is employed in *Xenopus* and 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*, and *hhex*) 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 in fish 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 (*nis* and *tg* (*thyroglobulin*)) (Sun et al., 2018). Particularly, Walter et al. (2019) identified a paralog of *dio3* (*dio3-b*) as being highly sensitive during early zebrafish development to both hyperthyroidism and hypothyroidism induced by exogenous THs or PTU, respectively. However, 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 of *myelin 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 gene *plp1b* (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 in *Xenopus*. 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 oligodendrocytes 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 of *synapsin 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 organophosphate flame retardant TDCPP,<fnref n="8" /> 3-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*, *a1-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. Indeed, the ontogenesis in fish with less pronounced transformations than the flatfish also involves TH (Rastorguev et al., 2016). Interestingly, pectoral fin 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

<footnote n="8">⁸ Tris (1, 3-dichloro-2-propyl) phosphate.</footnote>
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 with first-tier in chemico assays for determination of molecular initiating events (Stinckens et al., 2018). Coiling behaviour, another neurological-based endpoint, has been linked to thyroid receptor-β deficiency (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 devel-
opment 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 zebrafish thyroglobulin promoter allows for in
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 using *D. rerio* &
*X. laevis* embryos 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 dis-
ruption 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 neurobehaviour in vivo,
framing endpoints in AOPs using both in silico and in vitro assays 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 promising in
vitro high-throughput screening assays (Noyes et al., 2019).

Assessing the ecological impact of THADCs may only benefit from
added taxonomic breadth. Besides *X. laevis* and *D. rerio* studies retrieved
from 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, 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 for Fig. 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 at https://
doi.org/10.1016/j.mce.2020.110779.

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