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ERGO: Breaking Down the Wall between Human Health and Environmental Testing of Endocrine Disrupters

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ERGO (EndocRine Guideline Optimization) is the acronym of a European Union-funded research and innovation action, that aims to break down the wall between mammalian and non-mammalian vertebrate regulatory testing of endocrine disruptors (EDs), by identifying, developing and aligning thyroid-related biomarkers and endpoints (B/E) for the linkage of effects between vertebrate classes. To achieve this, an adverse outcome pathway (AOP) network covering various modes of thyroid hormone disruption (THD) in multiple vertebrate classes will be developed. The AOP development will be based on existing and new data from in vitro and in vivo experiments with fish, amphibians and mammals, using a battery of different THDs. This will provide the scientifically plausible and evidence-based foundation for the selection of B/E and assays in lower vertebrates, predictive of human health outcomes. These assays will be prioritized for validation at OECD (Organization for Economic Cooperation and Development) level. ERGO will re-think ED testing strategies from in silico methods to in vivo testing and develop, optimize and validate existing in vivo and early life-stage OECD guidelines, as well as new in vitro protocols for THD. This strategy will reduce requirements for animal testing by preventing duplication of testing in mammals and non-mammalian vertebrates and increase the screening capacity to enable more chemicals to be tested for ED properties.
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International Journal of
Molecular Sciences
Project Report
ERGO: Breaking Down the Wall between Human
Health and Environmental Testing of
Endocrine Disrupters
Henrik Holbech 1, *, Peter Matthiessen 2, Martin Hansen 3, Gerrit Schüürmann 4,5 ,
Dries Knapen 6, Marieke Reuver 7, Frédéric Flamant 8, Laurent Sachs 9, Werner Kloas 10,
Klara Hilscherova 11, Marc Leonard 12 , Jürgen Arning 13, Volker Strauss 14, Taisen Iguchi 15
and Lisa Baumann 16
1Department of Biology, University of Southern Denmark, 5230 Odense M, Denmark
2Matthiessen Consultancy, Dolfan Barn, Beulah LD5 4UE, UK; petermatthiessen12@gmail.com
3Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark;
martin.hansen@envs.au.dk
4
UFZ Department of Ecological Chemistry, Helmholtz Centre for Environmental Research, Permoserstraße 15,
04318 Leipzig, Germany; gerrit.schuurmann@ufz.de
5Institute of Organic Chemistry, Technical University Bergakademie Freiberg, Leipziger Straße 29,
09596 Freiberg, Germany
6Zebrafishlab, Department of Veterinary Sciences, University of Antwerp, 2610 Wilrijk, Belgium;
dries.knapen@uantwerpen.be
7AquaTT, Olympic House, Pleasants Street, D08 H67X Dublin, Ireland; marieke@aquatt.ie
8Functional Genomics of Thyroid Hormone Signaling Group, Institut de Génomique Fonctionnelle de Lyon
Ecole Normale Supérieure de Lyon, 69007 Lyon, France; Frederic.flamant@ens-lyon.fr
9UMR7221 Molecular Physiology and Adaption, Centre National de le Recherche Scientifique—Muséum
National d’Histoire Naturelle, CEDEX 05, 75231 Paris, France; laurent.sachs@mnhn.fr
10 Abt. Ökophysiologie und Aquakultur Leibniz-Institut für Gewässerökologie und Binnenfischerei,
12587 Berlin, Germany; werner.kloas@igb-berlin.de
11 RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic;
klara.hilscherova@recetox.muni.cz
12 Laboratoire Recherche Environnementale, L’ORÉAL Recherche & Innovation,
93601 Aulnay-sous-Bois, France; marc.leonard@rd.loreal.com
13 German Environment Agency (UBA), Section IV2.3 Chemicals, 06844 Dessau-Roßlau, Germany;
Juergen.arning@uba.de
14 BASF SE, Experimental Toxicology and Ecotoxicology, 67098 Ludwigshafen, Germany;
volker.strauss@basf.com
15 Graduate School of Nanobioscience, Yokohama City University, Yokohama 236-0027, Japan;
taiseni@hotmail.co.jp
16 Centre for Organismal Studies, University of Heidelberg, 69120 Heidelberg, Germany;
lisa.baumann@uni-heidelberg.de
*Correspondence: hol@biology.sdu.dk; Tel.: +45-65502770
Received: 31 March 2020; Accepted: 20 April 2020; Published: 22 April 2020
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Abstract:
ERGO (EndocRine Guideline Optimization) is the acronym of a European Union-funded
research and innovation action, that aims to break down the wall between mammalian and
non-mammalian vertebrate regulatory testing of endocrine disruptors (EDs), by identifying,
developing and aligning thyroid-related biomarkers and endpoints (B/E) for the linkage of eects
between vertebrate classes. To achieve this, an adverse outcome pathway (AOP) network covering
various modes of thyroid hormone disruption (THD) in multiple vertebrate classes will be developed.
The AOP development will be based on existing and new data from
in vitro
and
in vivo
experiments
with fish, amphibians and mammals, using a battery of dierent THDs. This will provide the
scientifically plausible and evidence-based foundation for the selection of B/E and assays in lower
Int. J. Mol. Sci. 2020,21, 2954; doi:10.3390/ijms21082954 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2020,21, 2954 2 of 18
vertebrates, predictive of human health outcomes. These assays will be prioritized for validation
at OECD (Organization for Economic Cooperation and Development) level. ERGO will re-think
ED testing strategies from in silico methods to
in vivo
testing and develop, optimize and validate
existing
in vivo
and early life-stage OECD guidelines, as well as new
in vitro
protocols for THD. This
strategy will reduce requirements for animal testing by preventing duplication of testing in mammals
and non-mammalian vertebrates and increase the screening capacity to enable more chemicals to be
tested for ED properties.
Keywords:
endocrine disruption; thyroid hormone disruption; AOP; adverse outcome pathway;
OECD; test guideline; integrated approach to testing and assessment; IATA; cross-species
extrapolation; biomarkers
1. Introduction
Recently, international workshops and projects arranged by the European Commission (EC)
and OECD (Organization for Economic Cooperation and Development) have identified gaps in the
testing of suspected endocrine disruptors (EDs) [
1
3
]. Regulators are therefore requesting better
tests and approaches for the assessment of the hazards and risks of EDs, to protect human health
and the environment. Based on workshop recommendations and identified gaps, the EC made
a call under Horizon 2020 Research and Innovation Actions (H2020-SC1-BHC-2018-2020, Topic:
SC1-BHC-27-2018: New testing and screening methods to identify endocrine disrupting chemicals) to
support research enabling the gaps to be filled. ERGO (acronym for EndocRine Guideline Optimization)
and 7 ‘sister’ projects signed consortium agreements with the EC in 2019 and formed the EURION
cluster [
4
]. EURION facilitates collaboration and data sharing between the 8 projects and prevents
research overlaps.
In ERGO, we address additional challenges identified by the EC and OECD. In both EU and
other international legislations, regulatory procedures for the identification and assessment of EDs
are separated for human health and the environment. Consequently, useful data obtained from
non-mammalian vertebrate tests (e.g., fish and amphibians) are disregarded, or not given sucient
weight, in human health assessments and vice versa, even though the endocrine system is highly
conserved among vertebrate classes [
5
11
]. The international workshops pointed out thyroid hormone
disruption (THD) as a focus area, because existing vertebrate
in vivo
tests are not environmentally
protective enough and validated in vitro tests for THD are not yet available.
THD has been linked with certain neurodevelopmental disorders, such as autism, attention-deficit/
hyperactivity disorder (ADHD), learning disabilities and mental retardation, including decreased
IQ and modified brain structure in children [
12
,
13
]. These disorders have been reported to increase
over the last four decades [
14
,
15
]. Meanwhile, wildlife and human contamination by anthropogenic
chemicals, including EDs, is well documented [
16
,
17
], and recommendations have been made to
more systematically evaluate the potential developmental toxicity of new and existing chemicals [
14
].
Attempts have been made to evaluate the societal cost of human and environmental exposure to
EDs, which have been controversially discussed in the scientific community [
18
20
]. In view of the
increasing emotional concerns in the media and public, the exposure to, and the resulting impact of,
EDs must be properly discussed under strict scientific standards.
Nonetheless, as reported by [
21
], even EU REACH (Registration, Evaluation, Authorization
and Restriction of Chemicals) dossiers of high tonnage chemicals (above 100 and 1000 tonnes/year)
are lacking measured developmental toxicity data. Often, animal tests have been waived through
read-across and the exploitation of existing data, but not by employing alternative test methods [
22
].
Additionally, ED-related endpoints and eects regarding the environment are still missing in REACH
standard data requirements. With respect to this, it is currently discussed to amend the REACH standard
Int. J. Mol. Sci. 2020,21, 2954 3 of 18
data sets, to explicitly cover the ED mode of actions and correlated adverse eects. To eciently cover
ED in the tiered REACH testing approach, it is of utmost importance to use human health data for
environmental assessment and vice versa. According to the OECD conceptual framework (CF) [
23
],
only
in vivo
animal tests are able to identify adverse eects induced by ED pathways. In addition to
the ethical aspects of using animals, such
in vivo
tests are lengthy and expensive. Moreover, testing
on animals (as defined by Dir. 2010/63/UE) is not allowed in Europe (and some other countries) for
the safety assessment of cosmetic ingredients and low tonnage chemicals (less than 10 tonnes/year),
which must rely on alternative methods. Consequently, these compounds can only be tested with
OECD conceptual framework (CF) level 2 (
in vitro
) and some level 3 tests (fish and amphibian early
life stages). The latter provide data about endocrine mechanisms, but are currently not designed to
detect population-relevant adverse eects (AE).
ERGO is a coordinated attempt to help fill the gaps in the field of THD for human health and
the environment. It will allow the identification of both disturbance of the thyroid axis and its
potential adverse eects in dierent vertebrate classes, including humans. ERGO is expected to
improve methodologies for using cell tests and fish and amphibian assays for the early screening of
substances. It will also develop new in silico models for predicting the internal dose of THDs to design
physiologically based toxicokinetic (PBTK) models and to link molecular initiating events (MIEs)
with AE, within an adverse outcome pathway (AOP) network. PBTK models enable quantitative
descriptions of absorption, distribution, metabolism, and the excretion of chemicals in biota, and
inform about how compound properties and physiological characteristics aect the chemical’s fate in
the organism [24].
ERGO is expected to increase basic knowledge on the detailed eects of TH disturbances.
The ERGO approach will be of significant interest for the safety assessment of existing chemicals
lacking endocrine and developmental toxicity data and new chemicals at an early stage of their
industrial development. This methodology should allow:
The simultaneous screening of chemicals for their potential human THD eects, as well as their
environmental impact with similar adverse eects on e.g., fish and amphibians.
Significantly reducing the requirement for vertebrate animal testing, thus complying with the
3Rs principle.
Assessment at the vitro scale providing new clues for automation and higher throughput screening
of chemicals, which would further reduce the cost of their assessment.
2. Concept
ERGO will provide evidence that bridging mammalian and non-mammalian testing for the
identification of EDs is justified for chemicals aecting endocrine axes across vertebrate classes.
The proof of concept of cross-vertebrate extrapolation of ED eects will be presented for the thyroid
system (Figure 1). In current relevant regulations, both in the EU and worldwide, the identification
of EDs and the follow-up in terms of hazard and risk assessment is separated between mammalian
and non-mammalian testing. The separation is historically based on general systemic toxicity testing,
where best practice has been, and still is, species- and class-specific assessment, because systemic
toxicity is recognized to be highly dependent on species- and class-specific absorption, distribution,
metabolism and excretion (ADME).
Int. J. Mol. Sci. 2020,21, 2954 4 of 18
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 17
Figure 1. ERGO (acronym for EndocRine Guideline Optimization) approach for testing and cross-
class extrapolation of THD (thyroid hormone disruption) effects between mammalian and non-
mammalian test guidelines. Based on existing information, chemicals with known molecular
initiating events (MIE), key event (KE), endocrine activity (EA) and adverse effect (AE) in mammals
will be investigated in silico, in vitro and in vivo, in fish and amphibians. A battery of biomarkers and
endpoints (B/E) will be tested for EA and AE confirmation in the in vivo studies. Biotransformation
data will be obtained when EA and/or AE cannot be confirmed. All existing and new data will be
used for AOP (adverse outcome pathway) network development. Chemicals with unknown effects
in mammals but known EA and AE in non-mammalian vertebrates will be investigated in silico, in
vitro and for differences in biotransformation among mammals and non-mammalian vertebrates.
Blue arrow: Research in ERGO, Grey arrow: Research beyond ERGO, Green arrow: confirmed cross-
class extrapolation, Red arrow: cross-class extrapolation not demonstrated.
The existing separation in ED testing between mammals and non-mammals is clearly outlined
in the OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupters, where all
in vivo testing is divided into mammalian and non-mammalian toxicology test guidelines [23]. There
is, however, increasing scientific and regulatory awareness of the value of extrapolating data between
vertebrate classes, and this is highlighted in several sections of the 2018 update of OECD guidance
document 150 [25]. Strong and increasing scientific evidence supports read-across between
mammals, fish and amphibians, and thus, ERGO will establish test systems with lower vertebrates
(fish and amphibians) for early warning and screening purposes, for not only environmental, but also
human health. To this end, a detailed understanding of the mechanisms underlying the interference
of THDs with the highly conserved vertebrate thyroid system is required to allow the comparison of
results across vertebrate classes [26,27]. Teleost fish, the phylogenetically oldest and largest group of
vertebrates, will be used to achieve this goal. Specifically, zebrafish (Danio rerio), the most popular
fish model for toxicological and vertebrate developmental research, is being used. A strong basis of
information on the regulation of specific developmental processes by THs [28,29] has made zebrafish
an important biomedical model for TH-related diseases, including obesity, cardiovascular diseases
and diabetes [30]. Thus, a comprehensive database on structure and function of the zebrafish thyroid
system is available; the ontogenetic patterns of thyroid receptors (TR) L-TRαL-Thraa, S-Thraa, Thrab
Figure 1.
ERGO (acronym for EndocRine Guideline Optimization) approach for testing and cross-class
extrapolation of THD (thyroid hormone disruption) eects between mammalian and non-mammalian
test guidelines. Based on existing information, chemicals with known molecular initiating events (MIE),
key event (KE), endocrine activity (EA) and adverse eect (AE) in mammals will be investigated in
silico,
in vitro
and
in vivo
, in fish and amphibians. A battery of biomarkers and endpoints (B/E) will
be tested for EA and AE confirmation in the
in vivo
studies. Biotransformation data will be obtained
when EA and/or AE cannot be confirmed. All existing and new data will be used for AOP (adverse
outcome pathway) network development. Chemicals with unknown eects in mammals but known
EA and AE in non-mammalian vertebrates will be investigated in silico,
in vitro
and for dierences in
biotransformation among mammals and non-mammalian vertebrates. Blue arrow: Research in ERGO,
Grey arrow: Research beyond ERGO, Green arrow: confirmed cross-class extrapolation, Red arrow:
cross-class extrapolation not demonstrated.
The existing separation in ED testing between mammals and non-mammals is clearly outlined
in the OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupters, where
all
in vivo
testing is divided into mammalian and non-mammalian toxicology test guidelines [
23
].
There is, however, increasing scientific and regulatory awareness of the value of extrapolating data
between vertebrate classes, and this is highlighted in several sections of the 2018 update of OECD
guidance document 150 [
25
]. Strong and increasing scientific evidence supports read-across between
mammals, fish and amphibians, and thus, ERGO will establish test systems with lower vertebrates
(fish and amphibians) for early warning and screening purposes, for not only environmental, but also
human health. To this end, a detailed understanding of the mechanisms underlying the interference
of THDs with the highly conserved vertebrate thyroid system is required to allow the comparison of
results across vertebrate classes [
26
,
27
]. Teleost fish, the phylogenetically oldest and largest group of
vertebrates, will be used to achieve this goal. Specifically, zebrafish (Danio rerio), the most popular
fish model for toxicological and vertebrate developmental research, is being used. A strong basis of
information on the regulation of specific developmental processes by THs [
28
,
29
] has made zebrafish
an important biomedical model for TH-related diseases, including obesity, cardiovascular diseases
and diabetes [
30
]. Thus, a comprehensive database on structure and function of the zebrafish thyroid
system is available; the ontogenetic patterns of thyroid receptors (TR) L-TRαL-Thraa, S-Thraa, Thrab
Int. J. Mol. Sci. 2020,21, 2954 5 of 18
and Thrb are fully described [
26
,
31
,
32
], and the function of the zebrafish thyroid system appears to be
fully comparable to that of higher vertebrates and humans. For instance, the molecular mechanisms
of thyroid organogenesis and the role of TR signaling during embryogenesis are well documented
for zebrafish and seem to be highly conserved between zebrafish and mammalian models [
26
,
31
].
(Neuro)developmental processes that are, at least partially, regulated by THs include eye development,
swim bladder inflation and fin formation during early embryonic development and the metamorphosis
from the larval to juvenile stage [
33
37
]. Exposure to THDs has been shown to aect these important
developmental processes, which makes them promising candidates for thyroid-related endpoints in
THD fish testing assays. For instance, inhibited dierentiation of paired fins was observed in zebrafish
exposed to dierent THDs during the transition from larval to juvenile stage [
37
]. Moreover, eye
development and the resulting visual performance were demonstrated to be disturbed by disruption of
the thyroid system with dierent THDs or thyroid-specific gene-knockouts [
33
,
36
,
38
41
]. For the impact
of THDs on the swim bladder inflation of zebrafish, a putative AOP network already exists (Figure 2,
AOP nrs. 155–159, http://aopwiki.org), which will be further used for the potential implementation of
this endpoint in fish THD testing. The use of zebrafish for screening for general toxicity and ED eects
is common practice, as evidenced by multiple OECD test guidelines (TGs) (e.g., 210, 229, 230, 234, 236).
However, none of these test systems include biomarkers/endpoints (B/E) addressing eects on the
(hypothalamus pituitary thyroid) HPT axis, which is one of the specific goals of ERGO. The inclusion
of thyroid endpoints in OECD fish TGs has recently been added as a project to the OECD Work Plan
for the Test Guidelines Programme (Project 2.64), and the AOP network on thyroperoxidase and/or
deiodinase inhibition, leading to impaired swim bladder inflation in fish during early life stages is part
of the OECD development program workplan (Project 1.35). We therefore expect that, after further
development of the AOP network, the addition of thyroid-related AOP-supported endpoints to specific
OECD TGs will be achievable within the timeframe of ERGO.
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 17
and Thrb are fully described [26,31,32], and the function of the zebrafish thyroid system appears to
be fully comparable to that of higher vertebrates and humans. For instance, the molecular
mechanisms of thyroid organogenesis and the role of TR signaling during embryogenesis are well
documented for zebrafish and seem to be highly conserved between zebrafish and mammalian
models [26,31]. (Neuro)developmental processes that are, at least partially, regulated by THs include
eye development, swim bladder inflation and fin formation during early embryonic development
and the metamorphosis from the larval to juvenile stage [3337]. Exposure to THDs has been shown
to affect these important developmental processes, which makes them promising candidates for
thyroid-related endpoints in THD fish testing assays. For instance, inhibited differentiation of paired
fins was observed in zebrafish exposed to different THDs during the transition from larval to juvenile
stage [37]. Moreover, eye development and the resulting visual performance were demonstrated to
be disturbed by disruption of the thyroid system with different THDs or thyroid-specific gene-
knockouts [33,36,3841]. For the impact of THDs on the swim bladder inflation of zebrafish, a
putative AOP network already exists (Figure 2, AOP nrs. 155-159, http://aopwiki.org), which will be
further used for the potential implementation of this endpoint in fish THD testing. The use of
zebrafish for screening for general toxicity and ED effects is common practice, as evidenced by
multiple OECD test guidelines (TGs) (e.g., 210, 229, 230, 234, 236). However, none of these test
systems include biomarkers/endpoints (B/E) addressing effects on the (hypothalamus pituitary
thyroid) HPT axis, which is one of the specific goals of ERGO. The inclusion of thyroid endpoints in
OECD fish TGs has recently been added as a project to the OECD Work Plan for the Test Guidelines
Programme (Project 2.64), and the AOP network on thyroperoxidase and/or deiodinase inhibition,
leading to impaired swim bladder inflation in fish during early life stages is part of the OECD
development program workplan (Project 1.35). We therefore expect that, after further development
of the AOP network, the addition of thyroid-related AOP-supported endpoints to specific OECD TGs
will be achievable within the timeframe of ERGO.
Figure 2. Putative AOP network for deiodinase (DIO) and thyroperoxidase (TPO) inhibition, leading
to impaired swim bladder inflation in fish. DIO inhibition causes triiodothyronine (T3) serum
decrease and reduced anterior swim bladder inflation, with reduced swimming performance and
survival as adverse consequences. https://aopwiki.org/aops/155-159.
Together with already existing literature data, ERGO will provide a comprehensive data set to
demonstrate that fish, i.e., zebrafish, are a fully adequate and effective model to assess THD effects
and extrapolate the results to other vertebrate classes including humans. Similar data sets are
currently being generated for amphibians, as over many years, the amphibian model organism
Xenopus (laevis and tropicalis) has contributed fundamentally to research in biomedicine,
neurobiology, physiology, molecular biology, cell biology, and developmental biology, making it one
of the best investigated animal models [42,43]. The frog model has provided major insight into in
vivo mechanisms of TH signaling [44], since TH-dependent developmental changes like
metamorphosis are directly observable and quantifiable. The extreme sensitivity and responsiveness
of amphibian metamorphosis to TH signaling is exploited in OECD TGs 231, 241 and 248.
Both at the molecular and morphological levels, metamorphosis in lower vertebrates bears
strong similarities with perinatal postembryonic development in mammals. In general, TH signaling
Figure 2.
Putative AOP network for deiodinase (DIO) and thyroperoxidase (TPO) inhibition, leading
to impaired swim bladder inflation in fish. DIO inhibition causes triiodothyronine (T3) serum decrease
and reduced anterior swim bladder inflation, with reduced swimming performance and survival as
adverse consequences. https://aopwiki.org/aops/155-159.
Together with already existing literature data, ERGO will provide a comprehensive data set to
demonstrate that fish, i.e., zebrafish, are a fully adequate and eective model to assess THD eects and
extrapolate the results to other vertebrate classes including humans. Similar data sets are currently
being generated for amphibians, as over many years, the amphibian model organism Xenopus (laevis
and tropicalis) has contributed fundamentally to research in biomedicine, neurobiology, physiology,
molecular biology, cell biology, and developmental biology, making it one of the best investigated
animal models [
42
,
43
]. The frog model has provided major insight into
in vivo
mechanisms of TH
signaling [
44
], since TH-dependent developmental changes like metamorphosis are directly observable
and quantifiable. The extreme sensitivity and responsiveness of amphibian metamorphosis to TH
signaling is exploited in OECD TGs 231, 241 and 248.
Both at the molecular and morphological levels, metamorphosis in lower vertebrates bears strong
similarities with perinatal postembryonic development in mammals. In general, TH signaling including
Int. J. Mol. Sci. 2020,21, 2954 6 of 18
TH transport across cell membranes, metabolism by deiodinases, and molecular mechanisms of gene
regulation (TH receptors, transcriptional cofactors, and chromatin remodeling) are conserved to a high
degree in humans, fish and amphibians (Figure 3) [27].
Figure 3.
Conservation of TH signaling in vertebrates. Processes from TH transport into the cell to
control of gene expression share homologous proteins and mechanisms in humans, fish and amphibians.
Homologous TH transporters enable TH entry into cells, where deiodinases function to activate or
deactivate it. Cytoplasmic TH binding proteins (CTHBPs) modulate cytoplasmic availability. In the
nucleus, the TH receptor (TR) forms a heterodimer with the retinoid X receptor (RXR) and binds to
DNA at TH response elements (THRE), where co-regulator complexes are recruited to alter the state of
chromatin, ultimately leading to induced expression of TH response genes. Modified from [27].
As outlined above, both EC and OECD have set a high priority in developing new approaches
for the predictive ED assessment fit for regulatory purposes. In this context, THD is known as key
challenge because of lacking information-rich
in vitro
approaches, as well as
in vitro
to
in vivo
and
environmental-human extrapolations.
ERGO was built in response to these needs and a respective EU H2020 call (see above). In the
ERGO consortium, there are several partners with long-lasting expertise with work on the endocrine
system of dierent vertebrates. As described above, evidence in the peer-reviewed literature supports
the conservation of large parts of the vertebrate endocrine system, and this evidence is growing fast,
particularly because of a focus on AOP development and increasing knowledge about ED modes of
actions (MOA), including in silico modelling, biotransformation and transcriptomics data. This proof
of conservation of the endocrine system on the one hand and the separated testing strategies on the
other was the basic motivation for ERGO to develop the AOP network based on a cross-vertebrate
class eects approach. The thyroid system was selected due to reasons already outlined, however
cross-talk investigations with other conserved endocrine axes like the hypothalamus pituitary gonadal
(HPG) axis could be included in the cross-class approach in the future as well [45].
3. Approach
The ERGO project was launched in January 2019 and consists of 15 partners from academia,
industry and regulatory bodies. ERGO is divided into eight work packages (WPs), supporting an
ecient research and outreach structure (see below).
An initial joint eort has been made to select
MIEs subject to ERGO in vivo research
MIEs subject to ERGO in vitro research
In vivo and in vitro ERGO reference compounds
Int. J. Mol. Sci. 2020,21, 2954 7 of 18
In addition to covering MIEs of primary THD relevance, the bioassay feasibility and the chemical
domain (see below) of the test compounds were taken into account. The resulting set of 28 ERGO
reference compounds covers the following MIEs: thyroid hormone receptor (THR), thyroid transport
protein (TTR), thyroid peroxidase (TPO), sodium-iodide-symporter (NIS), deiodinases I-III (DIO),
and thyroid binding globulin (TBG). Besides
in vitro
profiling of all ERGO reference compounds,
the following subset of six compounds has been selected for generating
in vivo
reference data:
ampicillin, carbamazepine, iopanoic acid, perchlorate, propylthiouracil and tetrabromobisphenol A
(see WP5). In the following, the WP implementation of the ERGO research work in the eight WPs
is outlined.
3.1. WP1 Coordination
Besides an ERGO-internal Project Oce responsible for the overall management, WP1 coordinates
our interaction with an international Scientific Advisory Board (SAB), with members from academia
and regulatory bodies from EU, the UK, North and South America. The SAB gives advice on scientific
issues and disseminate ERGO concepts and results to other communities and regulatory bodies. WP1
represents ERGO in the OECD, including the OECD Validation Management Group for Ecotoxicity
Testing (VMG-Eco) and OECD Validation Management Group for Non-Animal Testing (VMG-NA).
VMG-Eco has the role of discussing and validating new and updated TGs and biomarkers for the
environment. Partners in ERGO are Co-chairs of VMG-Eco and responsible for OECD TG project 2.64;
“Inclusion of thyroid endpoints in OECD fish Test Guidelines”.
3.2. WP2 Knowledge Management and Data Basing
To integrate the work performed within the dierent WPs and to align the scientific tasks with
the overarching strategy of the ERGO project, structured and ecient data management is crucial.
A searchable and well-structured electronic database (Figure 4), containing test data as well as literature
data and relevant meta information, supports decision making on relevant project issues, like the
selection of adequate case study chemicals at the beginning of the project. Additionally, it is essential
for the success of the project to involve all interested stakeholders from the beginning to integrate their
knowledge and needs. This is done by establishing a user reference group compiled of experts from
industry, regulatory bodies and scientists.
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 17
ERGO, SCREENED and ENDpoiNTs) and developmental neurotoxicity (ATHENA and
ENDpoiNTs).
Figure 4. ERGO data warehouse. Via a combination of existing data from data reviews and data
generated in ERGO WPs, ERGO will develop a comprehensive data warehouse composed of different
kinds of cross-vertebrate class, in silico, in vitro, in vivo samples and cell-lines samples combined with
many state-of-the-art analytical methods.
3.4. WP4 Modelling and Biotransformation
This WP addresses the chemical perspective of THD, building on existing literature and recent
research projects [4648]. The focus is on how chemical structure triggers THD MIEs and MOAs
within and across species, and how this can be predicted in silico, taking into account metabolic half-
lives and biotransformation pathways of reference substrates, and correspondingly informed
physiologically based toxicokinetic modelling (PBTK). The major deliverable will be an in silico
decision support system (DSS) for the predictive THD potential of chemical substances, featuring
qualitative and quantitative structure-activity relationships (QSARs), consensus modelling and
Bayesian statistics, to account for conflicting input information, as well as varying levels of
information and confidence.
The initial focal points were to populate our database with literature-known TH disruptors, to
characterize the chemical domain of our ERGO reference compounds as related to the EINECS
inventory (see below), to undertake computational docking with reference ligands, and to profile
biotransformation through S9 enzyme assays derived from rat hepatocytes (such as the S9 mix used
for the Ames test). Respective biotransformation studies include a fish-specific S9 enzyme assay
(OECD 319B) [49] and the results feed the PBTK modelling regarding metabolism.
3.5. WP5 Case studies for thyroid-related endpoints and biomarkers in ED test systems
By far, the largest and most experimental WP in ERGO. Based on an analysis of gaps in the
database on effects of THDs in vertebrates, ERGO WP5 will (1) identify suitable B/E for THD across
several vertebrate classes and, thus, (2) provide the experimental data required to implement THD
as an endpoint in existing or novel OECD TGs. Figure 5 outlines a potential case study with chemical
X in WP5. Such case studies are performed with a battery of THDs, with six different defined MOAs
(Appendix A): (1) NIS inhibition, (2) THR interaction, (3) TPO inhibition, (4) DIO inhibition, (5)
TTR/TBG interaction, (6) TH liver clearance. For each MOA, different model compounds have been
selected based on available information from literature, which provides sufficient evidence for THD-
specific MOAs of these compounds, without undesired side effects. The selected compounds are:
ampicillin (negative control), carbamazepine, iopanoic acid, perchlorate, propylthiouracil and
tetrabromobisphenol A. Each compound is tested and compared across all test systems, with
Figure 4.
ERGO data warehouse. Via a combination of existing data from data reviews and data
generated in ERGO WPs, ERGO will develop a comprehensive data warehouse composed of dierent
kinds of cross-vertebrate class, in silico,
in vitro
,
in vivo
samples and cell-lines samples combined with
many state-of-the-art analytical methods.
Int. J. Mol. Sci. 2020,21, 2954 8 of 18
3.3. WP3 Adverse Outcome Pathway (AOP) Network Development
Based on comparative data compilation and comprehensive scientific evidence, an AOP network
covering multiple modes of THD will be developed. Structuring all available scientific evidence
according to formal OECD AOP development principles and following the OECD review process will
support the prioritization of assays for identifying THDs across vertebrate classes for validation. Both
experimental data from literature (WP2) and new data generated by the consortium (WP4, 5, 6) are used.
As a first step, existing data on the TH regulation of eye development of fish have been summarized
and a new, hypothesized AOP network is currently being developed and regularly updated with new
data generated in ERGO experiments. Evidence will be added to existing AOPs (see Figure 2) and
new AOPs will be developed if gaps exist, e.g., interference with thyroid serum-binding protein as
a MIE. WP3 will amend the relevant AOPs in the AOP-Wiki to promote international collaboration
and visibility. Due to the principle of re-usability of KEs included in individual AOPs, this will de
facto lead to the construction of an AOP network in the AOP-Wiki.
In addition, ERGO leads the AOP working group (WG) of the EURION cluster. There are
currently around 50 individual WG members, with representatives from each of the eight cluster
projects. The mission of the AOP WG is twofold: to support and facilitate AOP-related activities in
the EURION projects, and to bring together AOP-structured information and data across projects.
The WG is organizing AOP training workshops as a function of needs within the projects, AOP
development workshops as data becomes available, and regular meetings and teleconferences to align
AOP-related activities and stimulate collaboration across projects. Examples of specific opportunities for
collaborative AOP development that have been identified include THD (ATHENA, ERGO, SCREENED
and ENDpoiNTs) and developmental neurotoxicity (ATHENA and ENDpoiNTs).
3.4. WP4 Modelling and Biotransformation
This WP addresses the chemical perspective of THD, building on existing literature and recent
research projects [
46
48
]. The focus is on how chemical structure triggers THD MIEs and MOAs within
and across species, and how this can be predicted in silico, taking into account metabolic half-lives and
biotransformation pathways of reference substrates, and correspondingly informed physiologically
based toxicokinetic modelling (PBTK). The major deliverable will be an in silico decision support
system (DSS) for the predictive THD potential of chemical substances, featuring qualitative and
quantitative structure-activity relationships (QSARs), consensus modelling and Bayesian statistics,
to account for conflicting input information, as well as varying levels of information and confidence.
The initial focal points were to populate our database with literature-known TH disruptors,
to characterize the chemical domain of our ERGO reference compounds as related to the EINECS
inventory (see below), to undertake computational docking with reference ligands, and to profile
biotransformation through S9 enzyme assays derived from rat hepatocytes (such as the S9 mix used for
the Ames test). Respective biotransformation studies include a fish-specific S9 enzyme assay (OECD
319B) [49] and the results feed the PBTK modelling regarding metabolism.
3.5. WP5 Case Studies for Thyroid-Related Endpoints and Biomarkers in ED Test Systems
By far, the largest and most experimental WP in ERGO. Based on an analysis of gaps in the
database on eects of THDs in vertebrates, ERGO WP5 will (1) identify suitable B/E for THD across
several vertebrate classes and, thus, (2) provide the experimental data required to implement THD as
an endpoint in existing or novel OECD TGs. Figure 5outlines a potential case study with chemical
X in WP5. Such case studies are performed with a battery of THDs, with six dierent defined
MOAs (Appendix A): (1) NIS inhibition, (2) THR interaction, (3) TPO inhibition, (4) DIO inhibition,
(5) TTR/TBG interaction, (6) TH liver clearance. For each MOA, dierent model compounds have
been selected based on available information from literature, which provides sucient evidence for
THD-specific MOAs of these compounds, without undesired side eects. The selected compounds
Int. J. Mol. Sci. 2020,21, 2954 9 of 18
are: ampicillin (negative control), carbamazepine, iopanoic acid, perchlorate, propylthiouracil and
tetrabromobisphenol A. Each compound is tested and compared across all test systems, with dierent
vertebrate classes and life stages. B/E are being assessed according to the AOP concept, ranging
from the molecular, over morphological, up to the physiological level. Special emphasis is put on
the identification of new THD-related B/E in fish, preferably in embryonic stages to comply with the
3R principle. Current focus of the experimental work in WP5 is on THD-induced disturbance of the
development of eyes and swim bladder in fish. The data basis for the latter is very broad, and a putative
AOP network has already been developed (see Figure 2). A similar data set on the eects THDs on
eye development is currently being collected from literature and experiments performed by dierent
ERGO partners. The first results show that the eye development could serve as a meaningful B/E in
already existing OECD TGs with fish, including early life stages. These new fish B/E are compared with
already established B/E from amphibian testing and potentially new B/E for amphibians are assessed
as well.
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 17
different vertebrate classes and life stages. B/E are being assessed according to the AOP concept,
ranging from the molecular, over morphological, up to the physiological level. Special emphasis is
put on the identification of new THD-related B/E in fish, preferably in embryonic stages to comply
with the 3R principle. Current focus of the experimental work in WP5 is on THD-induced disturbance
of the development of eyes and swim bladder in fish. The data basis for the latter is very broad, and
a putative AOP network has already been developed (see Figure 2). A similar data set on the effects
THDs on eye development is currently being collected from literature and experiments performed
by different ERGO partners. The first results show that the eye development could serve as a
meaningful B/E in already existing OECD TGs with fish, including early life stages. These new fish
B/E are compared with already established B/E from amphibian testing and potentially new B/E for
amphibians are assessed as well.
An in vitro bioassay battery is being set up to address identified cross-species priority molecular
initiating/key events and to support in vivo studies. The models for studying the prioritized
endpoints and a set of biomarkers include human/mammalian cell lines from thyroid, liver and
neural stem cells. The effect of 2D and 3D cultivation of the cells on TH balance relevant endpoints is
examined. The in vitro bioassay battery includes the assays listed in Figure 5.
Figure 5. Example of a potential case study in WP5 with compound X.
3.6. WP6 Mammalian endpoints and epidemiology
Current mammalian assays for THDs have been criticized for both ethical and technical reasons.
In compliance with the 3Rs principle, WP6 proposes to refine these assays to improve sensitivity and
precision, and thus to reduce the number of animals notably by increasing the precision of the
endpoint’s measurement. A distinction is made between endpoints which relate to the maintenance
of steady state levels of T4 and T3 in blood, and endpoints which represent the tissue response to T3.
These deserve special attention, because THDs can alter neurodevelopment and metabolism without
modifying the circulating levels of T4/T3. WP6 explores the possibilities currently offered by “omics”,
combining metabolomics (mass spectrometry), transcriptome analysis (RNAseq), and the genome-
wide analysis of chromatin occupancy (ChipSeq) to characterize T3 signaling and the AO of THD
exposure. A new transgenic mouse model (Cre:LoxP technology) to restrict the expression of T3
Fish
in vivo
test
Experiment: Fish early life stage test (OECD TG 210)
Analyses: TH levels (LC-MS), thyroid follicles (histology), swimbladder inflation
(morphometry), eye development (morphometry, histology,
immunofluorescence)
Fish
in vitro
/embryo test
Experiment: Fish embryo toxicity test (OECD TG 236)
Analyses: TH levels (LC-MS), thyroid follicles (histology, immunofluorescence),
posterior swimbladder chamber inflation (morphometry), eye development
(morphometry, his tology, immunofluorescence)
Amphibian
in vivo
test
Experiment: Amphibian metamorpho sis assay (OECD TG 231)
Analyses: Metamorphosis, TH levels (LC-MS), thyroid follicles (histology,
immunofluorescence), eye development (morphometry, histology,
immunofluorescence)
Mamm alian
in vitro
test
Experiments: TR transactivation; NIS, TPO, DIO -inhibition; T4-TTR/TBG
displacement; targe ted transcriptomics; novel pri oritized B/Es
Analyses: luminescence, fluore scence, absorbance assay, qPCR
Metabolomics assay
Experiment: Metabolome characterization of tissue homogenate and plasma
samples from
in v ivo
and cell experiments (ultra-high resolution mass spec.)
Analyses: Identify biomarkers and highlight metabolic pathways for TD
(advanced
in silico
workflows).
Genomics assay
Experiment: RNA and DNA is olation from samples of
in vivo
and cell experiments
Analyses: RNAseq, MethylCAPseq, Deseq2 (list of differentially expressed genes
and methylated DNA locus, expression and methylation profiles, molecular
pathways, system biology).
New
TD-related
endpoints
and
biomarkers
for
fish
Additional
TD-related
endpoints
and biomarkers
for amphibians
New
TD-related
biomarker
for mammals
Comparative
information on TD-
induced metabolic
changes in
vertebrates
New/Comparative
TD-related
molecular
pathways in
vertebrates
AOP & TG
develop-
ment
Case study: TD substance X
Figure 5. Example of a potential case study in WP5 with compound X.
An
in vitro
bioassay battery is being set up to address identified cross-species priority molecular
initiating/key events and to support
in vivo
studies. The models for studying the prioritized endpoints
and a set of biomarkers include human/mammalian cell lines from thyroid, liver and neural stem
cells. The eect of 2D and 3D cultivation of the cells on TH balance relevant endpoints is examined.
The in vitro bioassay battery includes the assays listed in Figure 5.
3.6. WP6 Mammalian Endpoints and Epidemiology
Current mammalian assays for THDs have been criticized for both ethical and technical reasons.
In compliance with the 3Rs principle, WP6 proposes to refine these assays to improve sensitivity
and precision, and thus to reduce the number of animals notably by increasing the precision of the
endpoint’s measurement. A distinction is made between endpoints which relate to the maintenance
of steady state levels of T4 and T3 in blood, and endpoints which represent the tissue response to
T3. These deserve special attention, because THDs can alter neurodevelopment and metabolism
Int. J. Mol. Sci. 2020,21, 2954 10 of 18
without modifying the circulating levels of T4/T3. WP6 explores the possibilities currently oered by
“omics”, combining metabolomics (mass spectrometry), transcriptome analysis (RNAseq), and the
genome-wide analysis of chromatin occupancy (ChipSeq) to characterize T3 signaling and the AO of
THD exposure. A new transgenic mouse model (Cre:LoxP technology) to restrict the expression of T3
receptors with mutation or tags [
50
] allows the study of T3 signaling in specific cell types with intact
tissues. An epidemiological study including the CELSPAC birth cohort with focus on a potential role
of exposure in THD-related developmental disorders will employ an AOP network-based strategy
and will be closely coordinated with activities within the European Human Biomonitoring Initiative
(HBM4EU).
3.7. WP7 Pre-Validation and Validation of Biomarkers and Endpoints
The most valuable tools in the regulation of chemicals are standardized TGs with sensitive B/E,
that address the concerns of specific hazardous properties of chemicals. Such test methods have been
developed for the evaluation of EDs in the regime of OECD, to ensure international standardization
and mutual acceptance of data. WP7 will support OECD TGs with new B/E for THD. B/E selected,
developed and tested in WP2,3,4,5 and 6 will only go into WP7 for validation or pre-validation if
they have been evaluated as suitable for the cross-class extrapolation of eects from non-mammalian
vertebrates to mammals. Dependent on the TRL (technological readiness level) of the B/E, either
a pre-validation will be performed within ERGO or an OECD validation including external participants
will be carried out.
3.8. WP8 Dissemination and Exploitation
WP8 ensures that the outcomes and achievements of ERGO are eectively transferred to target-
and end-users and that there is a measurable impact of ERGOs results on society. A dissemination and
exploitation plan (DEP) is integrated in the project design, in order to maximize exposure of the project’s
progression and contributions to science and society. A tailored knowledge transfer methodology is
used to identify potential scientific, industrial and societal applications of the project’s outputs across
a variety of sectors and ensure they achieve measurable impact. In coordination with the Innovation
Task Force and WP2, WP8 has established supporting protocols for the management of Intellectual
Property of ERGO’s results. ERGO has established a dedicated project website (www.ergo-project.eu)
as the main tool for promoting the project and disseminating the project’s objectives, work plan and
results to a wide audience, including all stakeholders and possible end-users. To ensure successful
promotion of the project and to sustain the interest of the target audience and attract new users, the
website’s content will be maintained, continuously updated and populated with new information
throughout the project’s lifetime. The website will remain active for five years after the end of the
project, to serve as a valuable public resource of research information on the subject and for promoting
the outputs of publicly funded research in the domain beyond the project’s lifetime. Social networking
is part of the ERGO communication strategy and a dedicated project Twitter account (@ERGO_EU)
was set up at the start of the project and is used to publicly “tweet” ERGO relevant information.
Project-related tweets are posted regularly in accordance with the H2020 Programme Guidance Social
media guide for EU funded R+I projects.
In addition, ERGO leads the EURION Communication working group and have set up and are
maintaining the cluster website and social media activities.
4. ERGO vs. EINECS Chemical Domain
An important issue regarding
in vitro
bioassays are factors controlling the bioavailability of test
substances. In this context, compound dissolution may be hampered by sorption (to vials as well as
to extracellular matrix) and volatilization, which in turn are driven by hydrophobicity and Henry’s
law constant, respectively. Moreover, these physicochemical properties aect both toxicokinetics and
toxicodynamics, considering the variation in compound anity for water-rich vs. water-poor tissues
Int. J. Mol. Sci. 2020,21, 2954 11 of 18
(compartments), pathways and receptor sites. From this viewpoint, it is of interest to profile the
physicochemical space covered by our ERGO reference compounds.
According to EPISuite [
51
] and ACD/Percepta [
52
] calculations (Figure 6), the approximate property
ranges of the ERGO reference set of compounds (Appendix A) are as follows: log K
ow
(hydrophobicity,
octanol-water partition coecient) from
5 to 8, log H(Henry’s law constant, [Pa m
3
mol
1
]) from
17
to 1, log S
w
(water solubility, [mol L
1
]) from
11 to 1), and pK
a
(dissociation constant), indicating
the predominant speciation at pH 7. This wide physicochemical profile addresses potential
in vitro
challenges regarding sorption, volatilization and resultant bioavailability.
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 17
m
3
mol
1
]) from 17 to 1, log S
w
(water solubility, [mol L
1
]) from 11 to 1), and pK
a
(dissociation
constant), indicating the predominant speciation at pH 7. This wide physicochemical profile
addresses potential in vitro challenges regarding sorption, volatilization and resultant bioavailability.
Figure 6. Physicochemical domain of 28 ERGO reference compounds in terms of log K
ow
(octanol-
water partition coefficient), log H (Henry’s law constant) and log S
w
(water solubility) as calculated
through EPISuite ([51], augmented by calculated pK
a
-driven [52] main speciation.
In Figure 7, the ERGO physicochemical space (red) is compared with the one of the EINECS
subset of 56703 compounds (grey), that have well-defined chemical structures and yield formally
valid EPISuite results. In this graph, the calculated properties of EINECS compounds outside
reasonable ranges (as specified in the right part of the figure) are collected at the respective property
edges (light grey), facilitating visual inspection of the ERGO vs. EINECS setting. The latter shows
that the 28 ERGO reference substances cover a significant portion of the EINECS domain regarding
bioavailability-related properties.
Figure 6.
Physicochemical domain of 28 ERGO reference compounds in terms of log K
ow
(octanol-water
partition coecient), log H(Henry’s law constant) and log S
w
(water solubility) as calculated through
EPISuite ([51], augmented by calculated pKa-driven [52] main speciation.
In Figure 7, the ERGO physicochemical space (red) is compared with the one of the EINECS
subset of 56703 compounds (grey), that have well-defined chemical structures and yield formally valid
EPISuite results. In this graph, the calculated properties of EINECS compounds outside reasonable
ranges (as specified in the right part of the figure) are collected at the respective property edges
(light grey), facilitating visual inspection of the ERGO vs. EINECS setting. The latter shows that
the 28 ERGO reference substances cover a significant portion of the EINECS domain regarding
bioavailability-related properties.
Although the physiochemical domain provides an important aspect of a given compound set,
it does not inform directly about the associated structural domain. For the latter, the atom-centered
fragment (ACF) approach [
53
,
54
] has been applied for the ERGO-EINECS comparison, considering all
72,520 EINECS compounds with defined chemical structures. After correcting for various structural
chemistry issues in EINECS, the result is as follows: one of our 28 ERGO reference compounds (DON)
is borderline outside, one is borderline inside (BDE47), three (PTU, MMI, HBCD) are inside EINECS
with however quite unique structural features, and 23 (82%) have structural features well covered by
EINECS. This probably reflects the fact that bioactive compounds may contain structural features which
are systematically dierent from industrial compounds. Moreover, it demonstrates that substances with
physiochemical properties fitting well to a certain chemical inventory (here: EINECS) may nevertheless
be outside the respective structural domain. It follows further that the ERGO reference compounds
cover both EINECS-typical and outside-EINECS structural features and highlights the importance in
addressing chemical structure when assessing chemical domain belongings.
Int. J. Mol. Sci. 2020,21, 2954 12 of 18
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 17
m
3
mol
1
]) from 17 to 1, log S
w
(water solubility, [mol L
1
]) from 11 to 1), and pK
a
(dissociation
constant), indicating the predominant speciation at pH 7. This wide physicochemical profile
addresses potential in vitro challenges regarding sorption, volatilization and resultant bioavailability.
Figure 6. Physicochemical domain of 28 ERGO reference compounds in terms of log K
ow
(octanol-
water partition coefficient), log H (Henry’s law constant) and log S
w
(water solubility) as calculated
through EPISuite ([51], augmented by calculated pK
a
-driven [52] main speciation.
In Figure 7, the ERGO physicochemical space (red) is compared with the one of the EINECS
subset of 56703 compounds (grey), that have well-defined chemical structures and yield formally
valid EPISuite results. In this graph, the calculated properties of EINECS compounds outside
reasonable ranges (as specified in the right part of the figure) are collected at the respective property
edges (light grey), facilitating visual inspection of the ERGO vs. EINECS setting. The latter shows
that the 28 ERGO reference substances cover a significant portion of the EINECS domain regarding
bioavailability-related properties.
Figure 7.
ERGO vs. EINECS physicochemical domain in terms of log K
ow
(octanol-water partition
coecient), log H(Henry’s law constant) and log S
w
(water solubility), as calculated through
EPISuite [
51
]. Among 100,102 EINECS entries, 72,520 have defined chemical structures, of which
56,703 compounds with formally valid EIPSuite results (grey) are compared to the 28 ERGO reference
compounds (red). EINECS compounds with calculated properties outside reasonable ranges are plotted
in the property edges (light grey).
5. Expected Impact
ERGO is expected to develop an IATA across mammalian (rodent) and non-mammalian (fish,
amphibian) vertebrates as proof-of-concept, to assess compounds for their THD potential through
a battery of AOP-targeted
in vivo
experiments,
in vitro
bioassays, omics and in silico information.
To this end, novel MOA-specific B/E and structure-activity relationships will be profiled for their
predictive THD information content, with particular attention for within-species vs. across-species
AOP networks and their MIEs and KEs. In particular, state-of-the-art molecular biology in combination
with
in vitro
research and its assessment through judiciously selected reference investigations
in vivo
will be complemented by in silico information of molecular-level THD action regarding protein-ligand
interaction and PBTK. Accordingly, major outcomes include:
Identification of novel THD-related endpoints for
in vivo
testing with non-mammalian vertebrates
MIE/KE-specific
in vitro
protocols to assess the potential of THDs for triggering respective AOPs
MIE/KE-specific omics profiles informing about AOP progress
Opportunities for AOP information across mammalian and non-mammalian vertebrate TH disruption
THD-relevant AOP crosstalk patterns, and
Structure-activity insight into AOP/THD-specific MIEs
These will serve as novel components of an overall IATA, augmented by a decision-support scheme
in order to help converting many-endpoint/method information into a coherent THD assessment
strategy. The respective proof-of-concept is envisaged to be ready for subsequent OECD validation,
thus fostering a refinement, reduction and replacement of
in vivo
THD testing, in line with current
needs to overcome practical 3R barriers in the REACH context. Furthermore, the results obtained here
can be used to support conclusions on ED properties of substances, e.g., under the biocidal and plant
protection framework without the need of extensive in vivo testing.
Int. J. Mol. Sci. 2020,21, 2954 13 of 18
6. Outreach and Implementation
During the course of the project, ERGO will ensure close collaboration and harmonization with
other European projects and initiatives on ED testing, e.g., the ongoing JRC work on THD
in vitro
testing or dierent EU Tender projects on optimization of existing OECD TGs. Based on the outcomes
of the knowledge management and transfer activities, ERGO will organize three focused workshops
that will aim to transfer project knowledge to priority target users. The first workshop will be targeted
at end users of OECD TGs, including contract laboratories and larger enterprises, intended to share
the results of ERGO and feed them into policy and regulatory processes. A second workshop will
be opened up to a broader range of actors involved in the regulatory process across Europe, sharing
ERGO knowledge outputs such as scientific findings, recommendations, and datasets, that could be
taken up and applied by others. A specific training session will be provided on how to use key outputs.
The third workshop will be a final project showcase event to share the achievements of the project to
a wide stakeholder base interested in hearing about the results and impacts of ERGO. Representatives
from EURION and any other projects and initiatives working on EDs will be invited to these events.
At the end of the project, a key achievements publication outlining the knowledge outputs generated
by ERGO, and the transfer activities that took place within the project, will be widely distributed for
outreach to wider society. It will include a roadmap for post project actions that may be needed to
maximize the impacts of the project.
7. Conclusions
The overall concept of ERGO is to improve the hazard and risk assessment of EDs for the protection
of human health and the environment, by introducing a paradigm shift in the scientific and regulatory
use of TG data. ERGO aims to break down the existing wall between mammalian and non-mammalian
vertebrate testing, by demonstrating that it is feasible to extrapolate eects of EDs across vertebrate
classes, i.e., an adverse eect on an endocrine-specific endpoint observed in a fish or amphibian
study will also raise concerns about a possible adverse eect in humans. Thyroid-related biomarkers
and apical endpoints suitable for the extrapolation of eects in fish and amphibians to humans and
other mammals (and vice versa) are investigated, evaluated and finally validated for inclusion in
existing or new OECD TGs. A cross-class AOP network will provide the scientifically plausible and
evidence-based foundation for the selection of B/E in lower vertebrate assays predictive of human
health outcomes. These assays will be prioritized in ERGO, in preparation for international validation
through OECD. ERGO will also develop and implement a novel concept of an AOP network-based
strategy for epidemiological and human exposure studies (e.g., from the workplace), allowing the
evaluation of mechanistically based associations of external and internal exposure with THD-related
health disorders. This strategy enables the prioritization of risk drivers and provides information for
the improved hazard and risk assessment of THD.
Author Contributions:
H.H., P.M., M.H., G.S., D.K., M.R., F.F., L.S., W.K., K.H., M.L., J.A., V.S., T.I. and L.B. have
contributed to the ERGO Horizon 2020 application and the content of the present project report. All authors have
read and agreed to the published version of the manuscript.
Funding:
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 13 of 17
taken up and applied by others. A specific training session will be provided on how to use key
outputs. The third workshop will be a final project showcase event to share the achievements of the
project to a wide stakeholder base interested in hearing about the results and impacts of ERGO.
Representatives from EURION and any other projects and initiatives working on EDs will be invited
to these events. At the end of the project, a key achievements publication outlining the knowledge
outputs generated by ERGO, and the transfer activities that took place within the project, will be
widely distributed for outreach to wider society. It will include a roadmap for post project actions
that may be needed to maximize the impacts of the project.
7. Conclusions
The overall concept of ERGO is to improve the hazard and risk assessment of EDs for the
protection of human health and the environment, by introducing a paradigm shift in the scientific
and regulatory use of TG data. ERGO aims to break down the existing wall between mammalian and
non-mammalian vertebrate testing, by demonstrating that it is feasible to extrapolate effects of EDs
across vertebrate classes, i.e., an adverse effect on an endocrine-specific endpoint observed in a fish
or amphibian study will also raise concerns about a possible adverse effect in humans. Thyroid-
related biomarkers and apical endpoints suitable for the extrapolation of effects in fish and
amphibians to humans and other mammals (and vice versa) are investigated, evaluated and finally
validated for inclusion in existing or new OECD TGs. A cross-class AOP network will provide the
scientifically plausible and evidence-based foundation for the selection of B/E in lower vertebrate
assays predictive of human health outcomes. These assays will be prioritized in ERGO, in preparation
for international validation through OECD. ERGO will also develop and implement a novel concept
of an AOP network-based strategy for epidemiological and human exposure studies (e.g., from the
workplace), allowing the evaluation of mechanistically based associations of external and internal
exposure with THD-related health disorders. This strategy enables the prioritization of risk drivers
and provides information for the improved hazard and risk assessment of THD.
Author Contributions: All authors; HH, PM, MH, GS, DK, MR, FF, LS, WK, KH, ML, JA, VS, TI and LB have
contributed to the ERGO Horizon 2020 application and the content of the present project report.
Funding:
This project has received funding from the European Union’s Horizon 2020 research and
innovation program, under grant agreement No. 825753 (ERGO). This output reflects only the
author’s view and the European Union cannot be held responsible for any use that may be
made of the information contained therein.
Acknowledgments: The authors would like to acknowledge the ERGO Scientific Advisory Board (SAB) for their
support and constructive input to the scientific tasks in ERGO. A special thanks to Harald Hasler-Sheetal for the
preparation of Figure 4.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
ADHD Attention Deficit Hyperactivity Disorder
ADME Absorption, Distribution, Metabolism and Excretion
AE Adverse Effect
AOP Adverse Outcome Pathway
B/E Biomarkers and Endpoints
CF Conceptual Framework
CTHBP Cytoplasmic Thyroid Hormone Binding Proteins
DEP Dissemination and Exploitation Plan
DIO Deiodinase
DSS Decision Support System
EA Endocrine Activity
EC European Commission
ED Endocrine Disruption/Disruptor
This project has received funding from the European Union’s Horizon 2020 research and innovation program,
under grant agreement No. 825753 (ERGO). This output reflects only the author’s view and the European Union
cannot be held responsible for any use that may be made of the information contained therein.
Acknowledgments:
The authors would like to acknowledge the ERGO Scientific Advisory Board (SAB) for their
support and constructive input to the scientific tasks in ERGO. A special thanks to Harald Hasler-Sheetal for the
preparation of Figure 4.
Int. J. Mol. Sci. 2020,21, 2954 14 of 18
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
ADHD Attention Deficit Hyperactivity Disorder
ADME Absorption, Distribution, Metabolism and Excretion
AE Adverse Eect
AOP Adverse Outcome Pathway
B/E Biomarkers and Endpoints
CF Conceptual Framework
CTHBP Cytoplasmic Thyroid Hormone Binding Proteins
DEP Dissemination and Exploitation Plan
DIO Deiodinase
DSS Decision Support System
EA Endocrine Activity
EC European Commission
ED Endocrine Disruption/Disruptor
MIE Molecular Initiating Event
EU European Union
HPG Hypothalamus Pituitary Gonadal
HPT Hypothalamus Pituitary Thyroid
JRC Joint Research Centre
KE Key Event
MIE Molecular Initiating Event
MOA Mode of Action
NIS Sodium-Iodide-Symporter
OECD Organization for Economic Cooperation and Development
PBTK Physiologically Based Toxicokinetic
QSAR Quantitative Structure-Activity Relationships
REACH Registration, Evaluation, Authorization and Restriction of Chemicals
RXR Retinoid X Receptor
SAB Scientific Advisory Board
TBG Thyroid Binding Globulin
TG Test Guideline
TH Thyroid Hormone
THD Thyroid Hormone Disruption/Disruptor
THRE Thyroid Hormone Responsive Element
TPO Thyroperoxidase
TR Thyroid Receptor
TRL Technological Readiness Level
TTR Thyroid Transport Protein
VMG-Eco OECD Validation Management Group for Ecotoxicity Testing
VMG-NA OECD Validation Management Group for Non-Animal Testing
WG Working Group
WP Work Package
Appendix A
Lists of compounds used in ERGO: chemicals selected for FET (fish embryo toxicity) testing, their abbreviation,
CAS number, MIE and indication for further testing in in vitro and in vivo tests.
Int. J. Mol. Sci. 2020,21, 2954 15 of 18
Abbreviation Chemical Name CAS# In Vivo/In Vitro
AMIO Amiodarone 19774-82-4
AMP Ampicillin 69-53-4 in vivo, in vitro
BP2 2,20-4,40-tetrahydroxy benzophenone 131-55-5 in vitro
BPA 2,2-Bis(4-hydroxyphenyl)propane (Bisphenol A) 80-05-7 in vitro
CBZ Carbamazepine 298-46-4 in vivo, in vitro
ETU Ethylene thiourea 96-45-7
IOP Iopanoic acid 96-83-3 in vivo, in vitro
MMI Methimazole 60-56-0
PCL Perchlorate 14797-73-0 in vivo, in vitro
PFBS Nonafluorobutane-1-sulfonic acid 375-73-5
PFOA Perfluorooctanoic acid 335-67-1 in vitro
PTU 6-propylthiouracil 51-52-5 in vivo, in vitro
SA Salicylic acid 69-72-7
SMX Sulfamethoxazol 723-46-6
T3 3,30,5-Triiodo-L-thyronine 6893-02-3 in vitro
T4 3,30,5,5”-Tetraiodo-L-thyronine 51-48-9 in vitro
TBBPA Tetrabromobisphenol A 79-94-7 in vivo, in vitro
TBP Tribromophenol 118-79-6
TCBPA Tetrachlorobisphenol A 79-95-8
TCS Triclosan 3380-34-5
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... A more sensitive study results in a lower effect value and is hence more protective when used as a key study in decisionmaking. One example of this is the rapid test development within research on endocrine disruption, and subsequent use of these studies in risk assessments [13,52]. Still, this use has not been straightforward. ...
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... EURION is a cluster of eight research projects, funded under the European Commission's Horizon 2020 Research and Innovation Programme, focusing on developing new testing and screening methods identifying EDs and studying their effects on metabolism Küblbeck et al., 2020;Legler et al., 2020), female reproductive toxicity (van Duursen et al., 2020), developmental neurotoxicity (Lupu et al., 2020) and thyroid-mediated toxicity (Holbech et al., 2020;Kortenkamp et al., 2020;Moroni et al., 2020). AOP development is an integral part of each of these projects (Street et al., 2021). ...
... The development of specific chemical safety testing is required (Browne et al., 2020) with special attention to neurological development Kortenkamp et al., 2020;O'Shaughnessy and Gilbert, 2020). Attention is also paid to break down the wall between mammalian and non-mammalian vertebrate regulatory testing (Couderq et al., 2020;Holbech et al., 2020). In addition to phenotypic or histological end-points, the use of molecular assays (transcripts, proteins or metabolites) will be more sensitive and most of all will allow detection before adverse effects occur (Fini et al., 2007;Kulkarni and Buchholz, 2013). ...
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Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and involve diverse chemical-receptor interactions that can perturb hormone signaling. The Organization for Economic Co-operation and Development has validated several EDC-receptor bioassays to detect endocrine active chemicals and has established guidelines for regulatory testing of EDCs. Focus on testing over the past decade has been initially directed to EATS modalities (estrogen, androgen, thyroid, and steroidogenesis) and validated tests for chemicals that exert effects through non-EATS modalities are less established. Due to recognition that EDCs are vast in their mechanisms of action, novel bioassays are needed to capture the full scope of activity. Here, we highlight the need for validated assays that detect non-EATS modalities and discuss major international efforts underway to develop such tools for regulatory purposes, focusing on non-EATS modalities of high concern (i.e., retinoic acid, aryl hydrocarbon receptor, peroxisome proliferator-activated receptor, and glucocorticoid signaling). Two case studies are presented with strong evidence amongst animals and human studies for non-EATS disruption and associations with wildlife and human disease. This includes metabolic syndrome and insulin signaling (case study 1) and chemicals that impact the cardiovascular system (case study 2). This is relevant as obesity and cardiovascular disease represent two of the most significant health-related crises of our time. Lastly, emerging topics related to EDCs are discussed, including recognition of crosstalk between the EATS and non-EATS axis, complex mixtures containing a variety of EDCs, adverse outcome pathways for chemicals acting through non-EATS mechanisms, and novel models for testing chemicals. Recommendations and considerations for evaluating non-EATS modalities are proposed. Moving forward, improved understanding of the non-EATS modalities will lead to integrated testing strategies that can be used in regulatory bodies to protect environmental, animal, and human health from harmful environmental chemicals.
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Many regulations are beginning to explicitly require investigation of a chemical's endocrine disrupting properties as a part of the safety assessment process, for substances already on or about to be placed on the market. Different jurisdictions are applying distinct approaches. However, all share a common theme requiring testing for endocrine activity and adverse effects, typically involving in vitro and in vivo assays on selected endocrine pathways. For ecotoxicological evaluation, in vivo assays can be performed across various animal species, including mammals, amphibians, and fish. Results indicating activity (i.e., that a test substance may interact with the endocrine system) from in vivo screens usually trigger further higher‐tier in vivo assays. Higher‐tier assays provide data on adverse effects on relevant endpoints over more extensive parts of the organism's life cycle. Both in vivo screening and higher‐tier assays are animal‐ and resource‐intensive and can be technically challenging to conduct. Testing large numbers of chemicals will inevitably result in the use of large numbers of animals, contradicting stipulations set out within many regulatory frameworks that animal studies be conducted as a last resort. Improved strategies are urgently required. In February 2020, the UK's National Centre for the 3Rs and the Health and Environmental Sciences Institute hosted a workshop (“Investigating Endocrine Disrupting Properties in Fish and Amphibians: Opportunities to Apply the 3Rs”). Over 50 delegates attended from North America and Europe, across academia, laboratories and consultancies, regulatory agencies, and industry. Challenges and opportunities in applying refinement and reduction approaches within the current animal test guidelines were discussed, and utilization of replacement/new approach methodologies, including in silico, in vitro, and embryo models, was explored. Efforts and activities needed to enable application of 3Rs approaches in practice were also identified. This article provides an overview of the workshop discussions and sets priority areas for follow‐up. This article is protected by copyright. All rights reserved.
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Recent regulatory testing programs have been designed to evaluate whether a chemical has the potential to interact with the endocrine system and could cause adverse effects. Some endocrine pathways are highly conserved among vertebrates, providing a potential to extrapolate data generated for one vertebrate taxonomic group to others (i.e., biological read‐across). To assess the potential for biological read‐across, we reviewed tools and approaches that support species extrapolation for fish, amphibians, birds, and reptiles. For each of the estrogen, androgen, thyroid, and steroidogenesis (EATS) pathways, we considered the pathway conservation across species and the responses of endocrine sensitive endpoints. Available data show a high degree of confidence in the conservation of the hypothalamus‐pituitary‐gonadal axis between fish and mammals and the hypothalamus‐pituitary‐thyroid axis between amphibians and mammals. Comparatively, there is less empirical evidence for the conservation of other EATS pathways between other taxonomic groups, but this may be due to limited data. While more information on sensitive pathways and endpoints would be useful, current developments in the use of molecular target sequencing similarity tools and thoughtful application of the adverse outcome pathway concept show promise for further advancement of read‐across approaches for testing EATS pathways in vertebrate ecological receptors. This article is protected by copyright. All rights reserved.
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Next-generation sequencing technologies have revolutionized the identification of disease-causing genes, accelerating the discovery of new mutations and new candidate genes for thyroid diseases. To face this flow of novel genetic information, it is important to have suitable animal models to study the mechanisms regulating thyroid development and thyroid hormone availability and activity. Zebrafish (Danio rerio), with its rapid external embryonic development, has been extensively used in developmental biology. To date, almost all of the components of the zebrafish thyroid axis have been characterized and are structurally and functionally comparable with those of higher vertebrates. The availability of transgenic fluorescent zebrafish lines allows the real-time analysis of thyroid organogenesis and its alterations. Transient morpholino-knockdown is a solution to silence the expression of a gene of interest and promptly obtain insights on its contribution during the development of the zebrafish thyroid axis. The recently available tools for targeted stable gene knockout have further increased the value of zebrafish to the study of thyroid disease. All of the reported zebrafish models can also be used to screen small compounds and to test new drugs and may allow the establishment of experimental proof of concept to plan subsequent clinical trials.
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Thyroid hormone (TH) signaling comprises TH transport across cell membranes, metabolism by deiodinases, and molecular mechanisms of gene regulation. Proper TH signaling is essential for normal perinatal development, most notably for neurogenesis and fetal growth. Knowledge of perinatal TH endocrinology needs improvement to provide better treatments for premature infants and endocrine diseases during gestation and to counteract effects of endocrine disrupting chemicals. Studies in amphibians have provided major insights to understand in-vivo mechanisms of TH signaling. The frog model boasts dramatic TH-dependent changes directly observable in free-living tadpoles with precise and easy experimental control of the TH response at developmental stages comparable to fetal stages in mammals. The hormones, their receptors, molecular mechanisms, and developmental roles of TH signaling are conserved to a high degree in humans and amphibians, such that with respect to developmental TH signaling "frogs are just little people that hop". The frog model is exceptionally illustrative of fundamental molecular mechanisms of in-vivo TH action involving TH receptors, transcriptional cofactors, and chromatin remodeling. This review highlights the current need, recent successes, and future prospects using amphibians as a model to elucidate molecular mechanisms and functional roles of TH signaling during post-embryonic development. This article is protected by copyright. All rights reserved.
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The Endocrine Society's first Scientific Statement in 2009 provided a wake-up call to the scientific community abouthow environmental endocrine-disrupting chemicals (EDCs) affect health and disease. Five years later, a substantially larger body of literature has solidified our understanding of plausible mechanisms underlying EDC actions and how exposures in animals and humans-especially during development-may lay the foundations for disease later in life. At this point in history, we have much stronger knowledge about how EDCs alter gene-environment interactions via physiological, cellular, molecular, andepigeneticchanges, therebyproducingeffects inexposedindividuals as well as theirdescendants. Causal links between exposure and manifestation of disease are substantiated by experimental animal models and are consistent with correlative epidemiological data in humans. There are several caveats because differences in how experimental animal work is conducted can lead to difficulties in drawing broad conclusions, and we must continue to be cautious about inferring causality in humans. In this second Scientific Statement, we reviewed the literature on a subset of topics for which the translational evidence is strongest: 1) obesity and diabetes; 2) female reproduction; 3) male reproduction; 4) hormone-sensitive cancers in females; 5) prostate; 6) thyroid; and 7) neurodevelopment and neuroendocrine systems. Our inclusion criteria for studies were those conducted predominantly in the past 5 years deemed to be of high quality based on appropriate negative and positive control groups or populations, adequate sample size and experimental design, and mammalian animal studies with exposure levels in arange that was relevant to humans. We also focused on studies using the developmental origins of health and disease model. No report was excluded based on a positive or negative effect of the EDC exposure. The bulk of the results across the board strengthen the evidence for endocrine health-related actions of EDCs. Based on this much more complete understanding of the endocrine principles by which EDCs act, including nonmonotonic dose-responses, low-dose effects, and developmental vulnerability, these findings canbemuchbetter translated tohumanhealth. Armedwith this information, researchers, physicians, andother healthcare providers can guide regulators and policymakers as they make responsible decisions.
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There is a growing recognition that application of mechanistic approaches to understand cross-species shared molecular targets and pathway conservation in the context of hazard characterization, provide significant opportunities in risk assessment (RA) for both human health and environmental safety. Specifically, it has been recognized that a more comprehensive and reliable understanding of similarities and differences in biological pathways across a variety of species will better enable cross-species extrapolation of potential adverse toxicological effects. Ultimately, this would also advance the generation and use of mechanistic data for both human health and environmental RA. A workshop brought together representatives from industry, academia and government to discuss how to improve the use of existing data, and to generate new NAMs data to derive better mechanistic understanding between humans and environmentally-relevant species, ultimately resulting in holistic chemical safety decisions. Thanks to a thorough dialogue among all participants, key challenges, current gaps and research needs were identified, and potential solutions proposed. This discussion highlighted the common objective to progress toward more predictive, mechanistically based, data-driven and animal-free chemical safety assessments. Overall, the participants recognized that there is no single approach which would provide all the answers for bridging the gap between mechanism-based human health and environmental RA, but acknowledged we now have the incentive, tools and data availability to address this concept, maximizing the potential for improvements in both human health and environmental RA.
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Teleosts are the most numerous class of living vertebrates. They exhibit great diversity in terms of morphology, developmental strategies, ecology and adaptation. In spite of this diversity, teleosts conserve similarities at molecular, cellular and endocrine levels. In the context of thyroidal systems, and as in the rest of vertebrates, thyroid hormones in fish regulate development, growth and metabolism by actively entering the nucleus and interacting with thyroid hormone receptors, the final sensors of this endocrine signal, to regulate gene expression. In general terms, vertebrates express the functional thyroid hormone receptors alpha and beta, encoded by two distinct genes (thra and thrb, respectively). However, different species of teleosts express thyroid hormone receptor isoforms with particular structural characteristics that confer singular functional traits to these receptors. For example, teleosts contain two thra genes and in some species also two thrb; some of the expressed isoforms can bind alternative ligands. Also, some identified isoforms contain deletions or large insertions that have not been described in other vertebrates and that have not yet been functionally characterized. As in amphibians, the regulation of some of these teleost isoforms coincides with the climax of metamorphosis and/or life transitions during development and growth. In this review, we aimed to gain further insights into thyroid signaling from a comparative perspective by proposing a systematic nomenclature for teleost thyroid hormone receptor isoforms and summarize their particular functional features when the information was available.
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Inflation of the posterior and/or anterior swim bladder are processes previously demonstrated to be thyroid-hormone regulated. We investigated whether inhibition of deiodinases, which convert thyroxine (T4) to the more biologically-active form, 3,5,3'-triiodothyronine (T3), would impact swim bladder inflation. Two experiments were conducted using a model deiodinase inhibitor, iopanoic acid (IOP). First, fathead minnow embryos were exposed to 0.6, 1.9, or 6.0 mg/L or control water until 6 days post-fertilization (dpf) at which time posterior swim bladder inflation was assessed. To examine anterior swim bladder inflation, a second study was conducted with 6 dpf larvae exposed to the same IOP concentrations until 21 dpf. Fish from both studies were sampled for T4/T3 measurements and gene transcription analyses. Incidence and length of inflated posterior swim bladders were significantly reduced in the 6.0 mg/L treatment at 6 dpf. Incidence of inflation and length of anterior swim bladder were significantly reduced in all IOP treatments at 14 dpf, but inflation recovered by 18 dpf. Throughout the larval study, whole body T4 concentrations increased and T3 concentrations decreased in all IOP treatments. Consistent with hypothesized compensatory responses, deiodinase-2 mRNA was up-regulated in the larval study, and thyroperoxidase mRNA was down-regulated in all IOP treatments in both studies. These results support the hypothesized adverse outcome pathways linking inhibition of deiodinase activity to impaired swim bladder inflation. This article is protected by copyright. All rights reserved.
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Thyroid hormones (THs) play important roles in vertebrates such as the control of the metabolism, development and seasonality. Given the pleiotropic effects of thyroid disorders (developmental delay, mood disorder, tachycardia, etc), THs signaling is highly investigated, specially using mammalian models. In addition, the critical role of TH in controlling frog metamorphosis has led to the use of Xenopus as another prominent model to study THs action. Nevertheless, animals regarded as non-model species can also improve our understanding of THs signaling. For instance, studies in amphioxus highlighted the role of Triac as a bona fide thyroid hormone receptor (TR) ligand. In this review, we discuss our current understanding of the THs signaling in the different taxa forming the metazoans (multicellular animals) group. We mainly focus on three actors of the THs signaling: the ligand, the receptor and the deiodinases, enzymes playing a critical role in THs metabolism. By doing so, we also pinpoint many key questions that remain unanswered. How can THs accelerate metamorphosis in tunicates and echinoderms while their TRs have not been yet demonstrated as functional THs receptors in these species? Do THs have a biological effect in insects and cnidarians even though they do not have any TR? What is the basic function of THs in invertebrate protostomia? These questions can appear disconnected from pharmacological issues and human applications, but the investigation of THs signaling at the metazoans scale can greatly improve our understanding of this major endocrinological pathway.
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Model animals are crucial to biomedical research. Among the commonly used model animals, the amphibian, Xenopus, has had tremendous impact because of its unique experimental advantages, cost effectiveness, and close evolutionary relationship with mammals as a tetrapod. Over the past 50 years the use of Xenopus has made possible many fundamental contributions to biomedicine, and it is a cornerstone of research in cell biology, developmental biology, evolutionary biology, immunology, molecular biology, neurobiology, and physiology. The prospects for Xenopus as an experimental system are excellent: Xenopus is uniquely well-suited for many contemporary approaches used to study fundamental biological and disease mechanisms. Moreover, recent advances in high throughput DNA sequencing, genome editing, proteomics, and pharmacological screening are easily applicable in Xenopus, enabling rapid functional genomics and human disease modeling at a systems level. This article is protected by copyright. All rights reserved.