AOP-Wiki

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<div id="title">
<h2>AOP ID and Title:</h2>
<div class="title">AOP 363: Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</div>
Short Title: TPOi retinal layer structure
</div>
<h2>Graphical Representation</h2>
<img src="https://aopwiki.org/system/dragonfly/production/2022/06/16/3s4pnizql_AOP_363.jpg" height="500" width="700" alt=""/>
<div id="authors">
<h2>Authors</h2>
Lisa Gölz [1]
Lisa Baumann [1]
Pauline Pannetier [1]
Lucia Vergauwen [2]
[1] University of Heidelberg, Centre for Organismal Studies, Aquatic Ecology and Toxicology, Im Neuenheimer Feld 504, 69120 Heidelberg, Germany
[2] Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium
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<div id="status">
<h2>Status</h2>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Author status</th>
<th scope="col">OECD status</th>
<th scope="col">OECD project</th>
<th scope="col">SAAOP status</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
~~<td>Open for comment. Do not cite</td>~~
~~<td></td>~~
~~<td></td>~~
~~<td></td>~~
<td>Open for citation & comment</td>
<td>Under Review</td>
<td>1.35</td>
<td>Included in OECD Work Plan</td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="abstract">
<h2>Abstract</h2>
Visual function is particularly important for survival, especially of developing life stages. Some chemicals, including thyroid hormone system disrupting chemicals (THSDCs), can impair eye development. The chain of events, from the molecular interaction of thyroid hormone system disruption (THSD) to the consequences at the level of vision, is not yet fully understood. The development of this AOP aims to contribute to filling these gaps and investigates how inhibition of the enzyme thyroperoxidase and resulting changes in thyroid hormone(TH) levels can lead to effects on the retinal layers and subsequently at the population level.
The focus of this AOP is on fish, as the largest amount of data is available for this taxonomic group. Data obtained with a variety of different techniques to induce or mimick TH synthesis disruption have been included (addressing KE 227: "Decreased thyroid hormone synthesis"), for example, exposure to THSDCs, generation of transgenic or mutant fish, microinjection, morpholino knockdown, thyroid ablation, etc. The resulting changes in hormone levels have been studied (KE 281: "Decreased thyroxine (T4) in serum" , KE 1003: "Decreased triiodothyronine (T3)"), as well as changes in the retinal layers (KE 1877: "Altered retinal layer structure"). These include e.g. cell size, cell layer structure, organisation and number of photoreceptors, pigmentation and information on morphological changes (e.g. cell shapes). At a higher level of biological organization, physiological and behavioural changes were investigated (AO 1643: "Altered visual function"), including e.g. optokinetic response, optomotor response, light response, etc. The present AOP is closely linked to AOPs 155-159 on THSD leading to impaired swim bladder inflation in fish, as well as AOP 297 on retinoic acid effects on eye development.
</div>
~~<div id="background">~~
~~<h3>Background</h3>~~
This AOP is based on data derived from several extensive literature searches.  First, data was collected on different biological levels: Results at the molecular level, data on hormone levels, data on the tissue level and on the behavioural/physiological level. In a next step, KEs and KERs were identified and defined and a more detailed search was initiated. While initially an AOP network including several different effects on eye development was considered, in a next step AOP 363 was selected and further refined, and again an intensive and very detailed final literature search was conducted. The search for bibliographic data was conducted online in "pubmed", "sciencedirect/Scopus" and "Web of Science". The initial search terms were: "(zebra-)fish", "eye development", "retina", "thyroid/hormone disorders", "visual behavio(u)r", "photoreceptors" and combinations of these terms. A very detailed manual search followed for the various KEs and KERs. Not only articles on chemical exposure of different animals were considered, but also more basic studies using other THSD induction techniques such as transgenic or mutant fish, microinjection, morpholino oligonucleotides, thyroidectomy, etc. The range of data that was assessed is wide, from gene expression and hormone levels to physiological and behavioural changes in different animals. In total, around 120 articles from this structured search were analysed in terms of experimental design and information on different biological levels. The majority of literature used fish, especially zebrafish (85%), which is why this AOP focuses on fish, but it can probably be applied to other vertebrate species as well.
<h2>AOP Development Strategy</h2>
<div id="context">
<h3>Context</h3>
This AOP is based on data derived from several extensive literature searches.  First, data was collected on different biological levels: Results at the molecular level, data on hormone levels, data on the tissue level and on the behavioural/physiological level. In a next step, KEs and KERs were identified and defined and a more detailed search was initiated. While initially an AOP network including several different effects on eye development was considered, in a next step AOP 363 was selected and further refined, and again an intensive and very detailed final literature search was conducted. The search for bibliographic data was conducted online in "pubmed", "sciencedirect/Scopus" and "Web of Science". The initial search terms were: "(zebra-)fish", "eye development", "retina", "thyroid/hormone disorders", "visual behavio(u)r", "photoreceptors" and combinations of these terms. A very detailed manual search followed for the various KEs and KERs. Not only articles on chemical exposure of different animals were considered, but also more basic studies using other THSD induction techniques such as transgenic or mutant fish, microinjection, morpholino oligonucleotides, thyroidectomy, etc. The range of data that was assessed is wide, from gene expression and hormone levels to physiological and behavioural changes in different animals. In total, around 120 articles from this structured search were analysed in terms of experimental design and information on different biological levels. The majority of literature used fish, especially zebrafish (85%), which is why this AOP focuses on fish, but it can probably be applied to other vertebrate species as well.
~~</div>~~
</div>
<div id="aop_summary">
<h2>Summary of the AOP</h2>
<h3>Events</h3>
<h3>Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sequence</th>
<th scope="col">Type</th>
<th scope="col">Event ID</th>
<th scope="col">Title</th>
<th scope="col">Short name</th>
</tr>
</thead>
<tbody>
<tr>
<td></td>
<td>MIE</td>
<td>279</td>
<td><a href="/events/279">Thyroperoxidase, Inhibition</a></td>
<td>Thyroperoxidase, Inhibition</td>
</tr>
<tr><td></td><td></td><td></td><td></td><td></td></tr>
<tr>
<td></td>
<td>KE</td>
<td>277</td>
<td><a href="/events/277">Thyroid hormone synthesis, Decreased</a></td>
<td>TH synthesis, Decreased</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>281</td>
<td><a href="/events/281"> Thyroxine (T4) in serum, Decreased</a></td>
<td>T4 in serum, Decreased</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1003</td>
<td><a href="/events/1003">Decreased, Triiodothyronine (T3)</a></td>
<td>Decreased, Triiodothyronine (T3)</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1877</td>
<td><a href="/events/1877">Altered, retinal layer structure</a></td>
<td>Altered, retinal layer structure</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1643</td>
<td><a href="/events/1643">Altered, Visual function</a></td>
<td>Altered, Visual function</td>
</tr>
<tr><td></td><td></td><td></td><td></td><td></td></tr>
<tr>
<td></td>
<td>AO</td>
<td>351</td>
<td><a href="/events/351">Increased Mortality</a></td>
<td>Increased Mortality</td>
</tr>
<tr>
<td></td>
<td>AO</td>
<td>360</td>
<td><a href="/events/360">Decrease, Population growth rate</a></td>
<td>Decrease, Population growth rate</td>
</tr>
</tbody>
</table>
</div>
<h3>Key Event Relationships</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Upstream Event</th>
<th scope="col">Relationship Type</th>
<th scope="col">Downstream Event</th>
<th scope="col">Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/relationships/309">Thyroperoxidase, Inhibition</a></td>
<td>adjacent</td>
<td>Thyroid hormone synthesis, Decreased</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/relationships/305">Thyroid hormone synthesis, Decreased</a></td>
<td>adjacent</td>
<td> Thyroxine (T4) in serum, Decreased</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/relationships/2038"> Thyroxine (T4) in serum, Decreased</a></td>
<td>adjacent</td>
<td>Decreased, Triiodothyronine (T3)</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/relationships/2373">Decreased, Triiodothyronine (T3)</a></td>
<td>adjacent</td>
<td>Altered, retinal layer structure</td>
<td>Moderate</td>
<td>Low</td>
</tr>
<tr>
<td><a href="/relationships/2374">Altered, retinal layer structure</a></td>
<td>adjacent</td>
<td>Altered, Visual function</td>
<td>High</td>
<td>Low</td>
</tr>
<tr>
<td><a href="/relationships/2375">Altered, Visual function</a></td>
<td>adjacent</td>
<td>Increased Mortality</td>
<td>Moderate</td>
<td>Low</td>
</tr>
<tr>
<td><a href="/relationships/2013">Increased Mortality</a></td>
<td>adjacent</td>
<td>Decrease, Population growth rate</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/366">Thyroperoxidase, Inhibition</a></td>
<td>non-adjacent</td>
<td> Thyroxine (T4) in serum, Decreased</td>
<td>High</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
<h3>Stressors</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Name</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Propylthiouracil</td>
<td>High</td>
</tr>
<tr>
<td>Methimazole</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="overall_assessment">
<h2>Overall Assessment of the AOP</h2>
<h3>Domain of Applicability</h3>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Embryo</td>
<td>High</td>
</tr>
<tr>
<td>Larvae</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
Taxonomic applicability:
The weight of evidence supporting the first linkage of this AOP between the MIE, TPO inhibition, and the KE of decreased TH synthesis, is strong and supported by more than three decades of research in animals including humans. Several papers have measured alterations in TPO and subsequent effects on TH synthesis (Cooper et al., 1982; Cooper et al.,1983; Divi and Doerge, 1994).
According to the evaluation of the empirical taxonomic domain of applicability (tDOA) of an adverse outcome pathway network for thyroid hormone system disruption (THSD) by Haigis et al., 2023, the level of confidence for a linkage between TPO inhibition and reduced thyroid hormone (TH) levels as well as for a linkage between THSD and altered visual function in general, was considered high for mammals and fish and moderate for amphibians. In addition, the level of confidence was considered moderate for birds for a linkage between THSD and altered visual function. This was supported by structural protein conservation analysis by Lalone et al., 2018 and Haigis et al., 2023. Structural protein conservation of mammalian, fish, amphibian, reptilian and avian TPO was found compared to the human (Homo sapiens) protein target using the U.S. Environmental Protection Agency’s Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS v6.0; seqapass.epa.gov/seqapass/) tool, while acknowledging the potential existence of interspecies differences in conservation. No empirical evidence linking TPO inhibition to THSD was found for reptiles and birds. Furthermore, no empirical evidence linking THSD to altered visual function was found for reptiles, but this taxon is considered part of the plausible tDOA based on the evaluation by Haigis et al., 2023. Scientific inference and the consideration of structural/functional conservation of organs, systems and pathways indicate that altered visual function can plausibly be measured and is plausibly linked to THSD.
What follows is a summary of evidence available on the taxonomic domain of applicability of specific parts of the AOP:
The weight of evidence supporting the first linkage of this AOP between the MIE, TPO inhibition, and the KE of decreased TH synthesis, is strong and supported by more than three decades of research in animals including humans. Several papers have measured alterations in TPO and subsequent effects on TH synthesis (Cooper et al., 1982; Cooper et al., 1983; Divi and Doerge, 1994).
Also for the next KER, it is widely accepted that TPO inhibition leads to declines in serum T4 levels in adult mammals. Strong qualitative and quantitative relationships exist between reduced TH synthesis and reduced serum T4 (Ekerot et al., 2013; Degon et al., 2008; Cooper et al., 1982; 1983; Leonard et al., 2016; Zoeller and Tan, 2007). Nevertheless, while a majority of the empirical evidence comes from work with laboratory rodents, there is a large amount of supporting data from humans (with anti-hyperthyroidism drugs including propylthiouracil and methimazole), some amphibian species (e.g., frog), fish species (e.g., zebrafish and fathead minnow), and some avian species (e.g, chicken) (Cooper et al. (1982; 1983); Hornung et al. (2010); Van Herck et al. (2013); Paul et al. (2013); Nelson et al. (2016); Alexander et al. (2017); Stinckens et al. (2020)).
Although the following KER (T4 in serum decreased leads to Triiodothyronine (T3) decreased) is also plausibly applicable across vertebrates, variation can be expected due to feedback/compensatory mechanisms that can also differ across species. In zebrafish and fathead minnow, several studies reported the evidence for a relationship between whole body T4 and T3 levels (Nelson et al., 2016; Stinckens et al., 2020, Wang et al., 2020).
The linkage between the MIE, decreased T3, and the KE of altered retinal layer structure, is evident in the different vertebrate classes. There is ample evidence that THs have an influence on the development of the retinal layer structure. Although there are some differences in eye structure between species, it is known that the retina follows the typical organisation of vertebrates. Within vertebrates, it consists of several layers such as the retinal pigment epithelium (RPE), photoreceptors, neurons and choroid. It is plausible to assume that TH levels are important for healthy eye development in all vertebrates.
TH receptors have a general function in different cell types of the vertebrate retina, they mediate specific events in retinal and photoreceptor development. The decrease of TH levels can lead to disturbances of the retinal layers, as shown by studies in various vertebrates such as fish species, rats, mice and humans (Baumann (2016), Komoike et al. (2013), Besson et al. (2020), Gamborino (2000), Houbrechts (2016), (Li et al. 2012)). In humans, hypothyroidism is also linked to impaired color vision (Racheva et al., 2020).
Life stage applicability:
This AOP considers effects of TPO inhibitors on the development of the retina during the embryo and larval life stage. In order to more specifically evaluate the life stage applicability of the impact of TPO inhibition on retinal layer structure and visual function leading to increased mortality, the timing of the ontogeny of the target organ needs to be matched to the timing of the ontogeny of the HPT-axis. Fish, amphibians and birds develop externally and rely on maternally transferred THs and TH machinery during the earliest stages of embryonic development. The first thyroid follicles in zebrafish appear around 55 hpf and endogenous T4 production has been observed at 72 hpf (Walter and others 2019). Since TPO is principally located in the thyroid follicles and responsible for the synthesis of TH which are released to circulation, important impacts on thyroidal TH synthesis due to TPO inhibition are not expected before 72 hpf. This hypothesis is in line with the observation that inflation of the posterior chamber of the swim bladder appears to be unaffected by TPO inhibition in zebrafish and fathead minnow (Nelson and others 2016; Stinckens and others 2016). We therefore hypothesize that effects on the retina are caused between activation of embryonic TH synthesis (around 72 hpf) and 5 dpf. In zebrafish, chemically-induced adverse effects on retinal layer structure are typically observed at 96 or 120 hpf. By 60 hpf, the different layers of the retina can be distinguished (Morris and Fadool 2005; Schmitt and Dowling 1999) but differentiation and maturation required for a functional retina continues until well beyond 84 hpf (Raymond and others 1995). For example, the first proper optokinetic response occurs around 4 dpf (Cohen et al., 2022). Based on these arguments, we identify early (< 72 hpf) processes that may not be (highly) sensitive to TPO inhibition and late (> 72 hpf) embryonic processes that are sensitive to TPO inhibition. Early processes can however be sensitive to other mechanisms of TH system disruption that impact on maternal THs, including deiodinase inhibition, since deiodinases are required to activate maternal T4 (Stinckens et al., 2016). While manu of the studies listed as evidence in this AOP are in line with this hypothesis, some studies raise uncertainties. For example, Reider and Connaughton (2014) observed reduction of ganglion cell layer thickness after exposing zebrafish embryos to MMI until 66 hpf and raising them afterwards in clean water until 72 hpf. It should be noted that there is still uncertainty about the exact timing of the activation of TH synthesis. The time window between 48 and 72 hpf has not been studied yet. Maternally transferred mRNA coding for TPO and NIS, as well as embryonic expression of thyroglobulin (Vergauwen et al., 2018) and the presence of the first thyroid follicle could allow for the start of TH synthesis between 55 and 72 hpf and therefore a corresponding sensitivity of developmental processes to TPO inhibition in this period.
There are potential, alternative pathways that may lead to altered retinal layer structure and that are not the subject of this AOP.  For example, TPO      expression has been observed locally in the eyes of mice (Li and others 2012), suggesting a potential role of local TH synthesis in eye development before the thyroid follicles become active. This process could be sensitive to TPO inhibition. There is currently insufficient information to evaluate this hypothesis. Among others, it is not clear whether TH synthesis can take place in the absence of a follicular epithelium. Additionally, TH-independent pathways may contribute to the impact on the retinal layer structure. For example, Komoike et al. (2013) suggested TH-independent apoptosis and Li et al. (2012) hypothesized that the inhibition of peroxidase activity in general could disrupt the formation of the extracellular matrix. Finally, TH system disruptors often act on multiple targets simultaneously, potentially affecting retinal layer structure through different TH dependent pathways (e.g., TPO and deiodinase inhibition by propylthiouracil).
Mammals on the other hand continuously receive maternal THs via the placenta during embryonic development. Therefore, exposure to inhibitors of TH synthesis is expected to have an effect on the earliest phases of embryonic development by inhibiting maternal TH synthesis (Klein and Mitchell, 1999; Klein et al., 2001, Elred et al. 2018).
Taken together, there is strong support for applicability of the current AOP to embryo-larval/embryo-foetal stages of vertebrates. Since the term 'eleutheroembryo' (stage starting at hatching and ending with free-feeding fish) is not applicable to all vertebrates, the terms 'embryo' and 'larvae' were selected to reflect this.
Sex applicability:
Fish species have different patterns of gonadal differentiation. Many species are undifferentiated gonochorists (e.g., zebrafish, fathead minnows), in which an indifferent gonad first develops into an ovary-like gonad which then further differentiates into either a mature ovary or a testis (Maack and Segner, 2003). Other fish species such as medaka are differentiated gonochorists where the indifferent gonad develops directly into an ovary or a testis. In both cases, in the early life stages in which the eyes develop, the gonads have not yet started to differentiate. For example, in zebrafish the eyes develop in the first 5 days of development and the gonads differentiate in the period around 20-50 dpf. In species such as zebrafish, even sex determination has not occurred by the time the eyes develop, since it is dependent on environmental factors. This means that in the life stages of interest for this AOP (embryo-larval), sex has not been established yet nor has gonad differentiation started. Therefore, sex is not assumed to be an important factor in determining the effect of TPO inhibitors on retinal structure development.
This does however not preclude the occurrence of sex dependent changes in eye structure during later life after gonadal differentiation. For example, Chen et al. (2018) exposed marine medaka to perfluorobutane sulfonate (PFBS) for an entire life cycle and this resulted in sex-dependent changes in eye water content and neurotransmitter levels in the eyes.
<h3>Essentiality of the Key Events</h3>
Essentiality means that a stressor can activate an AOP and its various KEs, and that cessation of this stressor can prevent this activation or lead to a recovery of the adverse effects. Certain studies, such as gene knockdown, recovery or knockout experiments, have been reviewed to evaluate this. Evidence for essentiality in this AOP can be classified as high. Direct evidence from specifically designed experimental studies illustrating essentiality is available for several KEs in the AOP. Especially the evidence of essentiality of decreased T3 levels for effects on the eyes is very important and strongly supports this AOP.
<h3>Weight of Evidence Summary</h3>
<ol>
<li>Biological plausibility:</li>
</ol>
Most of the KERs (309, 305, 366, 2374, 2375, 2013) were found to be highly biologically plausible. For example, TPO is known to be a key enzyme of the TH system and plays an important role in controlling important functions such as neuronal development, including eye development. Similarly, the thyroid hormone T4 is known to be activated to T3 by DIOs in the liver and other organs. Both T3 and T4 are present during retinal development (Roberts and others 2006), and key components such as DIOs (Heijlen and others 2013; summarized by Viets and others 2016), TH receptors (Gan and Flamarique 2010), and TPO (Li and others 2012) are also expressed in the vertebrate retina during retinal development. However, there are compensatory mechanisms that limit the impact on T3 levels, possibly through increased deiodinase activity or other feedback or compensatory mechanisms, as well as some gaps in knowledge. Therefore, the biological plausibility of KER 2038 and 2373 was determined to be moderate.
<ol start="2">
<li>Empirical support is moderate for most KERs in the AOP and low for the most upstream KERs.</li>
<li>Overall WoE ranges from moderate to high. As prescribed by the User's handbook, biological plausibility was given slightly more weight in this decision compared to empirical evidence.</li>
</ol>
<h3>Quantitative Consideration</h3>
The difficulties in generating quantitative data for this AOP may be due to the fact that both decreased and increased T3 levels affect the development of retinal structure, confirming that this process is under strict control of balanced TH levels, but also making it difficult to describe the quantitative relationship between T3 levels and altered retinal structure (Stinckens et al., 2020).
However, the combinations of some studies show some correlations:  For example, the study by Rehberger et al. (2018) shows a decrease in T3 and T4      with increasing PTU concentration in zebrafish embryos, Baumann et al. (2016) found both retinal malformations and behavioural abnormalities due to impaired visual performance in larvae at these concentrations (and at much higher concentrations). Baumann et al. (2016) also showed a correlation between increased TPO gene expression (measured as a fold change) and decreased RPE diameter with increasing PTU exposure in 5 dpf zebrafish.
There is quantitative data on KER1 (TPO, inhibition (KE 279) results in TH synthesis, reduced (KE 277), also. For example, Hassan et al (2017) quantified TH synthesis blocked by PTU and MMI in an in vitro TPO inhibition study to predict TH concentrations in rat serum. Similarly, Fisher et al. (2013) modelled the effect of TPO inhibition on serum TH concentrations during early development in rats. Haselman et al. (2020), in Xenopus laevis, demonstrated the temporal profiles of thyroid iodotyrosines (MIT/DIT) and iodothyronines (T4/T3), the products of TPO activity, after exposure to three different model TPO inhibitors (MMI, PTU, MBT) at different concentrations.
</div>
<div id="considerations_for_potential_applicaitons">
<h2>Considerations for Potential Applications of the AOP (optional)</h2>
THSDCs are increasingly recognized as a serious environmental problem for aquatic species, as well as for humans. Especially the THSD effects on (neuro-)developmental processes pose a risk to different vertebrate species. The current framework for assessment of THSD effects is separated between human and environmental health, and in the latter, it is restricted to amphibians. The implementation of thyroid-related endpoints into test guidelines using fish is urgently needed and is currently being addressed in different EU-funded research projects (Holbech et al., 2020) and in project 2.64 of the OECD TG work plan, “Inclusion of thyroid endpoints in OECD fish Test Guidelines”. Moreover, this testing gap has been recognized by OECD VMG-Eco in 2016 at two EU workshops, “Setting Priorities for Further Development and Validation of Test Methods and Testing Approaches'' and “Supporting the Organization of a Workshop on Thyroid Disruption” in 2017.
The present AOP provides strong evidence that eye development represents a very promising endpoint that could be implemented into existing OECD test guidelines that cover developmental phases of fish, such as the Fish Embryo Acute Toxicity (FET) test (OECD TG 236), the Fish Early Life Stage Toxicity (FELS) Test (OECD TG 210) and the Fish Sexual Development Test (FSDT, OECD TG 234). Especially the FET seems to be well suited for implementation of histopathological analyses of retinal structures for the detection of cellular changes that will ultimately result in decreased visual capacities and fitness impairment of exposed larvae. Combined with mechanistic analyses, such as gene expression or TH level measurements, a modified FET for detection of THSD in fish seems very promising for future THSD testing with fish. A major advantage is that a large part of the proposed endpoints in zebrafish can be assessed in embryonic life stages, which are considered “non-protected” alternatives to animal testing.
Consequently, based on AOP 363, together with other AOPs linking THSD to visual function that are under development (AOP 364, 365), we provide evidence that fish eye development, with focus on morphological and structural alterations, can be included as apical endpoint into fish endocrine disruption test guidelines for THSD. However, the TH-specificity of eye-related endpoints should be examined, since other signaling pathways, such as the estrogenic, retinoid, IGF-1 and aryl hydrocarbon receptor, can also affect eye development (Molla et al., 2019; Chen et al., 2020). Consequently, measurement of TH levels or performance of thyroid histopathology are required to support the causal link between the THSD mechanism and the observed effects in the eyes.
</div>
<div id="references">
<h2>References</h2>
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</div>
<div id="appendicies">
<h2>Appendix 1</h2>
<h3>List of MIEs in this AOP</h3>
<h4><a href="/events/279">Event: 279: Thyroperoxidase, Inhibition</a></h4>
<h5>Short Name: Thyroperoxidase, Inhibition</h5>
~~<h4>Key Event Component</h4>~~
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>iodide peroxidase activity</td>
<td>thyroid peroxidase</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/42">Aop:42 - Inhibition of Thyroperoxidase and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/119">Aop:119 - Inhibition of thyroid peroxidase leading to follicular cell adenomas and carcinomas (in rat and mouse)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/159">Aop:159 - Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/175">Aop:175 - Thyroperoxidase inhibition leading to altered amphibian metamorphosis</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/271">Aop:271 - Inhibition of thyroid peroxidase leading to impaired fertility in fish</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/364">Aop:364 - Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/365">Aop:365 - Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/402">Aop:402 - Thyroid peroxidase (TPO) inhibition leads to periventricular heterotopia formation in the developing rat brain</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/605">Aop:605 - Thyroid Peroxidase Inhibition Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>2(3H)-Benzothiazolethione</td></tr>
<tr><td>2-mercaptobenzothiazole</td></tr>
<tr><td>Ethylene thiourea</td></tr>
<tr><td>Mercaptobenzothiazole</td></tr>
<tr><td>Methimazole</td></tr>
<tr><td>Propylthiouracil</td></tr>
<tr><td>Resorcinol</td></tr>
<tr><td>Thiouracil</td></tr>
<tr><td>Ethylenethiourea</td></tr>
<tr><td>Amitrole</td></tr>
<tr><td>131-55-5</td></tr>
<tr><td>2,2',4,4'-Tetrahydroxybenzophenone</td></tr>
<tr><td>Daidzein</td></tr>
<tr><td>Genistein</td></tr>
<tr><td>4-Nonylphenol</td></tr>
<tr><td>4-propoxyphenol</td></tr>
<tr><td>Sulfamethazine</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Molecular</td></tr>
</tbody>
</table>
</div>
<h4>Cell term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Cell term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>thyroid follicular cell</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>thyroid follicle</td></tr>
</tbody>
</table>
</div>
~~<h3>Evidence for Perturbation by Stressor</h3>~~
~~<h4>Overview for Molecular Initiating Event</h4>~~
There is a wealth of information on the inhibition of TPO by drugs such as MMI and PTU, as well as environmental xenobiotics. In the landmark paper on TH system disruption by environmental chemicals, Brucker-Davis (1998) identified environmental chemicals that depressed TH synthesis by inhibiting TPO. Hurley (1998) listed TPO as a major target for thyroid tumor inducing pesticides. More recent work has tested over 1000 chemicals using a high-throughput screening assay (Paul-Friedman et al., 2016).
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>humans</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>pigs</td>
<td>Sus scrofa</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9823" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Xenopus laevis</td>
<td>Xenopus laevis</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>chicken</td>
<td>Gallus gallus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fathead minnow</td>
<td>Pimephales promelas</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td></td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Amphibia</td>
<td>Amphibia</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8292" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fish</td>
<td>fish</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Female</td>
<td>High</td>
</tr>
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Taxonomic:
This KE is plausibly applicable across vertebrates. TPO inhibition is a MIE conserved across taxa, with supporting data from experimental models and human clinical testing. This conservation is likely a function of the high degree of protein sequence similarity in the catalytic domain of mammalian peroxidases (Taurog, 1999). Ample data available for human, rat, and porcine TPO inhibition demonstrate qualitative concordance across these species (Schmultzer et al., 2007; Paul et al., 2013; Hornung et al., 2010). A comparison of rat TPO and pig TPO, bovine lactoperoxidase, and human TPO inhibition by genistein demonstrated good qualitative and quantitative (40–66%) inhibition across species, as indicated by quantification of MIT and DIT production (Doerge and Chang, 2002). Ealey et al. (1984) demonstrated peroxidase activity in guinea pig thyroid tissue using 3,3'-diaminobenzidine tetrahydrochloride (DAB) as a substrate that is oxidized by the peroxidase to form a brown insoluble reaction product. Formation of this reaction product was inhibited by 3-amino-1,2,4-triazole and the TPO inhibitor, methimazole (MMI). A comparative analysis of this action of MMI between rat- and human-derived TPO indicates concordance of qualitative response. Data also suggest an increased quantitative sensitivity to MMI in rats compared to humans (Vickers et al., 2012). Paul et al. (2013) tested 12 chemicals using the guaiacol assay using both porcine and rat thyroid microsomes. The authors concluded that there was an excellent qualitative concordance between rat and porcine TPO inhibition, as all chemicals that inhibited TPO in porcine thyroid microsomes also inhibited TPO in rat thyroid microsomes when tested within the same concentration range. In addition, these authors noted a qualitative concordance that ranged from 1.5 to 50-fold differences estimated by relative potency. Similarly, Takayama et al. (1986) found a very large species difference in potency for sulfamonomethoxine between cynomologus monkeys and rats.
This KE is plausibly applicable across vertebrates. TPO inhibition is a MIE conserved across taxa, with supporting data from experimental models and human clinical testing. This conservation is likely a function of the high degree of protein sequence similarity in the catalytic domain of mammalian peroxidases (Taurog, 1999). Ample data available for human, rat, and porcine TPO inhibition demonstrate qualitative concordance across these species (Schmultzer et al., 2007; Paul et al., 2013; Hornung et al., 2010). A comparison of rat TPO and pig TPO, bovine lactoperoxidase, and human TPO inhibition by genistein demonstrated good qualitative and quantitative (40–66%) inhibition across species, as indicated by quantification of monoiodotyrosine (MIT) and diiodotyrosine (DIT) production (Doerge and Chang, 2002). Ealey et al. (1984) demonstrated peroxidase activity in guinea pig thyroid tissue using 3,3'-diaminobenzidine tetrahydrochloride (DAB) as a substrate that is oxidized by the peroxidase to form a brown insoluble reaction product. Formation of this reaction product was inhibited by 3-amino-1,2,4-triazole and the TPO inhibitor, methimazole (MMI). A comparative analysis of this action of MMI between rat- and human-derived TPO indicates concordance of qualitative response. Data also suggest an increased quantitative sensitivity to MMI in rats compared to humans (Vickers et al., 2012). Paul et al. (2013) tested 12 chemicals using the guaiacol assay using both porcine and rat thyroid microsomes. The authors concluded that there was an excellent qualitative concordance between rat and porcine TPO inhibition, as all chemicals that inhibited TPO in porcine thyroid microsomes also inhibited TPO in rat thyroid microsomes when tested within the same concentration range. In addition, these authors noted a qualitative concordance that ranged from 1.5 to 50-fold differences estimated by relative potency. Similarly, Takayama et al. (1986) found a very large species difference in potency for sulfamonomethoxine between cynomologus monkeys and rats.
According to the evaluation of the empirical taxonomic domain of applicability (tDOA) of an adverse outcome pathway network for thyroid hormone system disruption (THSD) by Haigis et al., 2023, the level of confidence for a linkage between TPO inhibition and reduced thyroid hormone (TH) levels was considered high for mammals and fish (Dong et al., 2020, Fang et al., 2022, Hassan et al., 2020, Hornung et al., 2015, Jomaa et al., 2015, Nelson et al., 2016, O’Shaughnessy et al., 2018, Paul et al., 2013, Raldua and Babin, 2009, Ramhøj et al., 2021, 2022, Schmutzler et al., 2007, Stinckens et al., 2016, 2020, Thienpont et al., 2011, Tietge et al., 2013, Vickers et al., 2012, Wu et al., 2016) and moderate for amphibians (Haselman et al., 2020, Tietge et al., 2010, 2013). This was supported by structural protein conservation analysis by Lalone et al., 2018 and Haigis et al., 2023. Structural protein conservation of mammalian, fish, amphibian, reptilian and avian TPO was found compared to the human (Homo sapiens) protein target using the U.S. Environmental Protection Agency’s Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS v6.0; seqapass.epa.gov/seqapass/) tool, while acknowledging the potential existence of interspecies differences in conservation. No empirical evidence linking TPO inhibition to THSD was found for reptiles and birds.
Life stage:
Applicability to certain life stages may depend on the species and their dependence on maternally transferred THs during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf and not at 24 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TPO inhibition.
Applicability to certain life stages may depend on the species and their dependence on maternally transferred THs during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. Evidence supporting this hypothesis is obtained from a zebrafish TPO knockout line. In homozygous individuals TPO is inhibited from the embryonic developmental stage onwards, resulting in an abolished T4 production in thyroid follicles with phenotypical abnormalities such as reduced swim bladder inflation and growth retardation appearing at 20 dpf but not before 10 dpf (Fang et al., 2022). In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf and not at 24 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TPO inhibition.
Sex:
This KE is plausibly applicable to both sexes. The molecular components responsible for TH synthesis, including TPO, are identical in both sexes. Therefore inhibition of TPO is not expected to be sex-specific.
<h4>Key Event Description</h4>
Thyroperoxidase (TPO) is a heme-containing apical membrane protein within the follicular lumen of thyrocytes that acts as the enzymatic catalyst for thyroid hormone (TH) synthesis. TPO catalyzes several reactions in the thyroid gland, including: the oxidation of iodide; nonspecific iodination of tyrosyl residues of thyroglobulin (Tg); and the coupling of iodotyrosyls to produce Tg-bound monoiodotyrosine (MIT) and diiodotyrosine (DIT) (Divi et al., 1997; Kessler et al., 2008; Ruf et al., 2006; Taurog et al., 1996). The outcome of TPO inhibition is decreased synthesis of thyroxine (T4) and triiodothyronine (T3), a decrease in release of these hormones from the gland into circulation, and unless compensated, a consequent decrease in systemic concentrations of T4, and possibly T3. The primary product of TPO-catalyzed TH synthesis is T4 (Taurog et al., 1996; Zoeller et al., 2007) that would be peripherally or centrally deiodinated to T3.
It is important to note that TPO is a complex enzyme that has two catalytic cycles and is capable of iodinating multiple species (Divi et al., 1997). Alterations in all of these events are not covered by some of the commonly used assays that measure “TPO inhibition” (e.g., guaiacol and AmplexUltraRed, see below). Therefore, in the context of this AOP we are using TPO inhibition not in the classical sense, but instead to refer to the empirical data derived from the assays commonly used to investigate environmental chemicals.
It is important to note that TPO is a complex enzyme that has two catalytic cycles and is capable of iodinating multiple species (Divi et al., 1997). Alterations in all of these events are not covered by some of the commonly used assays that measure “TPO inhibition” (e.g., guaiacol and AmplexUltraRed, see below). Ususally just the first step of this series of events is covered by assays that measure TPO inhibition. Therefore, in the context of this AOP we are using TPO inhibition not in the classical sense, but instead to refer to the empirical data derived from the assays commonly used to investigate environmental chemicals. Therefore, in the context of this AOP we are using TPO inhibition not in the classical sense, but instead to refer to the empirical data derived from the assays commonly used to investigate environmental chemicals.
Figure 1      illustrates the enzymatic and nonenzymatic reactions mediated by TPO that result in the synthesis of thyroxine (T4) .
<span style="font-size:medium"><img src="blob:https://aopwiki.org/179e6970-1e8b-49c3-a87e-3b2feea07074" style="height:202px; width:454px" /></span></span>
<a href="https://aopwiki.org/system/dragonfly/production/2017/04/16/9jyfbzvr2o_Synthesis_figure.jpg"><img alt="" src="https://aopwiki.org/system/dragonfly/production/2017/04/16/9jyfbzvr2o_Synthesis_figure.jpg" style="height:330px; width:604px" /></a>
Inhibition of TPO can be reversible, with transient interaction between the enzyme and the chemical, or irreversible, whereby suicide substrates permanently inactivate the enzyme. Reversible and irreversible TPO inhibition may be determined by the chemical structure, may be concentration dependent, or may be influenced by other conditions, including the availability of iodine (Doerge and Chang, 2002).
Inhibition of TPO can be reversible, with transient interaction between the enzyme and the chemical, or irreversible, whereby suicide substrates permanently inactivate the enzyme. Reversible and irreversible (isoflavones such as genistein) TPO inhibition may be determined by the chemical structure, may be concentration dependent, or may be influenced by other conditions, including the availability of iodine (Doerge and Chang, 2002).
The ontogeny of TPO has been determined using both direct and indirect evidence in mammals.  Available evidence suggests the 11th to 12th fetal week as the beginning of functional TPO in humans. In rodents, TPO function begins late in the second fetal week, with the first evidence of T4 secretion on gestational day 17 (Remy et al., 1980). Thyroid-specific genes appear in the thyroid gland according to a specific temporal pattern; thyroglobulin (Tg), TPO (Tpo), and TSH receptor (Tshr) genes are expressed by gestational day 14 in rats, and the sodium iodide symporter, NIS (Nis), is expressed by gestational day 16 in rats. Maturation to adult function is thought to occur within a few weeks after parturition in rats and mice, and within the first few months in neonatal humans (Santisteban and Bernal, 2005).  Tg is first detected in human fetuses starting at 5th week of gestation and rises throughout gestation (Thorpe-Beeston et al., 1992), but iodine trapping and T4 production does not occur until around 10-12 weeks. Also, the dimerization of Tg, a characteristic of adult TH storage, is not found until much later in human gestation (Pintar, 2000). In rats, Tg immunoreactivity does not appear until day 15 of gestation (Fukiishi et al., 1982; Brown et al., 2000). The vast majority of research and knowledge on Tg is from mammals, although genomic orthologs are known for a variety of other species (Holzer et al., 2016). It is important to note that prior to the onset of fetal thyroid function, THs are still required by the developing fetus which until that time relies solely on maternal sources. Chemical-induced TPO inhibition can affect synthesis in the maternal gland and in the fetal gland.
The components of the TH system responsible for TH synthesis are highly conserved across vertebrates. In fish and amphibians TPO and NIS inhibition result in an expected decrease of TH synthesis (Hornung et al., 2010; Tietge et al., 2013; Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020) like in mammals. Although the TH system is highly conserved across vertebrates, there are some taxon-specific considerations.
Zebrafish and fathead minnows are oviparous fish species in which maternal THs are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic TH synthesis is initiated. Maternal transfer of THs to the eggs has been demonstrated in zebrafish (Walpita et al., 2007; Chang et al., 2012) and fathead minnows (Crane et al., 2004; Nelson et al., 2016).
Inhibition of TPO can only occur after activation of embryonic TH synthesis mediated by TPO. Endogenous transcription profiles of thyroid-related genes in zebrafish and fathead minnow showed that mRNA coding for TPO is maternally transferred in relatively high amounts with subsequent mRNA degradation followed by initiation of embryonic transcription around hatching (Vergauwen et al., 2018).
<h4>How it is Measured or Detected</h4>
There are no approved OECD or EPA guideline study protocols for measurement of TPO inhibition. However, there is an OECD scoping document on identification of chemicals that modulate TH signaling that provides details on a TPO assay (OECD, 2017).
From the early 1960's, microsomal fractions prepared from porcine thyroid glands and isolated porcine follicles were used as a source of TPO for inhibition experiments (Taurog, 2005). Microsomes from human goiter samples (Vickers et al., 2012) and rat thyroid glands (Paul et al., 2013; 2014; Paul-Friedman et al., 2016) have also been used as a source of TPO.
TPO activity has been measured for decades via indirect assessment by kinetic measurement of the oxidation of guaiacol (Chang & Doerge 2000; Hornung et al., 2010; Schmutzler et al., 2007).  This method is a low-throughput assay due to the very rapid kinetics of the guaiacol oxidation reaction. More recently, higher-throughput methods using commercial fluorescent and luminescent substrates with rodent, porcine, and human microsomal TPO have been developed (Vickers et al., 2012; Paul et al., 2013; 2014; Kaczur et al., 1997). This assay substitutes a pre-fluorescent substrate (Amplex UltraRed) for guaiacol, that when incubated with a source of peroxidase and excess hydrogen peroxidase, results in a stable fluorescent product proportional to TPO activity (Vickers et al., 2012). The stability of the fluorescent reaction product allows this assay to be used in a higher throughput format (Paul-Friedman et al., 2016). This approach is appropriate for high-throughput screening but does not elucidate the specific mechanism by which a chemical may inhibit TPO (Paul-Friedman et al., 2016), and as with most in vitro assays, is subject to various sources of assay interference (Thorne et al., 2010).
TPO activity has been measured for decades via indirect assessment by kinetic measurement of the oxidation of guaiacol (Chang & Doerge 2000; Hornung et al., 2010; Schmutzler et al., 2007).  This method is a low-throughput assay due to the very rapid kinetics of the guaiacol oxidation reaction. More recently, higher-throughput methods using commercial fluorescent and luminescent substrates with rodent, porcine, and human microsomal TPO have been developed (Vickers et al., 2012; Paul et al., 2013; 2014; Kaczur et al., 1997). This assay substitutes a pre-fluorescent substrate (Amplex UltraRed) for guaiacol, that when incubated with a source of peroxidase and excess hydrogen peroxidase, results in a stable fluorescent product proportional to TPO activity (Vickers et al., 2012). The stability of the fluorescent reaction product allows this assay to be used in a higher throughput format (Paul-Friedman et al., 2016). This approach is appropriate for high-throughput screening but does not elucidate the specific mechanism by which a chemical may inhibit TPO (Paul-Friedman et al., 2016), and as with most in vitro assays, is subject to various sources of assay interference (Thorne et al., 2010). Recombinant sources of TPO have also been used (e.g. Schmutzler et al., 2007; Dong et al., 2020)
HPLC has been used to measure the activity of TPO via formation of the precursors monoiodotyrosine (MIT), diiodotyrosine (DIT), and both T3 and T4, in a reaction mixture containing TPO, or a surrogate enzyme such as lactoperoxidase (Divi & Doerge 1994). The tools and reagents for this method are all available. However, HPLC or other analytical chemistry techniques make this a low throughput assay, depending on the level of automation. A primary advantage of this in vitro method is that it directly informs hypotheses regarding the specific mechanism by which a chemical may impact TH synthesis in vitro.
In fish, increases of TPO mRNA levels are often used as indirect evidence of TPO inhibition in in vivoexperiments (Baumann et al., 2016; Nelson et al., 2016; Wang et al., 2020).
<h4>References</h4>
Baumann L, Ros A, Rehberger K, Neuhauss SCF, Segner H. 2016. Thyroid disruption in zebrafish (danio rerio) larvae: Different molecular response patterns lead to impaired eye development and visual functions. Aquatic Toxicology. 172:44-55.
Brown RS, Shalhoub V, Coulter S, Alex S, Joris I, De Vito W, Lian J, Stein GS.  Developmental regulation of thyrotropin receptor gene expression in the fetal and neonatal rat thyroid: relation to thyroid morphology and to thyroid-specific gene expression.  Endocrinology. 2000 Jan;141(1):340-5.
Brucker-Davis F. 1998. Effects of environmental synthetic chemicals on thyroid function. Thyroid 8:827-856.
Campinho MA, Saraiva J, Florindo C, Power DM. 2014. Maternal thyroid hormones are essential for neural development in zebrafish. Molecular Endocrinology. 28(7):1136-1149.
Chang, H. C. and D. R. Doerge (2000) Dietary genistein inactivates rat thyroid peroxidase in vivo without an apparent hypothyroid effect. Toxicol Appl Pharmacol. 168:244-252.
Chang J, Wang M, Gui W, Zhao Y, Yu L, Zhu G. 2012. Changes in thyroid hormone levels during zebrafish development. Zoological Science. 29(3):181-184.
Crane HM, Pickford DB, Hutchinson TH, Brown JA. 2004. Developmental changes of thyroid hormones in the fathead minnow, pimephales promelas. General and Comparative Endocrinology. 139(1):55-60.
Divi, R. L., & Doerge, D. R. (1994). Mechanism-based inactivation of lactoperoxidase and thyroid peroxidase by resorcinol derivatives. Biochemistry 33(32), 9668–9674.
Divi, R. L., Chang, H. C., & Doerge, D. R. (1997). Anti-Thyroid Isoflavones from Soybean. Biochem. Pharmacol.  54(10), 1087–1096.
Doerge DR, Chang HC. Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo. J Chromatogr B Analy Technol Biomed Life Sci. 2002 Sep 25;777(1-2):269-79.
Dong, H.Y., Godlewska, M., Wade, M.G., 2020. A rapid assay of human thyroid peroxidase activity. Toxicology in Vitro 62
Ealey PA, Henderson B, Loveridge N.A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland. Histochem J. 1984 Feb;16(2):111-22.
Fang, Y., Wan, J. P., Zhang, R. J., Sun, F., Yang, L., Zhao, S. X., Dong, M., & Song, H. D. (2022). Tpo knockout in zebrafish partially recapitulates clinical manifestations of congenital hypothyroidism and reveals the involvement of TH in proper development of glucose homeostasis. General and Comparative Endocrinology, 323–324. https://doi.org/10.1016/j.ygcen.2022.114033
Fukiishi Y, Harauchi T, Yoshizaki T, Hasegawa Y, Eguchi Y.  Ontogeny of thyroid peroxidase activity in perinatal rats. Acta Endocrinol (Copenh). 1982 101(3):397-402.
Haigis A-C., Vergauwen L., LaLone C.A., Villeneuve D.L., O'Brien J.M., Knapen D. (2023). Cross-species applicability of an adverse outcome pathway network for thyroid hormone system disruption. Toxicol Sci. 195, 1-27.
Haselman, J. T., Olker, J. H., Kosian, P. A., Korte, J. J., Swintek, J. A., Denny, J. S., Nichols, J. W., Tietge, J. E., Hornung, M. W., and Degitz, S. J. (2020). Targeted pathway-based in vivo testing using thyroperoxidase inhibition to evaluate plasma thyroxine as a surrogate metric of metamorphic success in model amphibian Xenopus laevis. Toxicol. Sci. 175, 236–250
Hassan, I., El-Masri, H., Ford, J., Brennan, A., Handa, S., Paul Friedman, K., and Gilbert, M. E. (2020). Extrapolating in vitro screening assay data for thyroperoxidase inhibition to predict serum thyroid hormones in the rat. Toxicol. Sci. 173, 280–292.
Holzer G, Morishita Y, Fini JB, Lorin T, Gillet B, Hughes S, Tohmé M, Deléage G, Demeneix B, Arvan P, Laudet V. Thyroglobulin Represents a Novel Molecular Architecture of Vertebrates. J Biol Chem. 2016 Jun 16.
Hornung, M. W., Degitz, S. J., Korte, L. M., Olson, J. M., Kosian, P. a, Linnum, A. L., & Tietge, J. E. (2010). Inhibition of thyroid hormone release from cultured amphibian thyroid glands by methimazole, 6-propylthiouracil, and perchlorate. Toxicol Sci 118(1), 42–51.
Hornung, M. W., Kosian, P. A., Haselman, J. T., Korte, J. J., Challis, K., Macherla, C., Nevalainen, E., and Degitz, S. J. (2015). In vitro, ex vivo, and in vivo determination of thyroid hormone modulating activity of benzothiazoles. Toxicol. Sci. 146, 254–264.
Hurley PM. 1998. Mode of carcinogenic action of pesticides inducing thyroid follicular cell tumors in rodents. Environ Health Perspect 106:437-445.
Jomaa, B., De Haan, L. H. J., Peijnenburg, A. A. C. M., Bovee, T. F. H., Aarts, J. M. M. J. G., and Rietjens, I. M. C. M. (2015). Simple and rapid in vitro assay for detecting human thyroid peroxidase disruption. Altex 32, 191–200.
Kaczur, V., Vereb, G., Molnár, I., Krajczár, G., Kiss, E., Farid, N. R., & Balázs, C. (1997). Effect of anti-thyroid peroxidase (TPO) antibodies on TPO activity measured by chemiluminescence assay. Clin. Chem 43(8 Pt 1), 1392–6.
Kessler, J., Obinger, C., Eales, G., 2008. Factors influencing the study of peroxidase- generated iodine species and implications for thyroglobulin synthesis. Thyroid 18, 769–774.
Lalone, C. A., Villeneuve, D. L., Doering, J. A., Blackwell, B. R., Transue, T. R., Simmons, C. W., Swintek, J., Degitz, S. J., Williams, A. J., and Ankley, G. T. (2018). Evidence for cross species extrapolation of mammalian-based high-throughput screening assay results. Environ. Sci. Technol. 52, 13960–13971.
Nelson K, Schroeder A, Ankley G, Blackwell B, Blanksma C, Degitz S, Flynn K, Jensen K, Johnson R, Kahl M et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part i: Fathead minnow. Aquatic Toxicology. 173:192-203.
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<h3>List of Key Events in the AOP</h3>
<h4><a href="/events/277">Event: 277: Thyroid hormone synthesis, Decreased</a></h4>
<h5>Short Name: TH synthesis, Decreased</h5>
~~<h4>Key Event Component</h4>~~
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>thyroid hormone generation</td>
<td>thyroid hormone</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/42">Aop:42 - Inhibition of Thyroperoxidase and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/65">Aop:65 - XX Inhibition of Sodium Iodide Symporter and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/128">Aop:128 - Kidney dysfunction by decreased thyroid hormone</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/134">Aop:134 - Sodium Iodide Symporter (NIS) Inhibition and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/54">Aop:54 - Inhibition of Na+/I- symporter (NIS) leads to learning and memory impairment </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/159">Aop:159 - Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/175">Aop:175 - Thyroperoxidase inhibition leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/176">Aop:176 - Sodium Iodide Symporter (NIS) Inhibition leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/188">Aop:188 - Iodotyrosine deiodinase (IYD) inhibition leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/192">Aop:192 - Pendrin inhibition leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/193">Aop:193 - Dual oxidase (DUOX) inhibition leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/271">Aop:271 - Inhibition of thyroid peroxidase leading to impaired fertility in fish</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/364">Aop:364 - Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/365">Aop:365 - Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/119">Aop:119 - Inhibition of thyroid peroxidase leading to follicular cell adenomas and carcinomas (in rat and mouse)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/110">Aop:110 - Inhibition of iodide pump activity leading to follicular cell adenomas and carcinomas (in rat and mouse)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/402">Aop:402 - Thyroid peroxidase (TPO) inhibition leads to periventricular heterotopia formation in the developing rat brain</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/457">Aop:457 - Succinate dehydrogenase inhibition leading to increased insulin resistance through reduction in circulating thyroxine</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/459">Aop:459 - AhR activation in the thyroid leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/605">Aop:605 - Thyroid Peroxidase Inhibition Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>Propylthiouracil</td></tr>
<tr><td>Methimazole</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Cellular</td></tr>
</tbody>
</table>
</div>
<h4>Cell term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Cell term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>thyroid follicular cell</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>thyroid gland</td></tr>
</tbody>
</table>
</div>
~~<h3>Evidence for Perturbation by Stressor</h3>~~
~~<h4>Propylthiouracil</h4>~~
~~6-n-proylthiouracil is a common positive control for inhibition of TPO~~
~~~~
~~<h4>Methimazole</h4>~~
~~Methimazole is a very common positve control for inhibition of TPO~~
~~~~
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>human</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Xenopus laevis</td>
<td>Xenopus laevis</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fathead minnow</td>
<td>Pimephales promelas</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Sus scrofa</td>
<td>Sus scrofa</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9823" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
<tr>
<td>Female</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Taxonomic: This KE is plausibly applicable across vertebrates. Decreased TH synthesis resulting from TPO or NIS inhibition is conserved across vertebrate taxa, with in vivo evidence from humans, rats, amphibians, some fish species, and birds, and in vitro evidence from rat and porcine microsomes. Indeed, TPO and NIS mutations result in congenital hypothyroidism in humans (Bakker et al., 2000; Spitzweg and Morris, 2010), demonstrating the essentiality of TPO and NIS function toward maintaining euthyroid status. Though decreased serum T4 is used as a surrogate measure to indicate chemical-mediated decreases in TH synthesis, clinical and veterinary management of hyperthyroidism and Grave's disease using propylthiouracil and methimazole, known to decrease TH synthesis, indicates strong evidence for chemical inhibition of TPO (Zoeller and Crofton, 2005).
Taxonomic: This KE is plausibly applicable across vertebrates. Decreased TH synthesis resulting from TPO or NIS inhibition is conserved across vertebrate taxa, with in vivo evidence from humans, rats, amphibians, some fish species, and birds, and in vitro evidence from rat and porcine microsomes. Indeed, TPO and NIS mutations result in congenital hypothyroidism in humans (Bakker et al., 2000; Spitzweg and Morris, 2010), demonstrating the essentiality of TPO and NIS function toward maintaining euthyroid status. Though decreased serum T4 is used as a surrogate measure to indicate chemical-mediated decreases in TH synthesis, clinical and veterinary management of hyperthyroidism and Graves’ disease using propylthiouracil and methimazole, known to decrease TH synthesis, indicates strong evidence for chemical inhibition of TPO (Zoeller and Crofton, 2005).
Life stage: Applicability to certain life stages may depend on the species and their dependence on maternally transferred THs during the earliest phases of development. The earliest life stages of teleost fish (e.g., fathead minnow, zebrafish) rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). In externally developing fish species, decreases in TH synthesis can only occur after initiation of embryonic TH synthesis. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. Therefore, it is still uncertain when exactly embryonic TH synthesis is activated and thus when exactly this process becomes sensitive to disruption. In fathead minnows, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It currently remains unclear when exactly embryonic TH production is initiated in zebrafish.
Life stage: Applicability to certain life stages may depend on the species and their dependence on maternally transferred THs during the earliest phases of development. The earliest life stages of teleost fish (e.g., fathead minnow, zebrafish) rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). In externally developing fish species, decreases in TH synthesis can only occur after initiation of embryonic TH synthesis. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. TPO inhibition in a homozygous knockout line abolished the T4 production in thyroid follicles of mutant zebrafish with phenotypic abnormalities occurring from 20 dpf onwards but not before 10 dpf (Fang et al., 2022). Therefore, it is still uncertain when exactly embryonic TH synthesis is activated and thus when exactly this process becomes sensitive to disruption. In fathead minnows, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It currently remains unclear when exactly embryonic TH production is initiated in zebrafish.
Sex: The KE is plausibly applicable to both sexes. THs are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of TH levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in TH levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.
<h4>Key Event Description</h4>
The thyroid hormones (TH), triiodothyronine (T3) and thyroxine (T4) are thyrosine-based hormones. Synthesis of THs is regulated by thyroid-stimulating hormone (TSH) binding to its receptor and thyroidal availability of iodine via the sodium iodide symporter (NIS). Other proteins contributing to TH production in the thyroid gland, including thyroperoxidase (TPO), dual oxidase enzymes (DUOX), and pendrin are also necessary for iodothyronine production (Zoeller et al., 2007).
The thyroid hormones (TH), triiodothyronine (T3) and thyroxine (T4) are thyrosine-based hormones. Synthesis of THs is regulated by thyroid-stimulating hormone (TSH) binding to its receptor and thyroidal availability of iodine via the sodium iodide symporter (NIS). Other proteins contributing to TH production in the thyroid gland, including thyroperoxidase (TPO), dual oxidase enzymes (DUOX), and the transport protein pendrin are also necessary for iodothyronine production (Zoeller et al., 2007).
The production of THs in the thyroid gland and resulting serum concentrations are controlled by a negatively regulated feedback mechanism. Decreased T4 and T3 serum concentrations activates the hypothalamus-pituitary-thyroid (HPT) axis which upregulates thyroid-stimulating hormone (TSH) that acts to increase production of additional THs (Zoeller and Tan, 2007). This regulatory system includes: 1) the hypothalamic secretion of the thyrotropin-releasing hormone (TRH); 2) the thyroid-stimulating hormone (TSH) secretion from the anterior pituitary; 3) hormonal transport by the plasma binding proteins; 4) cellular uptake mechanisms at the tissue level; 5) intracellular control of TH concentrations by deiodinating mechanisms; 6) transcriptional function of the nuclear TH receptor; and 7) in the fetus, the transplacental passage of T4 and T3 (Zoeller et al., 2007).
TRH and the TSH primarily regulate the production of T4, often considered a “pro-hormone,” and to a lesser extent of T3, the transcriptionally active TH. Most of the hormone released from the thyroid gland into circulation is in the form of T4, while peripheral deiodination of T4 is responsible for the majority of circulating T3. Outer ring deiodination of T4 to T3 is catalyzed by the deiodinases 1 and 2 (DIO1 and DIO2), with DIO1 expressed mainly in liver and kidney, and DIO2 expressed in several tissues including the brain (Bianco et al., 2006). Conversion of T4 to T3 takes place mainly in the liver and kidney, but also in other target organs such as in the brain, the anterior pituitary, brown adipose tissue, thyroid and skeletal muscle (Gereben et al., 2008; Larsen, 2009).
In mammals, most evidence for the ontogeny of TH synthesis comes from measurements of serum hormone concentrations.  And, importantly, the impact of xenobiotics on fetal hormones must include the influence of the maternal compartment since a majority of fetal THs are derived from maternal blood early in fetal life, with a transition during mid-late gestation to fetal production of THs that is still supplemented by maternal THs. In humans, THs can be found in the fetus as early as gestational weeks 10-12, and concentrations rise continuously until birth. At term, fetal T4 is similar to maternal levels, but T3 remains 2-3 fold lower than maternal levels. In rats, THs can be detected in the fetus as early as the second gestational week, but fetal synthesis does not start until gestational day 17 with birth at gestational day 22-23. Maternal THs continue to supplement fetal production until parturition (see Howdeshell, 2002; Santisteban and Bernal, 2005 for review). The ontogeny of TPO inhibition during development by environmental chemicals represents a data gap.
In mammals, most evidence for the ontogeny of TH synthesis comes from measurements of serum hormone concentrations. And, importantly, the impact of xenobiotics on fetal hormones must include the influence of the maternal compartment since a majority of fetal THs are derived from maternal blood early in fetal life, with a transition during mid-late gestation to fetal production of THs that is still supplemented by maternal THs. In humans, THs can be found in the fetus as early as gestational weeks 10-12, and concentations rise continuously until birth. At term, fetal T4 is similar to maternal levels, but T3 remains 2-3 fold lower than maternal levels. In rats, THs can be detected in the fetus as early as the second gestational week, but fetal synthesis does not start until gestational day 17 with birth at gestational day 22-23. Maternal THs continue to supplement fetal production until parturition. (see Howdeshell, 2002; Santisteban and Bernal, 2005 for review). Due to the maternal factor, the life stage specific impact of TPO inhibition after exposure to environmental chemicals is complex (Ramhoj et al., 2022).
Decreased TH synthesis in the thyroid gland may result from several possible molecular-initiating events (MIEs) including: 1) Disruption of key catalytic enzymes or cofactors needed for TH synthesis, including TPO, NIS, or dietary iodine insufficiency. Theoretically, decreased synthesis of Tg could also affect TH production (Kessler et al., 2008; Yi et al., 1997). Mutations in genes that encode requisite proteins in the thyroid may also lead to impaired TH synthesis, including mutations in pendrin associated with Pendred Syndrome (Dossena et al., 2011), mutations in TPO and Tg (Huang and Jap 2015), and mutations in NIS (Spitzweg and Morris, 2010). 2) Decreased TH synthesis in cases of clinical hypothyroidism may be due to Hashimoto's thyroiditis or other forms of thyroiditis, or physical destruction of the thyroid gland as in radioablation or surgical treatment of thyroid lymphoma. 3) It is possible that TH synthesis may also be reduced subsequent to disruption of the negative feedback mechanism governing TH homeostasis, e.g. pituitary gland dysfunction may result in a decreased TSH signal with concomitant T3 and T4 decreases. 4) More rarely, hypothalamic dysfunction can result in decreased TH synthesis.
Increased fetal TH levels are also possible. Maternal Graves disease, which results in fetal thyrotoxicosis (hyperthyroidism and increased serum T4 levels), has been successfully treated by maternal administration of TPO inhibitors (c.f., Sato et al., 2014).
It should be noted that different species and different life stages store different amounts of TH precursors and iodine within the thyroid gland. Thus, decreased TH synthesis via transient iodine insufficiency or inhibition of TPO may not affect TH release from the thyroid gland until depletion of stored iodinated Tg. Adult humans may store sufficient Tg-DIT residues to serve for several months to a year of TH demand (Greer et al., 2002; Zoeller, 2004). Neonates and infants have a much more limited supply of less than a week.
While the TH system is highly conserved across vertebrates, there are some taxon-specific considerations.
Zebrafish and fathead minnows are oviparous fish species in which maternal THs are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic TH synthesis is initiated. Maternal transfer of THs to the eggs has been demonstrated in zebrafish (Walpita et al., 2007; Chang et al., 2012) and fathead minnows (Crane et al., 2004; Nelson et al., 2016).
Decreases in TH synthesis can only occur after initiation of embryonic TH synthesis. The components of the TH system responsible for TH synthesis are highly conserved across vertebrates and therefore interference with the same molecular targets compared to mammals can lead to decreased TH synthesis (TPO, NIS, etc.) in fish. Endogenous transcription profiles of thyroid-related genes in zebrafish and fathead minnow showed that mRNA coding for these genes is also maternally transferred and increasing expression of most transcripts during hatching and embryo-larval transition indicates a fully functional HPT axis in larvae (Vergauwen et al., 2018). Although the HPT axis is highly conserved, there are some differences between fish and mammals (Blanton and Specker, 2007; Deal and Volkoff, 2020). For example, in fish, corticotropin releasing hormone (CRH) often plays a more important role in regulating thyrotropin (TSH) secretion by the pituitary and thus TH synthesis compared to TSH-releasing hormone (TRH). Also, in most fish species thyroid follicles are more diffusely located in the pharyngeal region rather than encapsulated in a gland.
<h4>How it is Measured or Detected</h4>
Decreased TH synthesis is often implied by measurement of TPO and NIS inhibition measured clinically and in laboratory models as these enzymes are essential for TH synthesis. Rarely is decreased TH synthesis measured directly, but rather the impact of chemicals on the quantity of T4 produced in the thyroid gland, or the amount of T4 present in serum is used as a marker of decreased T4 release from the thyroid gland (e.g., Romaldini et al., 1988). Methods used to assess TH synthesis include, incorporation of radiolabeled tracer compounds, radioimmunoassay, ELISA, and analytical detection.
Recently, amphibian thyroid explant cultures have been used to demonstrate direct effects of chemicals on TH synthesis, as this model contains all necessary synthesis enzymes including TPO and NIS (Hornung et al., 2010). For this work THs was measured by HPLC/ICP-mass spectometry. Decreased TH synthesis and release, using T4 release as the endpoint, has been shown for thiouracil antihyperthyroidism drugs including MMI, PTU, and the NIS inhibitor perchlorate (Hornung et al., 2010).
Techniques for in vivo analysis of TH system disruption among other drug-related effects in fish were reviewed by Raldua and Piña (2014). TIQDT (Thyroxine-immunofluorescence quantitative disruption test) is a method that provides an immunofluorescent based estimate of thyroxine in the gland of zebrafish (Raldua and Babin, 2009; Thienpont et al., 2011; Jomaa et al., 2014; Rehberger et al., 2018).  Thienpont used this method with ~25 xenobiotics (e.g., amitrole, perchlorate, methimazole, PTU, DDT, PCBs). The method detected changes for all chemicals known to directly impact TH synthesis in the thyroid gland (e.g., NIS and TPO inhibitors), but not those that upregulate hepatic catabolism of T4. Rehberger et al. (2018) updated the method to enable simultaneous semi-quantitative visualization of intrafollicular T3 and T4 levels. Most often, whole body TH level measurements in fish early life stages are used as indirect evidence of decreased TH synthesis (Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020). Analytical determination of TH levels by LC-MS is becoming increasingly available (Hornung et al., 2015).
More recently, transgenic zebrafish with fluorescent thyroid follicles are being used to visualize the compensatory proliferation of the thyroid follicles following inhibition of TH synthesis among others (Opitz et al., 2012).
<h4>References</h4>
Bakker B, Bikker H, Vulsma T, de Randamie JS, Wiedijk BM, De Vijlder JJ. 2000. Two decades of screening for congenital hypothyroidism in The Netherlands: TPO gene mutations in total iodide organification defects (an update). The Journal of clinical endocrinology and metabolism.  85:3708-3712.
Bianco AC, Kim BW. (2006). Deiodinases: implications of the local control of thyroid hormone action. J Clin Invest. 116: 2571–2579.
Blanton ML, Specker JL. 2007. The hypothalamic-pituitary-thyroid (hpt) axis in fish and its role in fish development and reproduction. Crit Rev Toxicol. 37(1-2):97-115.
Campinho MA, Saraiva J, Florindo C, Power DM. 2014. Maternal thyroid hormones are essential for neural development in zebrafish. Molecular Endocrinology. 28(7):1136-1149.
Chang J, Wang M, Gui W, Zhao Y, Yu L, Zhu G. 2012. Changes in thyroid hormone levels during zebrafish development. Zoological Science. 29(3):181-184.
Crane HM, Pickford DB, Hutchinson TH, Brown JA. 2004. Developmental changes of thyroid hormones in the fathead minnow, pimephales promelas. General and Comparative Endocrinology. 139(1):55-60.
Deal CK, Volkoff H. 2020. The role of the thyroid axis in fish. Frontiers in Endocrinology. 11.
Dossena S, Nofziger C, Brownstein Z, Kanaan M, Avraham KB, Paulmichl M. (2011). Functional characterization of pendrin mutations found in the Israeli and Palestinian populations. Cell Physiol Biochem. 28: 477-484.Gereben B, Zavacki AM, Ribich S, Kim BW, Huang SA, Simonides WS, Zeöld A, Bianco AC. (2008). Cellular and molecular basis of deiodinase-regulated thyroid hormone signalling. Endocr Rev. 29:898–938.
Fang, Y., Wan, J. P., Zhang, R. J., Sun, F., Yang, L., Zhao, S. X., Dong, M., & Song, H. D. (2022). Tpo knockout in zebrafish partially recapitulates clinical manifestations of congenital hypothyroidism and reveals the involvement of TH in proper development of glucose homeostasis. General and Comparative Endocrinology, 323–324. https://doi.org/10.1016/j.ygcen.2022.114033
Gereben B, Zeöld A, Dentice M, Salvatore D, Bianco AC.  Activation and inactivation of thyroid hormone by deiodinases: local action with general consequences.  Cell Mol Life Sci. 2008 Feb;65(4):570-90
Greer MA, Goodman G, Pleus RC, Greer SE. Health effects assessment for environmental perchlorate contamination: the dose response for inhibition of thyroidal radioiodine uptake in humans. Environ Health Perspect. 2002. 110:927-937.
Hernandez-Mariano JA, Torres-Sanchez L, Bassol-Mayagoitia S, Escamilla-Nunez M, Cebrian ME, Villeda-Gutierrez EA, Lopez-Rodriguez G, Felix-Arellano EE, Blanco-Munoz J. 2017. Effect of exposure to p,p '-dde during the first half of pregnancy in the maternal thyroid profile of female residents in a mexican floriculture area. Environmental Research. 156:597-604.
Hornung MW, Degitz SJ, Korte LM, Olson JM, Kosian PA, Linnum AL, Tietge JE. 2010. Inhibition of thyroid hormone release from cultured amphibian thyroid glands by methimazole, 6-propylthiouracil, and perchlorate. Toxicol Sci 118:42-51.
Hornung MW, Kosian PA, Haselman JT, Korte JJ, Challis K, Macherla C, Nevalainen E, Degitz SJ. 2015. In vitro, ex vivo, and in vivo determination of thyroid hormone modulating activity of benzothiazoles. Toxicological Sciences. 146(2):254-264.
Howdeshell KL. 2002. A model of the development of the brain as a construct of the thyroid system. Environ Health Perspect. 110 Suppl 3:337-48.
Huang CJ and Jap TS. 2015. A systematic review of genetic studies of thyroid disorders in Taiwan. J Chin Med Assoc. 78: 145-153.
Jomaa B, Hermsen SAB, Kessels MY, van den Berg JHJ, Peijnenburg AACM, Aarts JMMJG, Piersma AH, Rietjens IMCM. 2014. Developmental toxicity of thyroid-active compounds in a zebrafish embryotoxicity test. Altex-Alternatives to Animal Experimentation. 31(3):303-317.
Kessler J, Obinger C, Eales G. Factors influencing the study of peroxidase-generated iodine species and implications for thyroglobulin synthesis. Thyroid. 2008 Jul;18(7):769-74. doi: 10.1089/thy.2007.0310
Larsen PR. (2009). Type 2 iodothyronine deiodinase in human skeletal muscle: new insights into its physiological role and regulation. J Clin Endocrinol Metab. 94:1893-1895.
Liu XS, Cai Y, Wang Y, Xu SH, Ji K, Choi K. 2019. Effects of tris(1,3-dichloro-2-propyl) phosphate (tdcpp) and triphenyl phosphate (tpp) on sex-dependent alterations of thyroid hormones in adult zebrafish. Ecotoxicology and Environmental Safety. 170:25-32.
Nelson K, Schroeder A, Ankley G, Blackwell B, Blanksma C, Degitz S, Flynn K, Jensen K, Johnson R, Kahl M et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part i: Fathead minnow. Aquatic Toxicology. 173:192-203.
Opitz R, Maquet E, Huisken J, Antonica F, Trubiroha A, Pottier G, Janssens V, Costagliola S. 2012. Transgenic zebrafish illuminate the dynamics of thyroid morphogenesis and its relationship to cardiovascular development. Developmental Biology. 372(2):203-216.
Opitz R, Maquet E, Zoenen M, Dadhich R, Costagliola S. 2011. Tsh receptor function is required for normal thyroid differentiation in zebrafish. Molecular Endocrinology. 25(9):1579-1599.
Power DM, Llewellyn L, Faustino M, Nowell MA, Bjornsson BT, Einarsdottir IE, Canario AV, Sweeney GE. 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. 130(4):447-459.
Raldua D, Babin PJ. 2009. Simple, rapid zebrafish larva bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function. Environmental Science & Technology. 43(17):6844-6850.
Raldua D, Pina B. 2014. In vivo zebrafish assays for analyzing drug toxicity. Expert Opinion on Drug Metabolism & Toxicology. 10(5):685-697.
Ramhoj, L., Svingen, T., Fradrich, C., Rijntjes, E., Wirth, E.K., Pedersen, K., Kohrle, J., Axelstad, M., 2022. Perinatal exposure to the thyroperoxidase inhibitors methimazole and amitrole perturbs thyroid hormone system signaling and alters motor activity in rat offspring. Toxicology Letters 354, 44-55.
Rehberger K, Baumann L, Hecker M, Braunbeck T. 2018. Intrafollicular thyroid hormone staining in whole-mount zebrafish (danio rerio) embryos for the detection of thyroid hormone synthesis disruption. Fish Physiology and Biochemistry. 44(3):997-1010.
Romaldini JH, Farah CS, Werner RS, Dall'Antonia Júnior RP, Camargo RS. 1988.  "In vitro" study on release of cyclic AMP and thyroid hormone in autonomously functioning thyroid nodules.  Horm Metab Res.20:510-2.
Ruuskanen S, Hsu BY. 2018. Maternal thyroid hormones: An unexplored mechanism underlying maternal effects in an ecological framework. Physiological and Biochemical Zoology. 91(3):904-916.
Santisteban P, Bernal J. Thyroid development and effect on the nervous system. Rev Endocr Metab Disord. 2005 Aug;6(3):217-28.
Spitzweg C, Morris JC. 2010. Genetics and phenomics of hypothyroidism and goiter due to NIS mutations. Molecular and cellular endocrinology. 322:56-63.
Stinckens E, Vergauwen L, Blackwell BR, Anldey GT, Villeneuve DL, Knapen D. 2020. Effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology. 54(10):6213-6223.
Stinckens E, Vergauwen L, Schroeder A, Maho W, Blackwell B, Witters H, Blust R, Ankley G, Covaci A, Villeneuve D et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part ii: Zebrafish. Aquatic Toxicology. 173:204-217.
Thienpont B, Tingaud-Sequeira A, Prats E, Barata C, Babin PJ, Raldúa D.  Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis.  Environ Sci Technol. 2011. 45(17):7525-32.
Vergauwen L, Cavallin JE, Ankley GT, Bars C, Gabriels IJ, Michiels EDG, Fitzpatrick KR, Periz-Stanacev J, Randolph EC, Robinson SL et al. 2018. Gene transcription ontogeny of hypothalamic-pituitary-thyroid axis development in early-life stage fathead minnow and zebrafish. General and Comparative Endocrinology. 266:87-100.
Wabukebunoti MAN, Firling CE. 1983. The prehatching development of the thyroid-gland of the fathead minnow, pimephales-promelas (rafinesque). General and Comparative Endocrinology. 49(2):320-331.
Walpita CN, Van der Geyten S, Rurangwa E, Darras VM. 2007. The effect of 3,5,3'-triiodothyronine supplementation on zebrafish (danio rerio) embryonic development and expression of iodothyronine deiodinases and thyroid hormone receptors. Gen Comp Endocrinol. 152(2-3):206-214.
Walter KM, Miller GW, Chen XP, Yaghoobi B, Puschner B, Lein PJ. 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (danio rerio). General and Comparative Endocrinology. 272:20-32.
Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347.
Yi X, Yamamoto K, Shu L, Katoh R, Kawaoi A. Effects of Propyithiouracil (PTU) Administration on the Synthesis and Secretion of Thyroglobulin in the Rat Thyroid Gland: A Quantitative Immuno-electron Microscopic Study Using Immunogold Technique. Endocr Pathol. 1997 Winter;8(4):315-325.
Zoeller RT, Crofton KM. 2005.  Mode of action: developmental thyroid hormone insufficiency--neurological abnormalities resulting from exposure to propylthiouracil. Crit Rev Toxicol. 35:771-81
Zoeller RT, Tan SW, Tyl RW. 2007. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical reviews in toxicology.  37:11-53.
Zoeller RT.  Interspecies differences in susceptibility to perturbation of thyroid hormone homeostasis requires a definition of "sensitivity" that is informative for risk analysis. Regul Toxicol Pharmacol. 2004 Dec;40(3):380.
<h4><a href="/events/281">Event: 281: Thyroxine (T4) in serum, Decreased</a></h4>
<h5>Short Name: T4 in serum, Decreased</h5>
~~<h4>Key Event Component</h4>~~
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>abnormal circulating thyroxine level</td>
<td>thyroxine</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/42">Aop:42 - Inhibition of Thyroperoxidase and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/54">Aop:54 - Inhibition of Na+/I- symporter (NIS) leads to learning and memory impairment </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/8">Aop:8 - Upregulation of Thyroid Hormone Catabolism via Activation of Hepatic Nuclear Receptors, and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/65">Aop:65 - XX Inhibition of Sodium Iodide Symporter and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/134">Aop:134 - Sodium Iodide Symporter (NIS) Inhibition and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/152">Aop:152 - Interference with thyroid serum binding protein transthyretin and subsequent adverse human neurodevelopmental toxicity </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/159">Aop:159 - Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/175">Aop:175 - Thyroperoxidase inhibition leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/176">Aop:176 - Sodium Iodide Symporter (NIS) Inhibition leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/194">Aop:194 - Hepatic nuclear receptor activation leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/366">Aop:366 - Competitive binding to thyroid hormone carrier protein transthyretin (TTR) leading to altered amphibian metamorphosis </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/367">Aop:367 - Competitive binding to thyroid hormone carrier protein thyroid binding globulin (TBG) leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/364">Aop:364 - Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/365">Aop:365 - Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/119">Aop:119 - Inhibition of thyroid peroxidase leading to follicular cell adenomas and carcinomas (in rat and mouse)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/110">Aop:110 - Inhibition of iodide pump activity leading to follicular cell adenomas and carcinomas (in rat and mouse)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/162">Aop:162 - Enhanced hepatic clearance of thyroid hormones leading to thyroid follicular cell adenomas and carcinomas in the rat and mouse</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/128">Aop:128 - Kidney dysfunction by decreased thyroid hormone</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/188">Aop:188 - Iodotyrosine deiodinase (IYD) inhibition leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/192">Aop:192 - Pendrin inhibition leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/193">Aop:193 - Dual oxidase (DUOX) inhibition leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/457">Aop:457 - Succinate dehydrogenase inhibition leading to increased insulin resistance through reduction in circulating thyroxine</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/458">Aop:458 - AhR activation in the liver leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/459">Aop:459 - AhR activation in the thyroid leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/605">Aop:605 - Thyroid Peroxidase Inhibition Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>Propylthiouracil</td></tr>
<tr><td>Methimazole</td></tr>
<tr><td>Perchlorate</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>serum</td></tr>
</tbody>
</table>
</div>
~~<h3>Evidence for Perturbation by Stressor</h3>~~
~~<h4>Propylthiouracil</h4>~~
~~6-n-propylthouracil is a classic positive control for inhibition of TPO~~
~~~~
~~<h4>Methimazole</h4>~~
~~Methimazole is a classic positive control for inhibition of TPO.~~
~~~~
~~<h4>Perchlorate</h4>~~
~~Perchlorate ion (ClO− ₄) is a classic positive control for inhibition of NIS~~
~~~~
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>human</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>chicken</td>
<td>Gallus gallus</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Xenopus laevis</td>
<td>Xenopus laevis</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fathead minnow</td>
<td>Pimephales promelas</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Sus scrofa</td>
<td>Sus scrofa</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9823" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Female</td>
<td>High</td>
</tr>
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Taxonomic: This KE is plausibly applicable across vertebrates and the overall evidence supporting taxonomic applicability is strong. THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in zebrafish development, embryo-to-larval transition and larval-to-juvenile transition (Thienpont et al., 2011; Liu and Chan, 2002), and amphibian and lamprey metamorphoses is well established (Manzon and Youson, 1997; Yaoita and Brown, 1990; Furlow and Neff, 2006). Their role as environmental messenger via exogenous routes in echinoderms confirms the hypothesis that these molecules are widely distributed among the living organisms (Heyland and Hodin, 2004). However, the role of THs in the different species depends on the expression and function of specific proteins (e.g receptors or enzymes) under TH control and may vary across species and tissues. As such, extrapolation regarding TH action across species and developmental stages should be done with caution.
With few exceptions, vertebrate species have circulating T4 (and T3) that are bound to transport proteins in blood. Clear species differences exist in serum transport proteins (Dohler et al., 1979; Yamauchi and Isihara, 2009). There are three major transport proteins in mammals; thyroid binding globulin (TBG), transthyretin (TTR), and albumin. In adult humans, the percent bound to these proteins is about 75, 15 and 10 percent, respectively (Schussler 2000).  In contrast, in adult rats the majority of THs are bound to TTR. Thyroid- binding proteins are developmentally regulated in rats. TBG is expressed in rats until approximately postnatal day (PND) 60, with peak expression occurring during weaning (Savu et al., 1989). However, low levels of TBG persist into adult ages in rats and can be experimentally induced by hypothyroidism, malnutrition, or caloric restriction (Rouaze-Romet et al., 1992). While these species differences impact TH half-life (Capen, 1997) and possibly regulatory feedback mechanisms, there is little information on quantitative dose-response relationships of binding proteins and serum hormones during development across different species. Serum THs are still regarded as the most robust measurable key event causally linked to downstream adverse outcomes.
Life stage: The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, T4 levels are not expected to decrease in response to exposure to inhibitors of TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH system disruptors.
Sex: The KE is plausibly applicable to both sexes. THs are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of TH levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in TH levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.
<h4>Key Event Description</h4>
All iodothyronines are derived from the modification of tyrosine molecules (Taurog, 2000). There are two biologically active thyroid hormones (THs) in serum, triiodothyronine (T3) and T4, and a few less active iodothyronines (rT3, 3,5-T2). T4 is the predominant TH in circulation, comprising approximately 80% of the TH excreted from the thyroid gland in mammals and is the pool from which the majority of T3 in serum is generated (Zoeller et al., 2007). As such, serum T4 changes usually precede changes in other serum THs.  Decreased thyroxine (T4) in serum results from one or more MIEs upstream and is considered a key biomarker of altered TH homeostasis (DeVito et al., 1999).
All iodothyronines are derived from the modification of tyrosine molecules (Taurog, 2000). There are two biologically active thyroid hormones (THs) in serum, triiodothyronine (T3) and T4, and a few less active iodothyronines, reverse T3 (rT3),  and 3,3'-Diiodothyronine (3,5-T2). T4 is the predominant TH in circulation, comprising approximately 80% of the TH excreted from the thyroid gland in mammals and is the pool from which the majority of T3 in serum is generated (Zoeller et al., 2007). As such, serum T4 changes usually precede changes in other serum THs. Decreased thyroxine (T4) in serum results from one or more MIEs upstream and is considered a key biomarker of altered TH homeostasis (DeVito et al., 1999).
Serum T4 is used as a biomarker of TH status because the circulatory system serves as the major transport and delivery system for TH delivery to tissues. The majority of THs in the blood are bound to transport proteins (Bartalena and Robbins, 1993). In serum, it is the unbound, or ‘free’ form of the hormone that is thought to be available for transport into tissues. Free hormones are approximately 0.03 and 0.3 percent for T4 and T3, respectively. There are major species differences in the predominant binding proteins and their affinities for THs (see below). However, there is broad agreement that changes in serum concentrations of THs is diagnostic of thyroid disease or chemical-induced disruption of thyroid homeostasis across vertebrates (DeVito et al., 1999; Miller et al., 2009; Zoeller et al., 2007; Carr and Patiño, 2011).
Normal serum T4 reference ranges can be species and lifestage specific. In rodents, serum THs are low in the fetal circulation, increasing as the fetal thyroid gland becomes functional on gestational day 17, just a few days prior to birth. After birth serum hormones increase steadily, peaking at two weeks, and falling slightly to adult levels by postnatal day 21 (Walker et al., 1980; Harris et al., 1978; Goldey et al., 1995; Lau et al., 2003). Similarly, in humans, adult reference ranges for THs do not reflect the normal ranges for children at different developmental stages, with TH concentrations highest in infants, still increased in childhood, prior to a decline to adult levels coincident with pubertal development (Corcoran et al. 1977; Kapelari et al., 2008).
In some frog species, there is an analogous peak in THs in tadpoles that starts around embryonic NF stage 56, peaks at stage 62 and the declines to lower levels by stage 56 (Sternberg et al., 2011; Leloup and Buscaglia, 1977).
Additionally, ample evidence is available from studies investigating responses to inhibitors of TH synthesis in fish. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.
<h4>How it is Measured or Detected</h4>
Serum T3 and T4 can be measured as free (unbound) or total (bound + unbound). Free hormone concentrations are clinically considered more direct indicators of T4 and T3 activities in the body, but in animal studies, total T3 and T4 are typically measured. Historically, the most widely used method in toxicology is the radioimmunoassay (RIA). The method is routinely used in rodent endocrine and toxicity studies. The ELISA method is commonly used as a human clinical test method. Analytical determination of iodothyronines (T3, T4, rT3, T2) and their conjugates, through methods employing HPLC, liquid chromatography, immuno luminescence, and mass spectrometry are less common, but are becoming increasingly available (Hornung et al., 2015; DeVito et al., 1999; Baret and Fert, 1989; Spencer, 2013; Samanidou V.F et al., 2000; Rathmann D. et al., 2015 ).  In fish early life stages most evidence for the ontogeny of TH synthesis comes from measurements of whole body TH levels using LC-MS techniques (Hornung et al., 2015) are increasingly used to accurately quantify whole body TH levels as a proxy for serum TH levels (Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020). It is important to note that TH concentrations can be influenced by a number of intrinsic and extrinsic factors (e.g., circadian rhythms, stress, food intake, housing, noise) (see for example, Döhler et al., 1979).
Serum T3 and T4 can be measured as free (unbound) or total (bound + unbound). Free hormone concentrations are clinically considered more direct indicators of T4 and T3 activities in the body, but in animal studies, total T3 and T4 are typically measured. Historically, the most widely used method in toxicology is the radioimmunoassay (RIA). The method is routinely used in rodent endocrine and toxicity studies. The ELISA method is commonly used as a human clinical test method. Analytical determination of iodothyronines (T3, T4, rT3, T2) and their conjugates, through methods employing HPLC, liquid chromatography, immuno luminescence, and mass spectrometry are less common, but are becoming increasingly available (Hornung et al., 2015; DeVito et al., 1999; Baret and Fert, 1989; Spencer, 2013; Samanidou V.F et al., 2000; Rathmann D. et al., 2015 ). In fish early life stages most evidence for the ontogeny of thyroid hormone synthesis comes from measurements of whole body thyroid hormone levels using LC-MS techniques (Hornung et al., 2015) which are increasingly used to accurately quantify whole body thyroid hormone levels as a proxy for serum thyroid hormone levels (Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020). It is important to note that thyroid hormones concentrations can be influenced by a number of intrinsic and extrinsic factors (e.g., circadian rhythms, stress, food intake, housing, noise) (see for example, Döhler et al., 1979).
Any of these measurements should be evaluated for the relationship to the actual endpoint of interest, repeatability, reproducibility, and lower limits of quantification using a fit-for-purpose approach (i.e., different regulatory needs will require different levels of confidence in the AOP). This is of particular significance when assessing the very low levels of THs present in fetal serum. Detection limits of the assay must be compatible with the levels in the biological sample.  All three of the methods summarized above would be fit-for-purpose, depending on the number of samples to be evaluated and the associated costs of each method. Both RIA and ELISA measure THs by an indirect methodology, whereas analytical determination is the most direct measurement available. All these methods, particularly RIA, are repeatable and reproducible.
Any of these measurements should be evaluated for the relationship to the actual endpoint of interest, repeatability, reproducibility, and lower limits of quantification using a fit-for-purpose approach. This is of particular significance when assessing the very low levels of TH present in fetal serum. Detection limits of the assay must be compatible with the levels in the biological sample. All three of the methods summarized above would be fit-for-purpose, depending on the number of samples to be evaluated and the associated costs of each method. Both RIA and ELISA measure THs by an indirect methodology, whereas analytical determination is the most direct measurement available. All these methods, particularly RIA, are repeatable and reproducible.
<h4>References</h4>
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Baret A. and Fert V.  T4 and ultrasensitive TSH immunoassays using luminescent enhanced xanthine oxidase assay. J Biolumin Chemilumin. 1989. 4(1):149-153
Bartalena L, Robbins J. Thyroid hormone transport proteins. Clin Lab Med. 1993 Sep;13(3):583-98. Bassett JH, Harvey CB, Williams GR. (2003). Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol Cell Endocrinol. 213:1-11.
Capen CC. Mechanistic data and risk assessment of selected toxic end points of the thyroid gland. Toxicol Pathol. 1997 25(1):39-48.
Carr JA, Patino R. 2011. The hypothalamus-pituitary-thyroid axis in teleosts and amphibians: Endocrine disruption and its consequences to natural populations. General and Comparative Endocrinology. 170(2):299-312.
Chang J, Wang M, Gui W, Zhao Y, Yu L, Zhu G. 2012. Changes in thyroid hormone levels during zebrafish development. Zoological Science. 29(3):181-184.
Cope RB, Kacew S, Dourson M. A reproductive, developmental and neurobehavioral study following oral exposure of tetrabromobisphenol A on Sprague-Dawley rats. Toxicology. 2015 329:49-59.
Corcoran JM, Eastman CJ, Carter JN, Lazarus L. (1977). Circulating thyroid hormone levels in children. Arch Dis Child. 52: 716-720.
Crane HM, Pickford DB, Hutchinson TH, Brown JA. 2006. The effects of methimazole on development of the fathead minnow, pimephales promelas, from embryo to adult. Toxicological Sciences. 93(2):278-285.
Crofton KM. Developmental disruption of thyroid hormone: correlations with hearing dysfunction in rats. Risk Anal. 2004 Dec;24(6):1665-71.
DeVito M, Biegel L, Brouwer A, Brown S, Brucker-Davis F, Cheek AO, Christensen R, Colborn T, Cooke P, Crissman J, Crofton K, Doerge D, Gray E, Hauser P, Hurley P, Kohn M, Lazar J, McMaster S, McClain M, McConnell E, Meier C, Miller R, Tietge J, Tyl R. (1999). Screening methods for thyroid hormone disruptors. Environ Health Perspect. 107:407-415.
Döhler KD, Wong CC, von zur Mühlen A (1979).   The rat as model for the study of drug effects on thyroid function: consideration of methodological problems.  Pharmacol Ther B. 5:305-18.
Eales JG. (1997). Iodine metabolism and thyroid related functions in organisms lacking thyroid follicles: Are thyroid hormones also vitaminsProc Soc Exp Biol Med. 214:302-317.
Furlow JD, Neff ES. (2006). A developmental switch induced by thyroid hormone: Xenopus laevis metamorphosis. Trends Endocrinol Metab. 17:40–47.
Goldey ES, Crofton KM. Thyroxine replacement attenuates hypothyroxinemia, hearing loss, and motor deficits following developmental exposure to Aroclor 1254 in rats. Toxicol Sci. 1998 45(1):94-10
Goldey ES, Kehn LS, Lau C, Rehnberg GL, Crofton KM.  Developmental exposure to polychlorinated biphenyls (Aroclor 1254) reduces circulating thyroid hormone concentrations and causes hearing deficits in rats. Tox Appl Pharmacol. 1995 135(1):77-88.
Harris AR, Fang SL, Prosky J, Braverman LE, Vagenakis AG.  Decreased outer ring monodeiodination of thyroxine and reverse triiodothyronine in the fetal and neonatal rat.  Endocrinology. 1978 Dec;103(6):2216-22
Hernandez-Mariano JA, Torres-Sanchez L, Bassol-Mayagoitia S, Escamilla-Nunez M, Cebrian ME, Villeda-Gutierrez EA, Lopez-Rodriguez G, Felix-Arellano EE, Blanco-Munoz J. 2017. Effect of exposure to p,p '-dde during the first half of pregnancy in the maternal thyroid profile of female residents in a mexican floriculture area. Environmental Research. 156:597-604.
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Hill RN, Crisp TM, Hurley PM, Rosenthal SL, Singh DV. Risk assessment of thyroid follicular cell tumors.  Environ Health Perspect. 1998 Aug;106(8):447-57.
Hornung MW, Kosian P, Haselman J, Korte J, Challis K, Macherla C, Nevalainen E, Degitz S (2015) In vitro, ex vivo and in vivo determination of thyroid hormone modulating activity of benzothiazoles. Toxicol Sci 146:254-264.
Hulbert AJ. Thyroid hormones and their effects: a new perspective. Biol Rev Camb Philos Soc. 2000 Nov;75(4):519-631. Review.
Kapelari K, Kirchlechner C, Högler W, Schweitzer K, Virgolini I, Moncayo R. 2008. Pediatric reference intervals for thyroid hormone levels from birth to adulthood: a retrospective study. BMC Endocr Disord. 8: 15.
Lau C, Thibodeaux JR, Hanson RG, Rogers JM, Grey BE, Stanton ME, Butenhoff JL, Stevenson LA.  Exposure to perfluorooctane sulfonate during pregnancy in rat and mouse. II: postnatal evaluation.  Toxicol Sci. 2003 Aug;74(2):382-92.
Leloup, J., and M. Buscaglia. La triiodothyronine: hormone de la métamorphose des amphibiens. CR Acad Sci 284 (1977): 2261-2263.
Liu J, Liu Y, Barter RA, Klaassen CD.: Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: a dose-response study. J Pharmacol Exp Ther 273, 977-85, 1994
Liu XS, Cai Y, Wang Y, Xu SH, Ji K, Choi K. 2019. Effects of tris(1,3-dichloro-2-propyl) phosphate (tdcpp) and triphenyl phosphate (tpp) on sex-dependent alterations of thyroid hormones in adult zebrafish. Ecotoxicology and Environmental Safety. 170:25-32.
Liu YW, Chan WK. 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation. 70(1):36-45.
Manzon RG, Youson JH. (1997). The effects of exogenous thyroxine (T4) or triiodothyronine (T3), in the presence and absence of potassium perchlorate, on the incidence of metamorphosis and on serum T4 and T3 concentrations in larval sea lampreys (Petromyzon marinus L.). Gen Comp Endocrinol. 106:211-220.
McClain RM. Mechanistic considerations for the relevance of animal data on thyroid neoplasia to human risk assessment. Mutat Res. 1995 Dec;333(1-2):131-42
Miller MD, Crofton KM, Rice DC, Zoeller RT.  Thyroid-disrupting chemicals: interpreting upstream biomarkers of adverse outcomes. Environ Health Perspect. 2009 117(7):1033-41
Morse DC, Wehler EK, Wesseling W, Koeman JH, Brouwer A. Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to polychlorinated biphenyls (Aroclor 1254). Toxicol Appl Pharmacol. 1996 Feb;136(2):269-79.
Nelson K, Schroeder A, Ankley G, Blackwell B, Blanksma C, Degitz S, Flynn K, Jensen K, Johnson R, Kahl M et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part i: Fathead minnow. Aquatic Toxicology. 173:192-203.
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Stinckens E, Vergauwen L, Schroeder A, Maho W, Blackwell B, Witters H, Blust R, Ankley G, Covaci A, Villeneuve D et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part ii: Zebrafish. Aquatic Toxicology. 173:204-217.
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Yen PM. (2001). Physiological and molecular basis of thyroid hormone action. Physiol Rev. 81:1097-1142.
Zoeller RT, Tan SW, Tyl RW. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Crit Rev Toxicol. 2007 Jan-Feb;37(1-2):11-53
Zoeller, R. T., R. Bansal, et al. (2005). "Bisphenol-A, an environmental contaminant that acts as a thyroid hormone receptor antagonist in vitro, increases serum thyroxine, and alters RC3/neurogranin expression in the developing rat brain." Endocrinology 146(2): 607-612.
<h4><a href="/events/1003">Event: 1003: Decreased, Triiodothyronine (T3)</a></h4>
<h5>Short Name: Decreased, Triiodothyronine (T3)</h5>
~~<h4>Key Event Component</h4>~~
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
~~<td>abnormal circulating hormone level</td>~~
<td>decreased triiodothyronine level</td>
<td>3,3',5'-triiodothyronine</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/155">Aop:155 - Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/156">Aop:156 - Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/157">Aop:157 - Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/158">Aop:158 - Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/189">Aop:189 - Type I iodothyronine deiodinase (DIO1) inhibition leading to altered amphibian metamorphosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/159">Aop:159 - Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/364">Aop:364 - Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/365">Aop:365 - Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/605">Aop:605 - Thyroid Peroxidase Inhibition Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fathead minnow</td>
<td>Pimephales promelas</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>African clawed frog</td>
<td>Xenopus laevis</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
Taxonomic: The overall evidence supporting taxonomic applicability is strong. With few exceptions vertebrate species have T3 and T4 that are mostly bound to transport proteins in blood as well as T3 and T4 in tissues. Therefore, the current key event is plausibly applicable to vertebrates in general. Clear species differences exist in transport proteins (Yamauchi and Isihara, 2009). Specifically, the majority of supporting data for TH decreases come from rat studies and have been measured mostly in serum. The predominant iodothyronine binding protein in rat serum is transthyretin (TTR). TTR demonstrates a reduced binding affinity for T4 when compared with thyroxine binding globulin (TBG), the predominant serum binding protein for T4 in humans. This difference in serum binding protein affinity for THs is thought to modulate serum half-life for T4; the half-life of T4 in rats is 12-24 hr, whereas the half-life in humans is 5-9 days (Capen, 1997). While these species differences impact hormone half-life, possibly regulatory feedback mechanisms, and quantitative dose-response relationships, measurement of decreased THs is still regarded as a measurable key event causatively linked to downstream adverse outcomes.
Taxonomic: The overall evidence supporting taxonomic applicability is strong. With few exceptions vertebrate species have T3 and T4 that are mostly bound to transport proteins in blood as well as T3 and T4 in tissues. Therefore, the current key event is plausibly applicable to vertebrates in general. Clear species differences exist in transport proteins (Yamauchi and Isihara, 2009). Specifically, the majority of supporting data for TH decreases come from rat studies and have been measured mostly in serum. The predominant iodothyronine binding protein in rat serum is transthyretin (TTR). TTR demonstrates a reduced binding affinity for T4 when compared with thyroxine binding globulin (TBG), the predominant serum binding protein for T4 in humans. This difference in serum binding protein affinity for THs is thought to modulate serum half-life for T4; the half-life of T4 in rats is 12-24 hr, whereas the half-life in humans is 5-9 days (Capen, 1997). While these species differences impact hormone half-life, possibly regulatory feedback mechanisms, and quantitative dose-response relationships, measurement of decreased THs is still regarded as a measurable key event causatively linked to downstream adverse outcomes.
Several studies have reported evidence of T3 decreases after exposure to TPO inhibitors and deiodinase inhibitors in early life stages of zebrafish (Stinckens et al., 2016; Stinckens et al., 2020; Wang et al., 2020) and fathead minnow (Nelson et al., 2016; Cavallin et al., 2017). Such measurements in fish early life stages are usually based on whole animal samples and do not allow for distinguishing between systemic and tissue TH alterations.
THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in amphibian and lamprey metamorphoses (Manzon and Youson, 1997; Yaoita and Brown, 1990) as well as fish development, embryo-to-larval transition and larval-to-juvenile transition (Thienpont et al., 2011; Liu and Chan, 2002) is well established. Their role as environmental messenger via exogenous routes in echinoderms confirms the hypothesis that these molecules are widely distributed among the living organisms (Heyland and Hodin, 2004). However, the role of TH in the different species may differ depending on the expression or function of specific proteins (e.g receptors or enzymes) that are related to TH function, and therefore extrapolation between species should be done with caution.
THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in amphibian and lamprey metamorphoses (Manzon and Youson, 1997; Yaoita and Brown, 1990) as well as fish development, embryo-to-larval transition and larval-to-juvenile transition (Thienpont et al., 2011; Liu and Chan, 2002) is well established. Their role as environmental messenger via exogenous routes in echinoderms confirms the hypothesis that these molecules are widely distributed among the living organisms (Heyland and Hodin, 2004). However, the role of TH in the different species may differ depending on the expression or function of specific proteins (e.g., receptors or enzymes) that are related to TH function, and therefore extrapolation between species should be done with caution.</span></p>
Life stage: THs are essential in all life stages, but decreases of TH levels are not applicable to all developmental phases. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, T4 levels are not expected to decrease in response to exposure to inhibitors of TH synthesis during these earliest stages of development. However, T3 levels are expected to decrease upon exposure to deiodinase inhibitors in any life stage, since maternal T4 needs to be activated to T3 by deiodinases similar to embryonically synthesized T4.
Life stage: THs are essential in all life stages, but decreases of TH levels are not applicable to all developmental phases. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, T4 levels are not expected to decrease in response to exposure to inhibitors of TH synthesis during these earliest stages of development. However, T3 levels are expected to decrease upon exposure to deiodinase inhibitors in any life stage, since maternal T4 needs to be activated to T3 by deiodinases similar to embryonically synthesized T4.
Sex: The KE is plausibly applicable to both sexes. THs are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of TH levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in TH levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.
Sex: The KE is plausibly applicable to both sexes. THs are essential in both sexes and the components of the HPT- axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of TH levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in TH levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.
<h4>Key Event Description</h4>
There are two biologically active thyroid hormones (THs), triiodothyronine (T3) and thyroxine (T4), and a few less active iodothyronines (rT3, 3,5-T2), which are all derived from the modification of tyrosine molecules (Hulbert, 2000). However, the plasma concentrations of the other iodothyronines are significantly lower than those of T3 and T4. The different iodothyronines are formed by the sequential outer or inner ring monodeiodination of T4 and T3 by the deiodinating enzymes, Dio1, Dio2, and Dio3 (Gereben et al., 2008). Deiodinase structure is considered to be unique, as THs are the only molecules in the body that incorporate iodide.
There are two biologically active thyroid hormones (THs), triiodothyronine (T3) and thyroxine (T4), and a few less active iodothyronines (rT3, 3,5-T2), which are all derived from the modification of tyrosine molecules (Hulbert, 2000). However, the plasma concentrations of the other iodothyronines are significantly lower than those of T3 and T4. The different iodothyronines are formed by the sequential outer or inner ring monodeiodination of T4 and T3 by the deiodinating enzymes, Dio1, Dio2, and Dio3 (Gereben et al., 2008). Deiodinase structure is considered to be unique, as THs are the only molecules in the body that incorporate iodide.
The circulatory system serves as the major transport and delivery system for THs from synthesis in the gland to delivery to tissues. The majority of THs in the blood are bound to transport proteins (Bartalena and Robbins, 1993). In humans, the major transport proteins are TBG (thyroxine binding globulin), TTR (transthyretin) and albumin. The percent bound to these proteins in adult humans is about 75, 15 and 10 percent, respectively (Schussler 2000). Unbound (free) hormones are approximately 0.03 and 0.3 percent for T4 and T3, respectively. In serum, it is the free form of the hormone that is active.
The circulatory system serves as the major transport and delivery system for THs from synthesis in the gland to delivery to tissues. The majority of THs in the blood are bound to transport proteins (Bartalena and Robbins, 1993). In humans, the major transport proteins are TBG (thyroxine binding globulin), TTR (transthyretin) and albumin. The percent bound to these proteins in adult humans is about 75, 15 and 10 percent, respectively (Schussler 2000). Unbound (free) hormones are approximately 0.03 and 0.3 percent for T4 and T3, respectively. In serum, it is the free form of the hormone that is active.
There are major species differences in the predominant binding proteins and their affinities for THs (see section below on Taxonomic applicability). However, there is broad agreement that changes in concentrations of THs is diagnostic of thyroid disease or chemical-induced disruption of thyroid homeostasis (Zoeller et al., 2007).
There are major species differences in the predominant binding proteins and their affinities for THs (see section below on Taxonomic applicability). However, there is broad agreement that changes in concentrations of THs is diagnostic of thyroid disease or chemical-induced disruption of thyroid homeostasis (Zoeller et al., 2007).
It is notable that the changes measured in the free TH concentration reflect mainly the changes in the serum transport proteins rather than changes in the thyroid status. These thyroid-binding proteins serve as hormonal storage which ensures their even and constant distribution in the different tissues, while they protect the most sensitive ones in the case of severe changes in thyroid availability, like in thyroidectomies (Obregon et al., 1981). Initially, it was believed that all of the effects of TH were mediated by the binding of T3 to the thyroid nuclear receptors (TRa and TRb), a notion which is now questionable due to the increasing evidence that support the non-genomic action of TH (Davis et al., 2010, Moeller et al., 2006). Many non-nuclear TH binding sites have been identified to date and they usually lead to rapid cellular response in TH-effects (Bassett et al., 2003). Four types of thyroid hormone signaling have been defined (Anyetei-Anum et al., 2018): type 1 is the canonical pathway in which liganded TR binds directly to DNA; type 2 describes liganded TR tethered to chromatin-associated proteins, but not bound to DNA directly; type 3 suggests that liganded TR can exert its function without recruitment to chromatin in either the nucleus or cytoplasm; and type 4 proposes that thyroid hormone acts at the plasma membrane or in the cytoplasm without binding TR, a mechanism of action that is emerging as a key component of thyroid hormone signaling.
It is notable that the changes measured in the free TH concentration reflect mainly the changes in the serum transport proteins rather than changes in the thyroid status. These thyroid-binding proteins serve as hormonal storage which ensures their even and constant distribution in the different tissues, while they protect the most sensitive ones in the case of severe changes in thyroid availability, like in thyroidectomies (Obregon et al., 1981). Initially, it was believed that all of the effects of TH were mediated by the binding of T3 to the thyroid nuclear receptors (TRa and TRb), a notion which is now questionable due to the increasing evidence that support the non-genomic action of TH (Davis et al., 2010, Moeller et al., 2006). Many non-nuclear TH binding sites have been identified to date and they usually lead to rapid cellular response in TH-effects (Bassett et al., 2003). Four types of thyroid hormone signaling have been defined (Anyetei-Anum et al., 2018): type 1 is the canonical pathway in which liganded TR binds directly to DNA; type 2 describes liganded TR tethered to chromatin-associated proteins, but not bound to DNA directly; type 3 suggests that liganded TR can exert its function without recruitment to chromatin in either the nucleus or cytoplasm; and type 4 proposes that thyroid hormone acts at the plasma membrane or in the cytoplasm without binding TR, a mechanism of action that is emerging as a key component of thyroid hormone signaling.
The production of THs in the thyroid gland and the circulation levels in the bloodstream are self-controlled by an efficiently regulated feedback mechanism across the Hypothalamus-Pituitary-Thyroid (HPT) axis. TH levels are regulated, not only in the plasma level, but also in the individual cell level, to maintain homeostasis. This is succeeded by the efficient regulatory mechanism of the thyroid hormone axis which consists of the following: (1) the hypothalamic secretion of the thyrotropin-releasing hormone (TRH), (2) the thyroid-stimulating hormone (TSH) secretion from the anterior pituitary, (3) hormonal transport by the plasma binding proteins, (4) cellular uptake mechanisms in the cell level, (5) intracellular control of TH concentrations by the deiodinating mechanism (6) transcriptional function of the nuclear TH receptor and (7) in the fetus, the transplacental passage of T4 and T3 (Cheng et al., 2010).
The production of THs in the thyroid gland and the circulation levels in the bloodstream are self-controlled by an efficiently regulated feedback mechanism across the Hypothalamus-Pituitary-Thyroid (HPT) axis. TH levels are regulated, not only in the plasma level, but also in the individual cell level, to maintain homeostasis. This is succeeded by the efficient regulatory mechanism of the thyroid hormone axis which consists of the following: (1) the hypothalamic secretion of the thyrotropin-releasing hormone (TRH), (2) the thyroid-stimulating hormone (TSH) secretion from the anterior pituitary, (3) hormonal transport by the plasma binding proteins, (4) cellular uptake mechanisms in the cell level, (5) intracellular control of TH concentration by the deiodinating mechanism (6) transcriptional function of the nuclear thyroid hormone receptor and (7) in the fetus, the transplacental passage of T4 and T3 (Cheng et al., 2010).
In regards to the brain, the TH concentration involves also an additional level of regulation, namely the hormonal transport through the Blood Brain Barrier (BBB) (Williams, 2008). The TRH and the TSH regulate the production of THs. Less T3 (the biologically more active TH) than T4 is produced by the thyroid gland. The rest of the required amount of T3 is produced by outer ring deiodination of T4 by the deiodinating enzymes D1 and D2 (Bianco et al., 2006), a process which takes place mainly in liver and kidneys but also in other target organs such as in the brain, the anterior pituitary, brown adipose tissue, thyroid and skeletal muscle (Gereben et al., 2008; Larsen, 2009). Both hormones exert their action in almost all tissues of mammals and they are acting intracellularly, and thus the uptake of T3 and T4 by the target cells is a crucial step of the overall pathway. The trans-membrane transport of TH is performed mainly through transporters that differ depending on the cell type (Hennemann et al., 2001; Friesema et al., 2005; Visser et al., 2008). Many transporter proteins have been identified to date. The monocarboxylate transporters (Mct8, Mct10) and the anion-transporting polypeptide (OATP1c1) show the highest degree of affinity towards TH (Jansen et al., 2005) and mutations in these genes have pathophysiological effects in humans (Bernal et al., 2015). Unlike humans with an MCT8 deficiency, MCT8 knockout mice do not have neurological impairment. One explanation for this discrepancy could be differences in expression of the T4 transporter OATP1C1 in the blood–brain barrier. This shows that cross-species differences in the importance of specific transporters may occur.    
In regards to the brain, the TH concentration involves also an additional level of regulation, namely the hormonal transport through the Blood Brain Barrier (BBB) (Williams, 2008). The TRH and the TSH regulate the production of thyroid hormones. Less T3 (the biologically more active TH) than T4 is produced by the thyroid gland. The rest of the required amount of T3 is produced by outer ring deiodination of T4 by the deiodinating enzymes D1 and D2 (Bianco et al., 2006), a process which takes place mainly in liver and kidneys but also in other target organs such as in the brain, the anterior pituitary, brown adipose tissue, thyroid and skeletal muscle (Gereben et al., 2008; Larsen, 2009). Both hormones exert their action in almost all tissues of mammals and they are acting intracellularly, and thus the uptake of T3 and T4 by the target cells is a crucial step of the overall pathway. The trans-membrane transport of TH is performed mainly through transporters that differ depending on the cell type (Hennemann et al., 2001; Friesema et al., 2005; Visser et al., 2008). Many transporter proteins have been identified to date. The monocarboxylate transporters (Mct8, Mct10) and the anion-transporting polypeptide (OATP1c1) show the highest degree of affinity towards TH (Jansen et al., 2005) and mutations in these genes have pathophysiological effects in humans (Bernal et al., 2015). Unlike humans with an MCT8 deficiency, MCT8 knockout mice do not have neurological impairment. One explanation for this discrepancy could be differences in expression of the T4 transporter OATP1C1 in the blood–brain barrier. This shows that cross-species differences in the importance of specific transporters may occur.
T3 and T4 have significant effects on normal development, neural differentiation, growth rate and metabolism (Yen, 2001; Brent, 2012; Williams, 2008), with the most prominent ones to occur during the fetal development and early childhood. The clinical features of hypothyroidism and hyperthyroidism emphasize the pleiotropic effects of these hormones on many different pathways and target organs. The thyroidal actions though are not only restricted to mammals, as their high significance has been identified also for other vertebrates, with the most well-studied to be the amphibian metamorphosis (Furlow and Neff, 2006). The importance of the thyroid-regulated pathways becomes more apparent in iodine deficient areas of the world, where a higher rate of cretinism and growth retardation has been observed and linked to decreased TH levels (Gilbert et al., 2012). Another very common cause of severe hypothyroidism in human is the congenital hypothyroidism, but the manifestation of these effects is only detectable in the lack of adequate treatment and is mainly related to neurological impairment and growth retardation (Glinoer, 2001), emphasizing the role of TH in neurodevelopment in all above cases. In adults, the thyroid-related effects are mainly linked to metabolic activities, such as deficiencies in oxygen consumption, and in the metabolism of the vitamin, proteins, lipids and carbohydrates, but these defects are subtle and reversible (Oetting and Yen, 2007). Blood tests to detect the amount of thyroid hormone (T4) and thyroid stimulating hormone (TSH) are routinely done for newborn babies for the diagnosis of congenital hypothyroidism at the earliest stage possible.
T3 and T4 have significant effects on normal development, neural differentiation, growth rate and metabolism (Yen, 2001; Brent, 2012; Williams, 2008), with the most prominent ones to occur during the fetal development and early childhood. The clinical features of hypothyroidism and hyperthyroidism emphasize the pleiotropic effects of these hormones on many different pathways and target organs. The thyroidal actions though are not only restricted to mammals, as their high significance has been identified also for other vertebrates, with the most well-studied to be the amphibian metamorphosis (Furlow and Neff, 2006). The importance of the thyroid-regulated pathways becomes more apparent in iodine deficient areas of the world, where a higher rate of cretinism and growth retardation has been observed and linked to decreased TH levels (Gilbert et al., 2012). Another very common cause of severe hypothyroidism in human is the congenital hypothyroidism, but the manifestation of these effects is only detectable in the lack of adequate treatment and is mainly related to neurological impairment and growth retardation (Glinoer, 2001), emphasizing the role of TH in neurodevelopment in all above cases. In adults, the thyroid-related effects are mainly linked to metabolic activities, such as deficiencies in oxygen consumption, and in the metabolism of the vitamin, proteins, lipids and carbohydrates, but these defects are subtle and reversible (Oetting and Yen, 2007). Blood tests to detect the amount of thyroid hormone (T4) and thyroid stimulating hormone (TSH) are routinely done for newborn babies for the diagnosis of congenital hypothyroidism at the earliest stage possible.
Although the components of the TH system as well as TH synthesis and action are highly conserved across vertebrates, there are some taxon-specific considerations.
Although the components of the thyroid hormone system as well as thyroid hormone synthesis and action are highly conserved across vertebrates, there are some taxon-specific considerations.
Although the HPT axis is highly conserved, there are some differences between fish and mammals (Blanton and Specker, 2007; Deal and Volkoff, 2020). For example, in fish, corticotropin releasing hormone (CRH) often plays a more important role in regulating thyrotropin (TSH) secretion by the pituitary and thus TH synthesis compared to TSH-releasing hormone (TRH). TTRs from fish have low sequence identity with human TTR, for example seabream TTR has 54% sequence identity with human TTR but the only amino acid difference within the thyroxine-binding site is the conservative substitution of Ser117 in human TTR to Thr117 in seabream TTR (Santos and Power, 1999; Yamauchi et al., 1999; Eneqvist et al., 2004). In vitro binding experiments showed that TH system disrupting chemicals bind with equal or weaker affinity to seabream TTR than to the human TTR with polar TH disrupting chemicals, in particular, showing a more than 500-fold lower affinity for seabream TTR compared to human TTR (Zhang et al., 2018).
Although the HPT axis is highly conserved, there are some differences between fish and mammals (Blanton and Specker, 2007; Deal and Volkoff, 2020). For example, in fish, corticotropin releasing hormone (CRH) often plays a more important role in regulating thyrotropin (TSH) secretion by the pituitary and thus thyroid hormone synthesis compared to TSH-releasing hormone (TRH). TTRs from fish have low sequence identity with human TTR, for example seabream TTR has 54% sequence identity with human TTR but the only amino acid difference within the thyroxine-binding site is the conservative substitution of Ser117 in human TTR to Thr117 in seabream TTR (Santos and Power, 1999; Yamauchi et al., 1999; Eneqvist et al., 2004). In vitro binding experiments showed that TH system disrupting chemicals bind with equal or weaker affinity to seabream TTR than to the human TTR with polar TH disrupting chemicals, in particular, showing a more than 500-fold lower affinity for seabream TTR compared to human TTR (Zhang et al., 2018).
Zebrafish and fathead minnow are oviparous fish species in which maternal THs are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic TH synthesis is initiated. Maternal transfer of THs, both T4 and T3, to the eggs has been demonstrated in zebrafish (Walpita et al., 2007; Chang et al., 2012) and fathead minnow (Crane et al., 2004; Nelson et al., 2016).
Zebrafish and fathead minnow are oviparous fish species in which maternal thyroid hormones are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic thyroid hormone synthesis is initiated. Maternal transfer of thyroid hormones, both T4 and T3, to the eggs has been demonstrated in zebrafish (Walpita et al., 2007; Chang et al., 2012) and fathead minnows (Crane et al., 2004; Nelson et al., 2016).
Several studies have reported evidence of T3 decreases after exposure to TPO inhibitors and deiodinase inhibitors in early life stages of zebrafish (Stinckens et al., 2016; Stinckens et al., 2020; Wang et al., 2020) and fathead minnow (Nelson et al., 2016; Cavallin et al., 2017).
Several studies have reported evidence of T3 decreases after exposure to TPO inhibitors and deiodinase inhibitors in early life stages of zebrafish (Stinckens et al., 2016; Stinckens et al., 2020; Wang et al., 2020) and fathead minnow (Nelson et al., 2016; Cavallin et al., 2017).
<h4>How it is Measured or Detected</h4>
T3 and T4 can be measured as free (unbound) or total (bound + unbound) in serum, or in tissues. Free hormones are considered more direct indicators of T4 and T3 activities in the body. The majority of T3 and T4 measurements are made using either RIA or ELISA kits. In animal studies, total T3 and T4 are typically measured as the concentrations of free hormone are very low and difficult to detect. Historically, the most widely used method in toxicology is RIA. The method is routinely used in rodent endocrine and toxicity studies. The ELISA method has become more routine in rodent studies. The ELISA method is      commonly used as a human clinical test method.
T3 and T4 can be measured as free (unbound) or total (bound + unbound) in serum, or in tissues. Free hormones are considered more direct indicators of T4 and T3 activities in the body. The majority of T3 and T4 measurements are made using either RIA or ELISA kits. In animal studies, total T3 and T4 are typically measured as the concentrations of free hormone are very low and difficult to detect.
Historically, the most widely used method in toxicology is RIA. The method is routinely used in rodent endocrine and toxicity studies. The ELISA method has become more routine in rodent studies. The ELISA method is commonly used as a human clinical test method.
Recently, analytical determination of iodothyronines (T3, T4, rT3, T2) and their conjugates through methods employing HPLC and mass spectrometry have become more common (DeVito et al., 1999; Miller et al., 2009; Hornung et al., 2015; Nelson et al., 2016; Stinckens et al., 2016).
Recently, analytical determination of iodothyronines (T3, T4, rT3, T2) and their conjugates through methods employing HPLC and mass spectrometry have become more common (DeVito et al., 1999; Miller et al., 2009; Hornung et al., 2015; Nelson et al., 2016; Stinckens et al., 2016).
Any of these measurements should be evaluated for fit-for-purpose, relationship to the actual endpoint of interest, repeatability, and reproducibility. All three of the methods summarized above would be fit-for-purpose, depending on the number of samples to be evaluated and the associated costs of each method. Both RIA and ELISA measure THs by      an indirect methodology, whereas analytical determination is the most direct measurement available. All of these methods, particularly RIA, are repeatable and reproducible.
Any of these measurements should be evaluated for fit-for-purpose, relationship to the actual endpoint of interest, repeatability, and reproducibility. All three of the methods summarized above would be fit-for-purpose, depending on the number of samples to be evaluated and the associated costs of each method. Both RIA and ELISA measure THs by a an indirect methodology, whereas analytical determination is the most direct measurement available. All of these methods, particularly RIA, are repeatable and reproducible.
In fish early life stages most evidence for the ontogeny of TH synthesis comes from measurements of whole body TH levels and using LC-MS techniques (Hornung et al., 2015) are increasingly used to accurately quantify whole body TH levels (Nelson et al., 2016; Stinckens et al., 2016, 2020).
In fish early life stages most evidence for the ontogeny of TH synthesis comes from measurements of whole-body TH levels and using LC-MS techniques (Hornung et al., 2015) are increasingly used to accurately quantify whole-body TH levels (Nelson et al., 2016; Stinckens et al., 2016, 2020).
<h4>References</h4>
Anyetei-Anum, C.S., Roggero, V.R., Allison, L.A., 2018. Thyroid hormone receptor localization in target tissues. Journal of Endocrinology 237, R19-R34.
Anyetei-Anum, C.S., Roggero, V.R., Allison, L.A., 2018. Thyroid hormone receptor localization in target tissues. Journal of Endocrinology 237, R19-R34.
Bartalena L, Robbins J.Thyroid hormone transport proteins.Clin Lab Med. 1993 Sep;13(3):583-98.</span></p>
Bartalena L, Robbins J.Thyroid hormone transport proteins.Clin Lab Med. 1993 Sep;13(3):583-98.
Bassett JH, Harvey CB, Williams GR. (2003). Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol Cell Endocrinol. 213:1-11.
Bassett JH, Harvey CB, Williams GR. (2003). Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol Cell Endocrinol. 213:1-11.
Bernal, J., Guadano-Ferraz, A., Morte, B., 2015. Thyroid hormone transporters-functions and clinical implications. Nature Reviews Endocrinology 11, 406-417.
Bernal, J., Guadano-Ferraz, A., Morte, B., 2015. Thyroid hormone transporters-functions and clinical implications. Nature Reviews Endocrinology 11, 406-417.
Bianco AC, Kim BW. (2006). Deiodinases: implications of the local control of thyroid hormone action. J Clin Invest. 116: 2571–2579.</span></p>
Bianco AC, Kim BW. (2006). Deiodinases: implications of the local control of thyroid hormone action. J Clin Invest. 116: 2571–2579.
Blanton ML, Specker JL. 2007. The hypothalamic-pituitary-thyroid (hpt) axis in fish and its role in fish development and reproduction. Crit Rev Toxicol. 37(1-2):97-115.</span></p>
Blanton ML, Specker JL. 2007. The hypothalamic-pituitary-thyroid (hpt) axis in fish and its role in fish development and reproduction. Crit Rev Toxicol. 37(1-2):97-115.
Brent GA. (2012). Mechanisms of thyroid hormone action. J Clin Invest. 122: 3035-3043.</span></p>
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Hornung, M.W., Kosian, P.A., Haselman, J.T., Korte, J.J., Challis, K., Macherla, C., Nevalainen, E., Degitz, S.J., 2015. In Vitro, Ex Vivo, and In Vivo Determination of Thyroid Hormone Modulating Activity of Benzothiazoles. Toxicological Sciences 146, 254-264.
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<h4><a href="/events/1877">Event: 1877: Altered, retinal layer structure</a></h4>
<h5>Short Name: Altered, retinal layer structure</h5>
~~<h4>Key Event Component</h4>~~
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>retina layer formation</td>
<td>retina</td>
<td>morphological change</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>Propylthiouracil</td></tr>
<tr><td>Methimazole</td></tr>
<tr><td>Perchlorate</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>eye</td></tr>
</tbody>
</table>
</div>
~~<h3>Evidence for Perturbation by Stressor</h3>~~
~~<h4>Propylthiouracil</h4>~~
6-n-propylthouracil is a classic positive control for inhibition of TPO.
~~~~
~~<h4>Methimazole</h4>~~
Methimazole is a classic positive control for inhibition of TPO.
~~~~
~~<h4>Perchlorate</h4>~~
Perchlorate ion (ClO− ₄) is a classic positive control for inhibition of NIS.
~~ ~~
~~~~
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Embryo</td>
<td>High</td>
</tr>
<tr>
<td>Larvae</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
Taxonomic applicability: In general, the eye structure is very conserved among vertebrates, but some differences exist with regard to shape and expression of the different retinal layers. Fig. 1 (from Richardson 2012) demonstrates the histology of the human vs the zebrafish eye. As in humans, the mature zebrafish retina consists of three nuclear layers separated by two plexiform layers. The photoreceptor rod and cone nuclei are located in the outer nuclear layer; the amacrine, horizontal, and Müller glial cell bodies are found in the inner nuclear layer and the ganglion cell bodies are placed in the ganglion cell layer. The plexiform layers connect these layers. In contrast to zebrafish, the human retina lacks UV-sensitive cones.
Other structural differences between species are mostly related to their lifestyle (e.g. nocturnal vs diurnal) (Bibliowicz 2011) and cannot be generalized for specific vertebrate classes.
<img alt="Ein Bild, das Text enthält.
Automatisch generierte Beschreibung" src="blob:https://aopwiki.org/588d0685-dcfd-418e-a35e-b68ac9560ad1" style="height:251px; width:453px" />
Life-stage applicability: Eye structure differs between life stages, as the different retinal layers do not develop at the same time and the eye itself grows with the organism. Eye development in zebrafish closely resembles the one in humans and other vertebrates. The eye develops from three different embryological tissues that form the specific structures of the eye, starting with the optic vesicle at 16 hpf, which further develops into the two-layered optic cup composed of the retinal neuroepithelium and pigmented epithelium until 20 hpf. Lens development begins as a lens placode that forms a solid lens mass by 22 hpf. Afterwards, the neuroectodermal layers of the optic vesicle invaginate ventrally by 24 hpf. By 48 hpf, zebrafish eye morphogenesis is almost complete with only retinal neurogenesis continuing. Retinal pigment epithelium flattening and final differentiation occurs around 27 hpf (Moreno-Marmol and others 2018). By 60 hpf, the different layers of the retina can be distinguished (Morris and Fadool 2005; Schmitt and Dowling 1999). Thereafter, further differentiation and maturation of the layers and cell types continues (Raymond and others 1995). For example, rods continue to mature until around 20 dpf (Morris and Fadool 2005). Impacts on retinal layer structure have been reported at 48, 66, 72, 96 and 120 hpf during zebrafish embryo-eleutheroembryo development (Baumann and others 2016; Komoike and others 2013; Reider and Connaughton 2014). Since the term 'eleutheroembryo' (stage starting at hatching and ending with free-feeding) is not available, the terms 'embryo' and 'larvae' were selected to reflect this.
Sex applicability: Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Effects on retinal layers during early development are therefore expected to be independent of sex.
At later life stages, however, sex dependency cannot be excluded. Sexual dimorphism of eye sclera surface exposure has been recently discovered (Danel et al. 2018; Danel et al., 2020). Danel et al. (2020) also found that women have rounder eye fissures and brighter irises compared to men. Maekawa et al. (2010) observed eye abnormalities such as microphthalmia and cataract in female mice but not in male mice when the fatty acid composition of the diet was changed during gestation. The authors hypothesized that this was due to differences in lipid metabolism. This suggests that effects of other factors on eye structure could also be sex- dependent in vertebrates.
Evidence for perturbation by stressor: Multiple studies demonstrate that eye development and its resulting structure can be disrupted by different stressors (reviewed for example by Chen 2020).
<h4>Key Event Description</h4>
The anatomy and histology of the eye are highly conserved among vertebrates. The cornea and lens refract and focus light onto the posterior chamber of the eye, the vitreous cavity, which is covered by the retina. The retina consists of three specialised layers of cells, the outermost of which is formed by photoreceptors that absorb light and transmit the subsequent neural signal to the innermost layers, which consist of neurons specialised in processing and transmitting this neural signal (Wässle and Riemann, 1978; Cameron and Carney, 2000 ; Rockhill et al, 2000; Fadool, 2003). The neurons of the innermost layer converge to form the optic nerve, which transmits visual information to the brain (Gestri et al., 2012). The retina has different types of photoreceptors, the cones, which are responsible for colour vision, and the rods, which enable vision in the dark or in very low light conditions. In adults, cones are distributed in the retina in a precise and very regular arrangement, forming a photoreceptor mosaic. The precise spatiotemporal pattern of maturation of cones may affect the organization of this mosaic, and THs appear to play a role in the coordination of this maturation process (Suzuki et al., 2013). In the fish retina, this arrangement is most evident in the outer nuclear layer where the position of each cone subtype is precisely arranged relative to the others (Fadool, 2003; Robinson et al., 1993) resulting in a highly ordered crystalline-like mosaic.
The retinal pigment epithelium (RPE) is important to maintain a healthy and functional retina (Strauss 2005). The strong connection between the RPE cells with the tight junction, creates a blood-retinal barrier to mediate the directional transport of ions, water and nutrients while removing waste products. Another key function of the RPE is to absorb excess light energy to protect the neural retina from phototoxicity (Plafker 2012). Phagocytosis of spilled photoreceptor outer segments (Lister 2002) is another function of the RPE to maintain balanced photoreceptor growth, which is important for their physiological function.
Studies that detect and measure altered retinal layer structure after exposure to THs or endocrine disruptors show, for example, altered cone cell number (Allison et al., 2006; Houbrechts et al., 2016; Vancamp et al., 2019), altered retinal cell number (Dong et al., 2014), or a general alteration of retinal morphology (Gamborino et al., 2001; Houbrechts et al., 2016; Komoike et al., 2013; Li et al., 2012; Reider & Connaughton, 2014), alteration of the RPE      (Baumann et al., 2016), abnormal cone differentiation (Duval & Allison, 2018; Suzuki et al., 2013; Viets et al., 2016) or prevention of the opsin switch (Gan & Flamarique, 2010; Raine & Hawryshyn, 2009). Especially the TH receptor TRβ seems to be a key regulator by determining the expression of photoreceptor development in the retina (Ng et al., 2010; Suzuki et al., 2013; Deveau et al., 2019, 2020).
<h4>How it is Measured or Detected</h4>
For assessment of eye structure and layers, mostly simple morphometric analyses based on histological sections are sufficient. This can either be electron microscopy for subcellular changes, or normal light microscopy for cellular changes. Specific antibody staining might help to identify the different retinal layers and photoreceptor types, but usually, they are easily distinguishable by normal histological staining (e.g. HE staining).
Measurement of cell layer diameter is the most popular and simple method to assess changes in eye structure and layers. Moreover, measurement of the pigmentation grade of the retinal pigment epithelium can be used to assess structural changes. Moreover, semi-quantitative assessment of severity grades of morphological changes can be assessed.
(reviewed in Chen 2020)
<h4>References</h4>
Allison, W. T., Dann, S. G., Veldhoen, K. M., & Hawryshyn, C. W. (2006). Degeneration and regeneration of ultraviolet cone photoreceptors during development in rainbow trout. Journal of Comparative Neurology, 499(5), 702–715. <a href="https://doi.org/10.1002/cne.21164">https://doi.org/10.1002/cne.21164</a>
Ali S, Champagne DL, Richardson MK. Behavioral profiling of zebrafish embryos exposed to a panel of 60 water-soluble compounds. Behav Brain Res. 2012;228(2):272-283. doi:10.1016/j.bbr.2011.11.020
Baumann, L., Ros, A., Rehberger, K., Neuhauss, S. C. F., & Segner, H. (2016). Thyroid disruption in zebrafish (Danio rerio) larvae: Different molecular response patterns lead to impaired eye development and visual functions.Aquatic Toxicology, 172, 44–55. <a href="https://doi.org/10.1016/j.aquatox.2015.12.015">https://doi.org/10.1016/j.aquatox.2015.12.015</a>
Baumann, L., Segner, H., Ros, A., Knapen, D., & Vergauwen, L. (2019). Thyroid Hormone Disruptors Interfere with Molecular Pathways of Eye Development and Function in Zebrafish. International Journal of Molecular Sciences, 20(7), 1543. <a href="https://doi.org/10.3390/ijms20071543">https://doi.org/10.3390/ijms20071543</a>
Bibliowicz J, Tittle RK, Gross JM. Toward a Better Understanding of Human Eye Disease: Insights from the Zebrafish, Danio Rerio. Vol 100.; 2011. doi:10.1016/B978-0-12-384878-9.00007-8
Cameron, D.A., Carney, L.H., 2000. Cell mosaic patterns in the native and regenerated inner retina of zebrafish: implications for retinal assembly. J. Comp. Neurol. 416, 356–367.
Danel DP, Wacewicz S, Kleisner K, Lewandowski Z, Kret ME, Zywiczynski P, Perea-Garcia JO. 2020. Sex differences in ocular morphology in Caucasian people: a dubious role of sexual selection in the evolution of sexual dimorphism of the human eye. Behavioral Ecology and Sociobiology 74(10).
Danel DP, Wacewicz S, Lewandowski Z, Zywiczynski P, Perea-Garcia JO. 2018. Humans do not perceive conspecifics with a greater exposed sclera as more trustworthy: a preliminary cross-ethnic study of the function of the overexposed human sclera. Acta Ethologica 21(3):203-208.
Dong, W., Macaulay, L. J., Kwok, K. W., Hinton, D. E., Ferguson, P. L., & Stapleton, H. M. (2014). The PBDE metabolite 6-OH-BDE 47 affects melanin pigmentation and THRβMRNA expression in the eye of zebrafish embryos. Endocrine Disruptors, 2(1), e969072. https://doi.org/10.4161/23273739.2014.969072
Duval, M. G., & Allison, W. T. (2018). Photoreceptor progenitors depend upon coordination of gdf6a, thrβ, and tbx2b to generate precise populations of cone photoreceptor subtypes. Investigative Ophthalmology and Visual Science, 59(15), 6089–6101. <a href="https://doi.org/10.1167/iovs.18-24461">https://doi.org/10.1167/iovs.18-24461</a>
Fadool, J.M., 2003. Development of a rod photoreceptor mosaic revealed in transgenic zebrafish. Dev. Biol. 258, 277–290.
Gamborino, M. J., Sevilla-Romero, E., Muñoz, A., Hernández-Yago, J., Renau-Piqueras, J., & Pinazo-Durán, M. D. (2001). Role of thyroid hormone in craniofacial and eye development using a rat model. Ophthalmic Research,33(5), 283–291. https://doi.org/10.1159/000055682
Gan, K. J., & Flamarique, I. N. (2010). Thyroid hormone accelerates opsin expression during early photoreceptor differentiation and induces opsin switching in differentiated TRα-expressing cones of the salmonid retina. Developmental Dynamics, 239(10), 2700–2713. <a href="https://doi.org/10.1002/dvdy.22392">https://doi.org/10.1002/dvdy.22392</a>
Gestri, G., Link, B. A., & Neuhauss, S. C. (2012). The visual system of zebrafish and its use to model
Houbrechts, A. M., Vergauwen, L., Bagci, E., Van houcke, J., Heijlen, M., Kulemeka, B., Hyde, D. R., Knapen, D., & Darras, V. M. (2016). Deiodinase knockdown affects zebrafish eye development at the level of gene expression, morphology and function. Molecular and Cellular Endocrinology, 424, 81–93. https://doi.org/10.1016/j.mce.2016.01.018
Komoike, Y., Matsuoka, M., & Kosaki, K. (2013). Potential teratogenicity of methimazole: Exposure of zebrafish embryos to methimazole causes similar developmental anomalies to human methimazole embryopathy. Birth Defects Research Part B - Developmental and Reproductive Toxicology, 98(3), 222–229. https://doi.org/10.1002/bdrb.21057
Li, Z., Ptak, D., Zhang, L., Walls, E. K., Zhong, W., & Leung, Y. F. (2012). Phenylthiourea specifically reduces zebrafish eye size. PLoS ONE,7(6), 1–14. <a href="https://doi.org/10.1371/journal.pone.0040132">https://doi.org/10.1371/journal.pone.0040132</a>
Lister JA. Development of pigment cells in the zebrafish embryo. Microsc Res Tech. 2002;58(6):435-441. doi:10.1002/jemt.10161
Moreno-Marmol T, Cavodeassi F, Bovolenta P. 2018. Setting Eyes on the Retinal Pigment Epithelium. Frontiers in Cell and Developmental Biology 6.
Morris AC, Fadool JM. 2005. Studying rod photoreceptor development in zebrafish. Physiology & Behavior 86(3):306-313.
Plafker SM, O'Mealey GB, Szweda LI. Mechanisms for countering oxidative stress and damage in retinal pigment epithelium. Int Rev Cell Mol Biol. 2012;298:135-77. doi: 10.1016/B978-0-12-394309-5.00004-3. PMID: 22878106; PMCID: PMC3564215.
Raine, J. C., & Hawryshyn, C. W. (2009). Changes in thyroid hormone reception precede SWS1 opsin downregulation in trout retina. Journal of Experimental Biology, 212(17), 2781–2786. <a href="https://doi.org/10.1242/jeb.030866">https://doi.org/10.1242/jeb.030866</a>
Raymond PA, Barthel LK, Curran GA. 1995. DEVELOPMENTAL PATTERNING OF ROD AND CONE PHOTORECEPTORS IN EMBRYONIC ZEBRAFISH. Journal of Comparative Neurology 359(4):537-550.
Reider, M., & Connaughton, V. P. (2014). Effects of low-dose embryonic thyroid disruption and rearing temperature on the development of the eye and retina in zebrafish. Birth Defects Research. Part B, Developmental and Reproductive Toxicology, 101(5), 347–354. <a href="https://doi.org/10.1002/bdrb.21118">https://doi.org/10.1002/bdrb.21118</a>
Rockhill, R.L., Euler, T., Masland, R.H., 2000. Spatial order within but not between types of retinal neurons. Proc. Natl. Acad. Sci. USA 97, 2303–2307.
Schmitt EA, Dowling JE. 1999. Early retinal development in the zebrafish, Danio rerio: Light and electron microscopic analyses. Journal of Comparative Neurology 404(4):515-536.
Suzuki, S. C., Bleckert, A., Williams, P. R., Takechi, M., Kawamura, S., & Wong, R. O. L. (2013). Cone photoreceptor types in zebrafish are generated by symmetric terminal divisions of dedicated precursors. Proceedings of the National Academy of Sciences, 110(37), 15109 LP – 15114.<a href="https://doi.org/10.1073/pnas.1303551110">https://doi.org/10.1073/pnas.1303551110</a>
Strauss O. The retinal pigment epithelium in visual function. Physiol Rev. 2005 Jul;85(3):845-81. doi: 10.1152/physrev.00021.2004. PMID: 15987797.
Vancamp, P., Bourgeois, N. M. A., Houbrechts, A. M., & Darras, V. M. (2019). Knockdown of the thyroid hormone transporter MCT8 in chicken retinal precursor cells hampers early retinal development and results in a shift towards more UV/blue cones at the expense of green/red cones. Experimental Eye Research, 178(September 2018), 135–147. https://doi.org/10.1016/j.exer.2018.09.018
Viets, K., Eldred, K. C., & Johnston, R. J. (2016). Mechanisms of Photoreceptor Patterning in Vertebrates and Invertebrates. Trends in Genetics, 32(10), 638–659. https://doi.org/10.1016/j.tig.2016.07.004
Wässle, H., Riemann, H.J., 1978. The mosaic of nerve cells in the mammalian retina. Proc. R. Soc. London B Biol. Sci. 200, 441–461.
<h4><a href="/events/1643">Event: 1643: Altered, Visual function</a></h4>
<h5>Short Name: Altered, Visual function</h5>
~~<h4>Key Event Component</h4>~~
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>vision trait</td>
<td>eye</td>
<td>functional change</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/297">Aop:297 - Inhibition of retinaldehyde dehydrogenase leads to population decline</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/399">Aop:399 - Inhibition of Fyna leading to increased mortality via decreased eye size (Microphthalmos)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/364">Aop:364 - Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/365">Aop:365 - Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>Propylthiouracil</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Organ</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>eye</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Embryo</td>
<td>High</td>
</tr>
<tr>
<td>Juvenile</td>
<td>Moderate</td>
</tr>
<tr>
<td>Larvae</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
Taxonomic applicability: Visual function decrease can be evaluated in a wide range of species including mammals, amphibians, fish and humans. Evaluation of these visual function modifications change according to the species and its environment.
Life-stage applicability: Vision plays a crucial role in the early life stages of most species, as timing of eye development and establishment of functional vision is essential for perception of food or avoidance of predators for example (Carvalho et al., 2002). The first visual responses based on retinal functionality appear around 70 hpf in zebrafish (Schmitt and Dowling 1999). It is plausible to assume that alterations of the eye structure would result in altered visual function across all life stages, but such alterations are most likely to occur during the development of the normal eye structure, which occurs in the embryo-eleutheroembryo phase. Some studies have also shown a decrease in vision related to age (Brastrom et al., 2019; Martínez-Roda et al., 2016; Segura et al., 2018) including on visual acuity, visual fields, colour vision and dark adaptation, are well documented (Hennelly et al, 1998).
Sex applicability: Sex does not seem relevant for most of the visual function decreases observed in different studies. Differences according to the sex of the individuals have however been reported concerning the basic visual capacities (e.g. color perception, contrast sensitivity, visual acuity, motion perception,...) but also concerning the frequency of certain diseases influencing these diminished visual functions, notably in humans (Vanston and Strother, 2017).
<h4>Key Event Description</h4>
The decrease in visual function can have different aspects, such as loss of chromatic vision, changes in eye movements, differences in sensitivity to light, but also changes in the retinal pigment epithelium (RPE) that may be related to a decrease in visual function (Strauss, 2005). The visual system is highly variable from one species to another, and this variability is a key factor influencing animal behaviour (Corral-López et al., 2017).
Decreases in these visual functions can have a strong impact on behaviour, leading to changes in individual response and abilities in the environment, including, for example, perception of food or avoidance of predators. Variation in the visual system can also influence learning tasks when visual stimuli are used (Corral-López et al., 2017).
Studies have detected visual impairments in fish at different temperatures (Babkiewicz et al., 2020) after treatment with the endocrine disruptor propylthiouracil (Baumann et al 2016 ), after chronic dietary selenomethionine exposure (Raine et al 2016), exposure to PCBs (Zhang et al, 2015) or deiodinase knockdown (Houbrechts et al 2016, Vancamp et al 2018).
<h4>How it is Measured or Detected</h4>
Measurements of visual function can be performed at the level of neuronal activity:
<ul>
<li>Electroretinography (Chrispell et al., 2015)</li>
<li>Analysis of neural activity in the optic tectum can be quantified as the ratio of phosphorylated extracellular signal–regulated kinase (ERK)      to total ERK in the optic tectum using immunofluorescent antibodies (Randlett et al., 2015, Dehnert et al., 2019).</li>
<li>Babkiewicz et al. (2020) used an advanced technique to display an artificial prey on a miniature OLED screen and use functional calcium imaging with light sheet microscopy to visualize a neural response in the optic tectum.</li>
</ul>
Other measurements are performed at the level of the eyes:
<ul>
<li>Opto Kinetic response, OKR (similar protocol for Rat/mice (Segura et al., 2018), fish (Zou et al., 2010) and humans (Kang and Wildsoet, 2016)). The OKR is a visually-mediated assay in which an individual will respond to alternating black and white stripes by exhibiting eye saccades, eye movements without coordinated body movements, in the same direction as rotating stripes. An eye saccade relies on the ability to rapidly move the eye from focusing on one external target to the next in a repeated manner (Magnuson et al., 2020). Optokinetic tracking has a robust performance and does not require training the animal, allowing for the quick assessment (and at earlier ages) of visual features such as visual acuity (VA) and contrast sensitivity (CS)11–14.      </li>
</ul>
Yet other studies use assessment of vision-related behaviours:
<ul>
<li>Opto Motor Reponses, OMR. OMR tracks the ability of fish to swim in the direction of a perceived motion when presented with a whole-field stimulus (Neuhauss, 2003), (Gould et al., 2017)).</li>
<li>Light-dark transition or vision startle response: reaction to change in light intensity (light sensitivity) (Brastrom et al., 2019)</li>
<li>Black-white preference test (Baumann et al., 2016)</li>
</ul>
<ul>
<li>Diverse Mobility assay including Tracking, touch-evoked escape-response assays, Swirl assays, locomotion assay, swimming activity, phototactic swimming activity assay, induced locomotor response (LLR) (Baumann et al., 2016; Gao et al., 2015; Zhao et al., 2014, Dehnert et al., 2019).</li>
</ul>
<h4>References</h4>
Baumann, L., Ros, A., Rehberger, K., Neuhauss, S. C. F., & Segner, H. (2016). Thyroid disruption in zebrafish (Danio rerio) larvae: Different molecular response patterns lead to impaired eye development and visual functions. Aquatic Toxicology, 172, 44–55. https://doi.org/10.1016/j.aquatox.2015.12.015
Babkiewicz, E., Bazała, M., Urban, P., Maszczyk, P., Markowska, M., & Maciej Gliwicz, Z. (2020). The effects of temperature on the proxies of visual detection of Danio rerio larvae: observations from the optic tectum. Biology Open, 9(7). https://doi.org/10.1242/BIO.047779
Brastrom, L.K., Scott, C.A., Dawson, D. V., Slusarski, D.C., 2019. A High-Throughput Assay for Congenital and Age-Related Eye Diseases in Zebrafish. Biomedicines 7, 28. https://doi.org/10.3390/biomedicines7020028
Carvalho, P.S.M., Noltie, D.B., Tillitt, D.E., 2002. Ontogenetic improvement of visual function in the medaka Oryzias latipes based on an optomotor testing system for larval and adult fish. Anim. Behav. 64, 1–10. https://doi.org/10.1006/anbe.2002.3028
Chrispell JD, Rebrik TI, Weiss ER. 2015. Electroretinogram Analysis of the Visual Response in Zebrafish Larvae. Jove-Journal of Visualized Experiments(97).
Corral-López, A., Garate-Olaizola, M., Buechel, S.D., Kolm, N., Kotrschal, A., 2017. On the role of body size, brain size, and eye size in visual acuity. Behav. Ecol. Sociobiol. 71. https://doi.org/10.1007/s00265-017-2408-z
Dehnert GK, Karasov WH, Wolman MA. 2019. 2,4-Dichlorophenoxyacetic acid containing herbicide impairs essential visually guided behaviors of larval fish. Aquatic Toxicology 209:1-12.
Gao, D., Wu, M., Wang, C., Wang, Y., Zuo, Z., 2015. Chronic exposure to low benzo[a]pyrene level causes neurodegenerative disease-like syndromes in zebrafish (Danio rerio). Aquat. Toxicol.
Gould, C. J., Wiegand, J. L., & Connaughton, V. P. (2017). Acute developmental exposure to 4-hydroxyandrostenedione has a long-term effect on visually-guided behaviors. Neurotoxicology and Teratology, 64, 45–49. https://doi.org/10.1016/j.ntt<a href="https://doi.org/10.1016/j.ntt.2017.10.003">.</a>2017.10.003
Hennelly, M. L., Barbur, J. L., Edgar, D. F., & Woodward, E. G. (1998). The effect of age on the light scattering characteristics of the eye. Ophthalmic and Physiological Optics, 18(2), 197–203. https://doi.org/10.1046/j.1475-1313.1998.00333.x
Houbrechts, A. M., Vergauwen, L., Bagci, E., Van houcke, J., Heijlen, M., Kulemeka, B., Hyde, D. R., Knapen, D., & Darras, V. M. (2016). Deiodinase knockdown affects zebrafish eye development at the level of gene expression, morphology and function. Molecular and Cellular Endocrinology, 424, 81–93.      https://doi.org/10.1016/j.mce.2016.01.018
Kang, P., & Wildsoet, C. F. (2016). Acute and short-term changes in visual function with multifocal soft contact lens wear in young adults. Contact Lens and Anterior Eye, 39(2), 133–140. https://doi.org/10.1016/j.clae.2015.09.004
Magnuson, J., Bautista, N., Lucero, J., Lund, A., Xu, E. G., Schlenk, D., Burggren, W., & Roberts, A. P. (2020). Exposure to crude oil induces retinal apoptosis and impairs visual function in fish. Environmental Science & Technology. https://doi.org/10.1021/acs.est.9b07658
Martínez-Roda, J. A., Vilaseca, M., Ondategui, J. C., Aguirre, M., & Pujol, J. (2016). Effects of aging on optical quality and visual function. Clinical and Experimental Optometry, 99(6), 518–525. https://doi.org/10.1111/cxo.12369
Neuhauss, S. C. F. (2003). Behavioral genetic approaches to visual system development and function in zebrafish. Journal of Neurobiology, 54(1), 148–160. https://doi.org/10.1002/neu.10165
Raine, J. C., Lallemand, L., Pettem, C. M., & Janz, D. M. (2016). Effects of Chronic Dietary Selenomethionine Exposure on the Visual System of Adult and F1 Generation Zebrafish (Danio rerio). Bulletin of Environmental Contamination and Toxicology, 97(3), 331–336. https://doi.org/10.1007/s00128-016-1849-9
Randlett O, Wee CL, Naumann EA, Nnaemeka O, Schoppik D, Fitzgerald JE, Portugues R, Lacoste AMB, Riegler C, Engert F et al. . 2015. Whole-brain activity mapping onto a zebrafish brain atlas. Nature Methods 12(11):1039-1046.
Schmitt, E. A., & Dowling, J. E. (1994). Early‐eye morphogenesis in the zebrafish, Brachydanio rerio. Journal of Comparative Neurology, 344(4), 532–542. https://doi.org/10.1002/cne.903440404
Segura, F., Arines, J., Sánchez-Cano, A., Perdices, L., Orduna-Hospital, E., Fuentes-Broto, L., & Pinilla, I. (2018). Development of optokinetic tracking software for objective evaluation of visual function in rodents. Scientific Reports, 8(1), 1–11. https://doi.org/10.1038/s41598-018-28394-x
Strauss, O. (2005). The retinal pigment epithelium in visual function. Physiological Reviews, 85(3), 845–881.https://doi.org/10.1152/physrev.00021.2004
Vancamp, P., Bourgeois, N. M. A., Houbrechts, A. M., & Darras, V. M. (2019). Knockdown of the thyroid hormone transporter MCT8 in chicken retinal precursor cells hampers early retinal development and results in a shift towards more UV/blue cones at the expense of green/red cones. Experimental Eye Research,178(September 2018), 135–147. https://doi.org/10.1016/j.exer.2018.09.018
Zhang, X., Hong, Q., Yang, L., Zhang, M., Guo, X., Chi, X., & Tong, M. (2015). PCB1254 exposure contributes to the abnormalities of optomotor responses and influence of the photoreceptor cell development in zebrafish larvae. Ecotoxicology and Environmental Safety, 118, 133–138. https://doi.org/10.1016/j.ecoenv.2015.04.026
Zhao, J., Xu, T., & Yin, D. Q. (2014). Locomotor activity changes on zebrafish larvae with different 2,2’,4,4’-tetrabromodiphenyl ether (PBDE-47) embryonic exposure modes. Chemosphere, 94, 53–61. https://doi.org/10.1016/j.chemosphere.2013.09.010
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<h3>List of Adverse Outcomes in this AOP</h3>
<h4><a href="/events/351">Event: 351: Increased Mortality</a></h4>
<h5>Short Name: Increased Mortality</h5>
~~<h4>Key Event Component</h4>~~
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mortality</td>
<td></td>
<td>increased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/16">Aop:16 - Acetylcholinesterase inhibition leading to acute mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/96">Aop:96 - Axonal sodium channel modulation leading to acute mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/104">Aop:104 - Altered ion channel activity leading impaired heart function</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/113">Aop:113 - Glutamate-gated chloride channel activation leading to acute mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/160">Aop:160 - Ionotropic gamma-aminobutyric acid receptor activation mediated neurotransmission inhibition leading to mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/161">Aop:161 - Glutamate-gated chloride channel activation leading to neurotransmission inhibition associated mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/138">Aop:138 - Organic anion transporter (OAT1) inhibition leading to renal failure and mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/177">Aop:177 - Cyclooxygenase 1 (COX1) inhibition leading to renal failure and mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/186">Aop:186 - unknown MIE leading to renal failure and mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/312">Aop:312 - Acetylcholinesterase Inhibition leading to Acute Mortality via Impaired Coordination & Movement</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/320">Aop:320 - Binding of SARS-CoV-2 to ACE2 receptor leading to acute respiratory distress associated mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/155">Aop:155 - Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/156">Aop:156 - Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/157">Aop:157 - Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/158">Aop:158 - Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/159">Aop:159 - Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Multi Organ Failure involving Acute Respiratory Distress Syndrome (ARDS)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/364">Aop:364 - Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/365">Aop:365 - Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/399">Aop:399 - Inhibition of Fyna leading to increased mortality via decreased eye size (Microphthalmos)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/413">Aop:413 - Oxidation and antagonism of reduced glutathione leading to mortality via acute renal failure</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/410">Aop:410 - GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/450">Aop:450 - Inhibition of AChE and activation of CYP2E1 leading to sensory axonal peripheral neuropathy and mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/536">Aop:536 - Estrogen receptor agonism leading to reduced survival and population growth due to renal failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/564">Aop:564 - DBDPE-induced inhibition of mitochondrial complex Ⅰ leading to population decline via neurotoxicity and metabotoxicity.</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/605">Aop:605 - Thyroid Peroxidase Inhibition Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Population</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>all species</td>
<td>all species</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
All living things are susceptible to mortality.
<h4>Key Event Description</h4>
Increased mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time.
<h4>How it is Measured or Detected</h4>
Mortality of animals is generally observed as cessation of the heart beat, breathing (gill or lung movement) and locomotory movements. Mortality is typically measured by observation. Depending on the size of the organism, instruments such as microscopes may be used. The reported metric is mostly the mortality rate: the number of deaths in a given area or period, or from a particular cause.
Depending on the species and the study setup, mortality can be measured:
<ul>
<li>in the lab by recording mortality during exposure experiments</li>
<li>in dedicated setups simulating a realistic situation such as mesocosms or drainable ponds for aquatic species</li>
<li>in the field, for example by determining age structure after one capture, or by capture-mark-recapture efforts. The latter is a method commonly used in ecology to estimate an animal population's size where it is impractical to count every individual.</li>
</ul>
<h4>Regulatory Significance of the AO</h4>
Increased mortality is one of the most common regulatory assessment endpoints, along with reduced growth and reduced reproduction.
<h4><a href="/events/360">Event: 360: Decrease, Population growth rate</a></h4>
<h5>Short Name: Decrease, Population growth rate</h5>
~~<h4>Key Event Component</h4>~~
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>population growth rate</td>
<td>population of organisms</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/23">Aop:23 - Androgen receptor agonism leading to reproductive dysfunction (in repeat-spawning fish)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/25">Aop:25 - Aromatase inhibition leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/29">Aop:29 - Estrogen receptor agonism leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/30">Aop:30 - Estrogen receptor antagonism leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/100">Aop:100 - Cyclooxygenase inhibition leading to reproductive dysfunction via inhibition of female spawning behavior</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/122">Aop:122 - Prolyl hydroxylase inhibition leading to reproductive dysfunction via increased HIF1 heterodimer formation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/123">Aop:123 - Unknown MIE leading to reproductive dysfunction via increased HIF-1alpha transcription</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/155">Aop:155 - Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/156">Aop:156 - Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/157">Aop:157 - Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/158">Aop:158 - Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/159">Aop:159 - Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/101">Aop:101 - Cyclooxygenase inhibition leading to reproductive dysfunction via inhibition of pheromone release</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/102">Aop:102 - Cyclooxygenase inhibition leading to reproductive dysfunction via interference with meiotic prophase I /metaphase I transition</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/63">Aop:63 - Cyclooxygenase inhibition leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/103">Aop:103 - Cyclooxygenase inhibition leading to reproductive dysfunction via interference with spindle assembly checkpoint</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/292">Aop:292 - Inhibition of tyrosinase leads to decreased population in fish</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/310">Aop:310 - Embryonic Activation of the AHR leading to Reproductive failure, via epigenetic down-regulation of GnRHR</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/16">Aop:16 - Acetylcholinesterase inhibition leading to acute mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/312">Aop:312 - Acetylcholinesterase Inhibition leading to Acute Mortality via Impaired Coordination & Movement</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/334">Aop:334 - Glucocorticoid Receptor Agonism Leading to Impaired Fin Regeneration</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/336">Aop:336 - DNA methyltransferase inhibition leading to population decline (1)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/337">Aop:337 - DNA methyltransferase inhibition leading to population decline (2)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/338">Aop:338 - DNA methyltransferase inhibition leading to population decline (3)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/339">Aop:339 - DNA methyltransferase inhibition leading to population decline (4)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/340">Aop:340 - DNA methyltransferase inhibition leading to transgenerational effects (1)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/341">Aop:341 - DNA methyltransferase inhibition leading to transgenerational effects (2)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/289">Aop:289 - Inhibition of 5α-reductase leading to impaired fecundity in female fish</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/297">Aop:297 - Inhibition of retinaldehyde dehydrogenase leads to population decline</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/346">Aop:346 - Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/326">Aop:326 - Thermal stress leading to population decline (3)</a></td>~~
~~<td>AdverseOutcome</td>~~
~~</tr>~~
~~<tr>~~
~~<td><a href="/aops/325">Aop:325 - Thermal stress leading to population decline (2)</a></td>~~
~~<td>AdverseOutcome</td>~~
~~</tr>~~
~~<tr>~~
~~<td><a href="/aops/324">Aop:324 - Thermal stress leading to population decline (1)</a></td>~~
~~<td>AdverseOutcome</td>~~
~~</tr>~~
~~<tr>~~
<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/349">Aop:349 - Inhibition of 11β-hydroxylase leading to decresed population trajectory </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/348">Aop:348 - Inhibition of 11β-Hydroxysteroid Dehydrogenase leading to decreased population trajectory </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/376">Aop:376 - Androgen receptor agonism leading to male-biased sex ratio</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/386">Aop:386 - Deposition of ionizing energy leads to leading to population decline via inhibition of photosynthesis</a></td>~~
<td><a href="/aops/386">Aop:386 - Deposition of ionizing energy leading to population decline via inhibition of photosynthesis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/387">Aop:387 - Deposition of ionising energy leading to population decline via mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/388">Aop:388 - Deposition of ionising energy leading to population decline via programmed cell death</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/389">Aop:389 - Oxygen-evolving complex damage leading to population decline via inhibition of photosynthesis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/364">Aop:364 - Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/365">Aop:365 - Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/399">Aop:399 - Inhibition of Fyna leading to increased mortality via decreased eye size (Microphthalmos)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/410">Aop:410 - GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/216">Aop:216 - Deposition of energy leading to population decline via DNA strand breaks and follicular atresia</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/238">Aop:238 - Deposition of energy leading to population decline via DNA strand breaks and oocyte apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/299">Aop:299 - Deposition of energy leading to population decline via DNA oxidation and follicular atresia</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/311">Aop:311 - Deposition of energy leading to population decline via DNA oxidation and oocyte apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/444">Aop:444 - Ionizing radiation leads to reduced reproduction in Eisenia fetida via reduced spermatogenesis and cocoon hatchability</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/138">Aop:138 - Organic anion transporter (OAT1) inhibition leading to renal failure and mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/177">Aop:177 - Cyclooxygenase 1 (COX1) inhibition leading to renal failure and mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/97">Aop:97 - 5-hydroxytryptamine transporter (5-HTT; SERT) inhibition leading to population decline</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/203">Aop:203 - 5-hydroxytryptamine transporter inhibition leading to decreased reproductive success and population decline</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/218">Aop:218 - Inhibition of CYP7B activity leads to decreased reproductive success via decreased locomotor activity</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/219">Aop:219 - Inhibition of CYP7B activity leads to decreased reproductive success via decreased sexual behavior</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/323">Aop:323 - PPARalpha Agonism Leading to Decreased Viable Offspring via Decreased 11-Ketotestosterone</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/536">Aop:536 - Estrogen receptor agonism leading to reduced survival and population growth due to renal failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/540">Aop:540 - Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/564">Aop:564 - DBDPE-induced inhibition of mitochondrial complex Ⅰ leading to population decline via neurotoxicity and metabotoxicity.</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/567">Aop:567 - Binding to plastoquinone B site leading to decreased population growth rate via photosystem II inhibition</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/592">Aop:592 - DBDPE-induced DNA strand breaks and LDH activity inhibition leading to population growth rate decline via energy metabolism disrupt and apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/605">Aop:605 - Thyroid Peroxidase Inhibition Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Population</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>all species</td>
<td>all species</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>Not Specified</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Not Specified</td>
</tr>
</tbody>
</table>
</div>
Consideration of population size and changes in population size over time is potentially relevant to all living organisms.
<h4>Key Event Description</h4>
A population can be defined as a group of interbreeding organisms, all of the same species, occupying a specific space during a specific time (Vandermeer and Goldberg 2003, Gotelli 2008).  As the population is the biological level of organization that is often the focus of ecological risk assessments, population growth rate (and hence population size over time) is important to consider within the context of applied conservation practices.
If N is the size of the population and t is time, then the population growth rate (dN/dt) is proportional to the instantaneous rate of increase, r, which measures the per capita rate of population increase over a short time interval. Therefore, r, is a difference between the instantaneous birth rate (number of births per individual per unit of time; b) and the instantaneous death rate (number of deaths per individual per unit of time; d) [Equation 1]. Because  r is an instantaneous rate, its units can be changed via division.  For example, as there are 24 hours in a day, an r of 24 individuals/(individual x day) is equal to an r of 1 individual/(individual/hour) (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020).
Equation 1:  r = b - d
This key event refers to scenarios where r < 0 (instantaneous death rate exceeds instantaneous birth rate).
Examining r in the context of population growth rate:
● A population will decrease to extinction when the instantaneous death rate exceeds the instantaneous birth rate (r < 0).
● The smaller the value of r below 1, the faster the population will decrease to zero.
● A population will increase when resources are available and the instantaneous birth rate exceeds the instantaneous death rate (r > 0)
● The larger the value that r exceeds 1, the faster the population can increase over time
● A population will neither increase or decrease when the population growth rate equals 0 (either due to N = 0, or if the per capita birth and death rates are exactly balanced).  For example, the per capita birth and death rates could become exactly balanced due to density dependence and/or to the effect of a stressor that reduces survival and/or reproduction (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020).
Effects incurred on a population from a chemical or non-chemical stressor could have an impact directly upon birth rate (reproduction) and/or death rate (survival), thereby causing a decline in population growth rate.
● Example of direct effect on r:  Exposure to 17b-trenbolone reduced reproduction (i.e., reduced b) in the fathead minnow over 21 days at water concentrations ranging from 0.0015 to about 41 mg/L (Ankley et al. 2001; Miller and Ankley 2004).
Alternatively, a stressor could indirectly impact survival and/or reproduction.
● Example of indirect effect on r:  Exposure of non-sexually differentiated early life stage fathead minnow to the fungicide prochloraz has been shown to produce male-biased sex ratios based on gonad differentiation, and resulted in projected change in population growth rate (decrease in reproduction due to a decrease in females and thus recruitment) using a population model. (Holbech et al., 2012; Miller et al. 2022)
Density dependence can be an important consideration:
● The effect of density dependence depends upon the quantity of resources present within a landscape.  A change in available resources could increase or decrease the effect of density dependence and therefore cause a change in population growth rate via indirectly impacting survival and/or reproduction.
● This concept could be thought of in terms of community level interactions whereby one species is not impacted but a competitor species is impacted by a chemical stressor resulting in a greater availability of resources for the unimpacted species.  In this scenario, the impacted species would experience a decline in population growth rate. The unimpacted species would experience an increase in population growth rate (due to a smaller density dependent effect upon population growth rate for that species).
Closed versus open systems:
● The above discussion relates to closed systems (there is no movement of individuals between population sites) and thus a declining population growth rate cannot be augmented by immigration.
● When individuals depart (emigrate out of a population) the loss will diminish population growth rate.
Population growth rate applies to all organisms, both sexes, and all life stages.
<h4>How it is Measured or Detected</h4>
Population growth rate (instantaneous growth rate) can be measured by sampling a population over an interval of time (i.e. from time t = 0 to time t = 1).  The interval of time should be selected to correspond to the life history of the species of interest (i.e. will be different for rapidly growing versus slow growing populations). The population growth rate, r, can be determined by taking the difference (subtracting) between the initial population size, Nt=0 (population size at time t=0), and the population size at the end of the interval, Nt=1 (population size at time t = 1), and then subsequently dividing by the initial population size.
Equation 2:  r = (Nt=1 - Nt=0) / Nt=0
The diversity of forms, sizes, and life histories among species has led to the development of a vast number of field techniques for estimation of population size and thus population growth over time (Bookhout 1994, McComb et al. 2021).
● For stationary species an observational strategy may involve dividing a habitat into units. After setting up the units, samples are performed throughout the habitat at a select number of units (determined using a statistical sampling design) over a time interval (at time t = 0 and again at time t = 1), and the total number of organisms within each unit are counted. The numbers recorded are assumed to be representative for the habitat overall, and can be used to estimate the population growth rate within the entire habitat over the time interval.
● For species that are mobile throughout a large range, a strategy such as using a mark-recapture method may be employed (i.e. tags, bands, transmitters) to determine a count over a time interval (at time = 0 and again at time =1).
Population growth rate can also be estimated using mathematical model constructs (for example, ranging from simple differential equations to complex age or stage structured matrix projection models and individual based modeling approaches), and may assume a linear or nonlinear population increase over time (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020). The AOP framework can be used to support the translation of pathway-specific mechanistic data into responses relevant to population models and output from the population models, such as changing (declining) population growth rate, can be used to assess and manage risks of chemicals (Kramer et al. 2011). As such, this translational capability can increase the capacity and efficiency of safety assessments both for single chemicals and chemical mixtures (Kramer et al. 2011).
Some examples of modeling constructs used to investigate population growth rate:
● A modeling construct could be based upon laboratory toxicity tests to determine effect(s) that are then linked to the population model and used to estimate decline in population growth rate.  Miller et al. (2007) used concentration–response data from short term reproductive assays with fathead minnow (Pimephales promelas) exposed to endocrine disrupting chemicals in combination with a population model to examine projected alterations in population growth rate.
● A model construct could be based upon a combination of effects-based monitoring at field sites (informed by an AOP) and a population model.  Miller et al. (2015) applied a population model informed by an AOP to project declines in population growth rate for white suckers (Catostomus commersoni) using observed changes in sex steroid synthesis in fish exposed to a complex pulp and paper mill effluent in Jackfish Bay, Ontario, Canada. Furthermore, a model construct could be comprised of a series of quantitative models using KERs that culminates in the estimation of change (decline) in population growth rate.
● A quantitative adverse outcome pathway (qAOP) has been defined as a mathematical construct that models the dose–response or response–response relationships of all KERs described in an AOP (Conolly et al. 2017, Perkins et al. 2019). Conolly et al. (2017) developed a qAOP using data generated with the aromatase inhibitor fadrozole as a stressor and then used it to predict potential population‐level impacts (including decline in population growth rate). The qAOP modeled aromatase inhibition (the molecular initiating event) leading to reproductive dysfunction in fathead minnow (Pimephales promelas) using 3 computational models: a hypothalamus–pituitary–gonadal axis model (based on ordinary differential equations) of aromatase inhibition leading to decreased vitellogenin production (Cheng et al. 2016), a stochastic model of oocyte growth dynamics relating vitellogenin levels to clutch size and spawning intervals (Watanabe et al. 2016), and a population model (Miller et al. 2007).
● Dynamic energy budget (DEB) models offer a methodology that reverse engineers stressor effects on growth, reproduction, and/or survival into modular characterizations related to the acquisition and processing of energy resources (Nisbet et al. 2000, Nisbet et al. 2011).  Murphy et al. (2018) developed a conceptual model to link DEB and AOP models by interpreting AOP key events as measures of damage-inducing processes affecting DEB variables and rates.
● Endogenous Lifecycle Models (ELMs), capture the endogenous lifecycle processes of growth, development, survival, and reproduction and integrate these to estimate and predict expected fitness (Etterson and Ankley, 2021).  AOPs can be used to inform ELMs of effects of chemical stressors on the vital rates that determine fitness, and to decide what hierarchical models of endogenous systems should be included within an ELM (Etterson and Ankley, 2021).
<h4>Regulatory Significance of the AO</h4>
Maintenance of sustainable fish and wildlife populations (i.e., adequate to ensure long-term delivery of valued ecosystem services) is a widely accepted regulatory goal upon which risk assessments and risk management decisions are based.
<h4>References</h4>
<ul>
<li>Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, Henry TR, Denny JS, Leino RL, Wilson VS, Cardon MD, Hartig PC, Gray LE. 2003. Effects of the androgenic growth promoter 17b-trenbolone on fecundity and reproductive endocrinology of the fathead minnow. Environ. Toxicol. Chem. 22: 1350–1360.</li>
<li>Bookhout TA. 1994. Research and management techniques for wildlife and habitats. The Wildlife Society, Bethesda, Maryland. 740 pp.</li>
<li>Caswell H. 2001. Matrix Population Models. Sinauer Associates, Inc., Sunderland, MA, USA</li>
<li>Cheng WY, Zhang Q, Schroeder A, Villeneuve DL, Ankley GT, Conolly R.  2016.  Computational modeling of plasma vitellogenin alterations in response to aromatase inhibition in fathead minnows. Toxicol Sci 154: 78–89.</li>
<li>Conolly RB, Ankley GT, Cheng W-Y, Mayo ML, Miller DH, Perkins EJ, Villeneuve DL, Watanabe KH. 2017. Quantitative adverse outcome pathways and their application to predictive toxicology. Environ. Sci. Technol. 51:  4661-4672.</li>
<li>Etterson MA, Ankley GT.  2021.  Endogenous Lifecycle Models for Chemical Risk Assessment. Environ. Sci. Technol. 55:  15596-15608. </li>
<li>Gotelli NJ, 2008. A Primer of Ecology. Sinauer Associates, Inc., Sunderland, MA, USA.</li>
<li>Holbech H, Kinnberg KL, Brande-Lavridsen N, Bjerregaard P, Petersen GI, Norrgren L, Orn S, Braunbeck T, Baumann L, Bomke C, Dorgerloh M, Bruns E, Ruehl-Fehlert C, Green JW, Springer TA, Gourmelon A. 2012 Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 155:  407–415.</li>
<li>Kramer VJ, Etterson MA, Hecker M, Murphy CA, Roesijadi G, Spade DJ, Stromberg JA, Wang M, Ankley GT.  2011.  Adverse outcome pathways and risk assessment: Bridging to population level effects.  Environ. Toxicol. Chem. 30, 64-76.</li>
<li>McComb B, Zuckerberg B, Vesely D, Jordan C.  2021.  Monitoring Animal Populations and their Habitats: A Practitioner's Guide.  Pressbooks, Oregon State University, Corvallis, OR Version 1.13, 296 pp. </li>
<li>Miller DH, Villeneuve DL, Santana Rodriguez KG, Ankley GT. 2022.  A multidimensional matrix model for predicting the effect of male biased sex ratios on fish populations. Environmental Toxicology and Chemistry 41(4): 1066-1077.</li>
<li>Miller DH, Villeneuve DL, Santana Rodriguez KJ, Ankley GT. 2022.  A multidimensional matrix model for predicting the effect of male biased sex ratios on fish populations. Environmental Toxicology and Chemistry 41(4): 1066-1077.</li>
<li>Miller DH, Tietge JE, McMaster ME, Munkittrick KR, Xia X, Griesmer DA, Ankley GT. 2015. Linking mechanistic toxicology to population models in forecasting recovery from chemical stress: A case study from Jackfish Bay, Ontario, Canada. Environmental Toxicology and Chemistry 34(7):  1623-1633.</li>
<li>Miller DH, Jensen KM, Villeneuve DE, Kahl MD, Makynen EA, Durhan EJ, Ankley GT. 2007. Linkage of biochemical responses to population-level effects: A case study with vitellogenin in the fathead minnow (Pimephales promelas). Environ Toxicol Chem 26:  521–527.</li>
<li>Miller DH, Ankley GT. 2004. Modeling impacts on populations: Fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17b-trenbolone as a case study. Ecotox Environ Saf 59: 1–9.</li>
<li>Murphy CA, Nisbet RM, Antczak P, Garcia-Reyero N, Gergs A, Lika K, Mathews T, Muller EB, Nacci D, Peace A, Remien CH, Schultz IR, Stevenson LM, Watanabe KH.  2018.  Incorporating suborganismal processes into dynamic energy budget models for ecological risk assessment.  Integrated Environmental Assessment and Management 14(5):  615–624.</li>
<li>Murray DL, Sandercock BK (editors).  2020.  Population ecology in practice.  Wiley-Blackwell, Oxford UK, 448 pp.</li>
<li>Nisbet RM, Jusup M, Klanjscek T, Pecquerie L.  2011.  Integrating dynamic energy budget (DEB) theory with traditional bioenergetic models.  The Journal of Experimental Biology 215: 892-902.</li>
<li>Nisbet RM, Muller EB, Lika K, Kooijman SALM. 2000. From molecules to ecosystems through dynamic energy budgets. J Anim Ecol 69:  913–926.</li>
<li>Perkins EJ,  Ashauer R, Burgoon L, Conolly R, Landesmann B,, Mackay C, Murphy CA, Pollesch N, Wheeler JR, Zupanic A, Scholzk S.  2019.  Building and applying quantitative adverse outcome pathway models for chemical hazard and risk assessment.  Environmental Toxicology and Chemistry 38(9): 1850–1865. </li>
<li>Vandermeer JH, Goldberg DE. 2003.  Population ecology: first principles.  Princeton University Press, Princeton NJ, 304 pp.</li>
<li>Villeneuve DL, Crump D, Garcia-Reyero N, Hecker M, Hutchinson TH, LaLone CA, Landesmann B, Lattieri T, Munn S, Nepelska M, Ottinger MA, Vergauwen L, Whelan M. Adverse outcome pathway (AOP) development 1: Strategies and principles. Toxicol Sci. 2014: 142:312–320</li>
<li>Watanabe KH, Mayo M, Jensen KM, Villeneuve DL, Ankley GT, Perkins EJ.  2016.  Predicting fecundity of fathead minnows (Pimephales promelas) exposed to endocrine‐disrupting chemicals using a MATLAB(R)‐based model of oocyte growth dynamics. PLoS One 11:  e0146594.</li>
</ul>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/309">Relationship: 309: Thyroperoxidase, Inhibition leads to TH synthesis, Decreased</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/42">Inhibition of Thyroperoxidase and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>adjacent</td>
<td>High</td>
<td>Low</td>
</tr>
<tr>
<td><a href="/aops/159">Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>adjacent</td>
<td>High</td>
<td>Low</td>
</tr>
<tr>
<td><a href="/aops/271">Inhibition of thyroid peroxidase leading to impaired fertility in fish</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/175">Thyroperoxidase inhibition leading to altered amphibian metamorphosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/363">Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/364">Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/365">Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/119">Inhibition of thyroid peroxidase leading to follicular cell adenomas and carcinomas (in rat and mouse)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>human</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Xenopus laevis</td>
<td>Xenopus laevis</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fathead minnow</td>
<td>Pimephales promelas</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
<tr>
<td>Female</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic: This KER is plausibly applicable across vertebrates. Inhibition of TPO activity is widely accepted to directly impact TH synthesis. This is true for both rats and humans, as well as some fishes, frogs and birds. Most of the data supporting a causative relationship between TPO inhibition and altered TH synthesis is derived from animal studies, in vitro thyroid microsomes from rats or pigs, and a limited number of human ex vivo (Nagasaka and Hidaka, 1976; Vickers et al., 2012) and clinical studies. There are data to support that gene mutations in TPO result in congenital hypothyroidism, underscoring the essential role of TPO in human TH synthesis.
Life stage: Applicability to certain life stages may depend on the species and their dependence on maternally transferred THs during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnow, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH system disruptors.
Life stage: Applicability to certain life stages may depend on the species and their dependence on maternally transferred THs during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. Evidence supporting this hypothesis is obtained from a zebrafish TPO knockout line. In homozygous individuals TPO is inhibited from the embryonic developmental stage onwards, resulting in an abolished T4 production in thyroid follicles with phenotypical abnormalities such as reduced swim bladder inflation and growth retardation appearing at 20 dpf but not before 10 dpf (Fang et al., 2022). In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnow, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH system disruptors.
Sex: The KE is plausibly applicable to both sexes. Thyroid hormones are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of thyroid hormone levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in thyroid hormone levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.
<h4>Key Event Relationship Description</h4>
Thyroperoxidase (TPO) is a heme-containing apical membrane protein within the follicular lumen of thyrocytes that acts as the enzymatic catalyst for thyroid hormone (TH) synthesis (Taurog, 2005) across vertebrates. Two commonly used reference chemicals, propylthiouracil (PTU) and methimazole (MMI), are drugs that inhibit the ability of TPO to: a) activate iodine and transfer it to thyroglobulin (Tg) (Davidson et al., 1978); and, b) couple thyroglobulin (Tg)-bound iodotyrosyls to produce Tg-bound thyroxine (T4) and triiodothyronine (T3) (Taurog, 2005).
<h4>Evidence Supporting this KER</h4>
The weight of evidence supporting a direct linkage between the MIE, TPO inhibition, and the KE of decreased TH synthesis, is strong and supported by more than three decades of research in animals, including humans (Cooper et al., 1982; Cooper et al.,1983; Divi and Doerge, 1994).
The weight of evidence supporting a direct linkage between the MIE, TPO inhibition, and the KE of decreased TH synthesis, is strong and supported by more than three decades of research in animals, including humans (Cooper et al., 1982; Cooper et al.,1983; Divi and Doerge, 1994; Fang et al., 2022).
Biological Plausibility
The biological plausibility for this KER is rated Strong. TPO is the only enzyme capable of de novo synthesis of TH. TPO catalyzes several reactions, including the oxidation of iodide, nonspecific iodination of tyrosyl residues of thyroglobulin (Tg) to form monoiodotyrosyl (MIT) or diiodotyrosyl (DIT) residues, and the coupling of these Tg-bound iodotyrosyls to produce Tg-bound T3 and T4 (Divi and Doerge, 1994; Kessler et al., 2008; Ruf et al., 2006; Taurog et al., 1996, 2005). Therefore, inhibition of TPO activity is widely accepted to directly impact TH synthesis.
Empirical Evidence
Empirical support for this KER is strong. There are several papers that have measured alterations in TPO and subsequent effects on TH synthesis across vertebrates. Taurog et al. (1996) showed decreased guicaol activity, decreased bound I125, and subsequent decreases in newly formed T3 and T4 per molecule of Tg, following exposure to PTU, MMI and some antibiotics.  There is important evidence in mammals. Following in vivo exposure to PTU in rats (Cooper et al., 1982; 1983), there are concentration and time-dependent decreases in thyroid protein bound iodine and serum T4 and T3 that recovered one month after cessation of PTU exposure.  In addition, measures of thyroidal iodine content were highly correlated with intra-thyroidal PTU concentration. Vickers et al. (2012) demonstrated dose- and time- dependent inhibition of TPO activity in both human and rat thyroid homogenates exposed to MMI. Hassan et al. (2017, 2020) and Handa et al. (2021) predicted the level of THs in serum after treatment with PTU and MMI in rats. They developed a quantitative model by comparing dose- response data.
Tietge et al (2010) showed decreases in thyroidal T4 following MMI exposure in Xenopus.  Also in Xenopus, Haselman et al (2020) showed decreases in thyroidal iodotyrosines (MIT/DIT) and iodothyronines (T4/T3) following exposure to MMI. Doerge et al (1998) showed that a tryphenylmethane dye, malachite green, inhibited TPO and lowered thyroxine production. A recent paper used a series of benzothiazoles and showed TPO inhibition (guicaol assay) and inhibition of TSH stimulated thyroxine release from Xenopus thyroid gland explant cultures (Hornung et al., 2015).
Additionally, evidence is available from studies investigating responses to TPO inhibitors in fish. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. Several other studies have also shown that chemically induced Inhibition of TPO results in reduced TH synthesis in zebrafish (Van der Ven et al., 2006; Raldua and Babin, 2009; Liu et al., 2011; Thienpont et al., 2011; Rehberger et al., 2018). A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.
Additionally, evidence is available from studies investigating responses to TPO inhibitors in fish. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. Several other studies have also shown that chemically induced Inhibition of TPO results in reduced TH synthesis in zebrafish (Van der Ven et al., 2006; Raldua and Babin, 2009; Liu et al., 2011; Thienpont et al., 2011; Rehberger et al., 2018). Using a TPO knockout line Fang et al. (2022) showed that TPO inhibition abolished the T4 synthesis in 7 dpf zebrafish mutants. A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.
Temporal Evidence: In mammals, the temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005). The impact of decreased TPO activity on TH synthesis is similar across all ages in mammals. Good evidence for the temporal relationship of the KER comes from thyroid system modeling (e.g., Degon et al., 2008; Fisher et al., 2013) using data from studies of iodine deficiency and chemicals that inhibit NIS. In addition, there is ample evidence of the temporal impacts of TPO inhibition on TH synthesis, using ex vivo and in vitro measures that demonstrate the time course of inhibition following chemical exposures, including some data from human thyroid microsomes and ex vivo thyroid slices (Vickers et al., 2012). Future work is needed that measures both TPO inhibition and TH production during development.
In oviparous fish such as zebrafish and fathead minnow, the nature of this KER depends on the life stage since the earliest stages of embryonic development rely on maternal THs transferred to the eggs. Embryonic TH synthesis is activated later during embryo-larval development. (See Domain of applicability)
Dose-Response Evidence: Dose-response data is available from a number of studies in mammals that correlate TPO inhibition with decreased TH production measured using a variety of endpoints including iodine organification (e.g., Taurog et al., 1996), inhibition of guicaol oxidation in thyroid microsomes (e.g., Doerge and Chang, 2002), and direct measure of thyroid gland T4 concentrations (e.g., Hornung et al., 2015). However, there is a lack of dose-response data from developmental studies showing direct linkages from TPO inhibition to thyroidal TH synthesis.
Uncertainties and Inconsistencies
While it is clear that TPO inhibition will lead to altered TH synthesis, there is a need for data that will inform quantitative modeling of the relationship between TPO inhibition and the magnitude of effects on TH synthesis.
Data from studies on genistein highlight this uncertainty. Doerge and colleagues have demonstrated that for this compound up to 80% TPO inhibition did not result in decreased serum T4 in rats (Doerge and Chang, 2002). This is not consistent with other prototypical TPO inhibitors (e.g., PTU, MMI). Genistein is however a well-known phytoestrogen and the observed inconsistency may be the result of feedback mechanisms resulting from its estrogenic effect.
<h4>Quantitative Understanding of the Linkage</h4>
In Xenopus laevis, Haselman et al.(2020) demonstrated temporal profiles of thyroidal iodotyrosines (MIT/DIT) and iodothyronines (T4/T3), the products of TPO activity, following exposure to three different model TPO inhibitors (MMI, PTU, MBT) at multiple concentrations. This study established that, in Xenopus, measurable decreases in the products of TPO activity can occur as early as 2 days of exposure during pro-metamorphosis. However, despite consistent profiles of some iodo-species across chemicals, other iodo-species showed inconsistent profiles across chemicals. This highlights the multiple mechanisms of TPO (iodination and coupling) and differential susceptibility to inhibition of those mechanisms depending on the chemical's type of interaction with TPO. The most consistent concentration-response relationship across chemicals and over time was demonstrated by thyroidal T4, which is the most relevant product to subsequent key events. At the highest concentrations tested for each chemical, thyroidal T4 was below detection by 7 days of exposure across all three TPO inhibitors. Keeping in mind that the thyroid gland has follicular lumen space where thyroglobulin/T4 is stored until proteolysis and release to the blood, full inhibition of TPO would result in a delayed measurable response due to the time it takes to deplete stored hormones. Regardless of the delay, the results from this study imply full inhibition of TPO by each of these three chemicals at the highest test concentrations, but would require chemical residue analysis and/or toxicokinetic modeling to relate cellular/tissue concentrations at the site of TPO catalysis to levels of inhibition via Michaelis-Menten kinetic descriptions.
Profiles of thyroidal iodinated species demonstrated by Haselman et al. (2020) across three different TPO inhibitors suggests that a high level of TPO inhibition must occur in order to elicit responses in subsequent key events. Although the level of TPO inhibition is not directly quantifiable from this study, these data suggest that at least 90-100% inhibition was occurring since circulating T4 was not detectable at 10 days of exposure to the highest concentrations of MMI and MBT. However, additional efforts would be necessary to determine the minimum level of TPO inhibition that leads to a measurable decrease in thyroidal T4 and subsequently circulating T4.  Furthermore, Hassan et al. (2017, 2020) and Handa et al. (2021) predicted the level of THs in serum after treatment with PTU and MMI in rats. They developed a quantitative model by comparing dose- response data.
Response-response relationship
There are only a limited number of studies where both TPO inhibition and iodine organification have been measured in vivo, and there is not enough data available to make any definitive quantitative correlations. One in vivo study in rats exposed to the TPO inhibitor genistein found no in vivo impact on serum TH concentrations, even when TPO was inhibited up to 80% (Chang and Doerge, 2000). Genistein is however a well-known phytoestrogen and the observed inconsistency may be the result of feedback mechanisms resulting from its estrogenic effect.
Given that this is an MIE to KE relationship, there is only one response to evaluate in the relationship. Decreased TH synthesis, as measured by responses of iodinated species in the thyroid gland, is the result of TPO inhibition, which cannot be measured directly in vivo.
Time-scale
In vivo, evaluations of TPO inhibition are limited to evaluation of the iodinated species, or products of TPO activity, present in the thyroid gland at a particular time. However, as stated previously, any measurable response in these iodinated species is not a discreet assessment of TPO activity given that the gland maintains storage of hormone in the follicular lumen space and any alteration of TPO activity would be detected once the stores begin to be depleted. In Xenopus laevis, Haselman et al. (2020) showed a decrease in thyroidal iodinated species after only 2 days of exposure to potent TPO inhibitor MMI during thyroid-mediated metamorphosis and within 4 days for PTU and MBT, both model TPO inhibitors. In zebrafish, Walter et al. (2019) reported a similar time frame, namely a decrease in T4 levels at 72 hpf after starting the exposure to PTU at 0-2 hpf. It should be noted that the time-scale is probably depending on the developmental stage and whether the embryo is capable of thyroid hormone synthesis, rather than on the exposure duration.
Known modulating factors
Iodine availability will impact the ability of TPO to iodinate tyrosine residues on thyroglobulin. Iodine availability to TPO can be impacted in a number of ways. First, environmental availability of iodine can vary greatly depending on whether and how much iodine exists in surface waters for aquatic organisms (gill respirators) and in the diets of both terrestrial and aquatic organisms. Second, somewhat regardless of iodine availability through environmental uptake (i.e., barring extremely high iodine exposure), iodine is actively transported into the thyroid follicular cell from the blood via sodium-iodide symporter (NIS), which has been shown to be susceptible to inhibition by, for example, perchlorate. As such, iodine availability to TPO is mediated by functional NIS. Finally, iodine is not fully available to TPO on the apical surface of the thyroid follicular cell until it is transported through the apical membrane by pendrin, an anion exchange protein - mutations or inhibition of pendrin could affect iodine availability to TPO.
Hydrogen peroxide is also needed by TPO to mediate the oxidation of iodide, which is produced locally by dual oxidase (DUOX). A mutation or inhibition of DUOX will impact local production of H2O2 leading to lower oxidizing potential of TPO and less organification of iodide.
Known Feedforward/Feedback loops influencing this KER
Thyroid stimulating hormone (TSH) released from the pituitary positively regulates the synthesis and release of thyroid hormones from the thyroid gland. As such, when TPO is inhibited and thyroid hormone synthesis is decreased, lower systemic levels of hormone cause feedback from the pituitary via TSH to upregulate a number of processes in the thyroid gland as a means of compensation, including (but not limited to) enhanced gene expression of NIS and thyrocyte cell proliferation (Tietge et al., 2010; Haselman et al., 2020).
<h4>References</h4>
Chang HC, Doerge DR. Dietary genistein inactivates rat thyroid peroxidase in vivo without an apparent hypothyroid effect. Toxicol Appl Pharmacol 168:244–252 (2000).
Chang, J., Wang, M., Gui, W., Zhao, Y., Yu, L., Zhu, G., 2012. Changes in Thyroid Hormone Levels during Zebrafish Development. Zoological Science 29, 181-184.
Cooper DS, Kieffer JD, Halpern R, Saxe V, Mover H, Maloof F, Ridgway EC (1983) Propylthiouracil (PTU) pharmacology in the rat. II. Effects of PTU on thyroid function. Endocrinology 113:921-928.
Cooper DS, Saxe VC, Meskell M, Maloof F, Ridgway EC. Acute effects of propylthiouracil (PTU) on thyroidal iodide organification and peripheral iodothyronine deiodination: correlation with serum PTU levels measured by radioimmunoassay. J Clin Endocrinol Metab. 1982 54(1):101-7.
Crane HM, Pickford DB, Hutchinson TH, Brown JA. 2006. The effects of methimazole on development of the fathead minnow, pimephales promelas, from embryo to adult. Toxicological Sciences. 93(2):278-285.
Davidson, B., Soodak, M., Neary, J.T., Strout, H.V., and Kieffer, J.D. (1978). The irreversible inactivation of thyroid peroxidase by methylmercaptoimidazole, thiouracil, and propylthiouracil in vitro and its relationship to in vivo findings. Endocrinology 103:871–882.
Divi, R. L., and Doerge, D. R. (1994). Mechanism-based inactivation of lactoperoxidase and thyroid peroxidase by resorcinol derivatives. Biochemistry 33(32), 9668-74.
Doerge DR, Chang HC, Divi RL, Churchwell Mechanism for inhibition of thyroid peroxidase by leucomalachite green. Chem Res Toxicol. 1998 11(9):1098-104.
Doerge DR, Chang HC.  Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo.  J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Sep 25;777(1-2):269-79
Fang, Y., Wan, J. P., Zhang, R. J., Sun, F., Yang, L., Zhao, S. X., Dong, M., & Song, H. D. (2022). Tpo knockout in zebrafish partially recapitulates clinical manifestations of congenital hypothyroidism and reveals the involvement of TH in proper development of glucose homeostasis. General and Comparative Endocrinology, 323–324. https://doi.org/10.1016/j.ygcen.2022.114033
Handa S, Hassan I, Gilbert M, El-Masri H. 2021. Mechanistic Computational Model for Extrapolating In Vitro Thyroid Peroxidase (TPO) Inhibition Data to Predict Serum Thyroid Hormone Levels in Rats. Toxicological Sciences 183(1):36-48.
Haselman, J.T., Olker, J.H., Kosian, P.A., Korte, J.J., Swintek, J.A., Denny, J.S., Nichols, J.W., Tietge, J.E., Hornung, M.W. and Degitz, S.J., 2020. Targeted pathway-based in vivo testing using thyroperoxidase inhibition to evaluate plasma thyroxine as a surrogate metric of metamorphic success in model amphibian Xenopus laevis. Toxicological Sciences, 175(2), pp.236-250.
Hassan I, El-Masri H, Ford J, Brennan A, Handa S, Friedman KP, Gilbert ME. 2020. Extrapolating In Vitro Screening Assay Data for Thyroperoxidase Inhibition to Predict Serum Thyroid Hormones in the Rat. Toxicological Sciences 173(2):280-292.
Hassan I, El-Masri H, Kosian PA, Ford J, Degitz SJ, Gilbert ME. 2017. Neurodevelopment and Thyroid Hormone Synthesis Inhibition in the Rat: Quantitative Understanding Within the Adverse Outcome Pathway Framework. Toxicological Sciences 160(1):57-73.
Hernandez-Mariano JA, Torres-Sanchez L, Bassol-Mayagoitia S, Escamilla-Nunez M, Cebrian ME, Villeda-Gutierrez EA, Lopez-Rodriguez G, Felix-Arellano EE, Blanco-Munoz J. 2017. Effect of exposure to p,p '-dde during the first half of pregnancy in the maternal thyroid profile of female residents in a mexican floriculture area. Environmental Research. 156:597-604.
Hornung MW, Kosian PA, Haselman JT, Korte JJ, Challis K, Macherla C, Nevalainen E, Degitz SJ.  In Vitro, Ex Vivo, and In Vivo Determination of Thyroid Hormone Modulating Activity of Benzothiazoles.Toxicol Sci. 2015 146(2):254-64.
Kessler, J., Obinger, C., and Eales, G. (2008). Factors influencing the study of peroxidase-generated iodine species and implications for thyroglobulin synthesis. Thyroid 18(7), 769-74, 10.1089/thy.2007.0310.
Liu CS, Zhang XW, Deng J, Hecker M, Al-Khedhairy A, Giesy JP, Zhou BS. 2011. Effects of prochloraz or propylthiouracil on the cross-talk between the hpg, hpa, and hpt axes in zebrafish. Environmental Science & Technology. 45(2):769-775.
Liu XS, Cai Y, Wang Y, Xu SH, Ji K, Choi K. 2019. Effects of tris(1,3-dichloro-2-propyl) phosphate (tdcpp) and triphenyl phosphate (tpp) on sex-dependent alterations of thyroid hormones in adult zebrafish. Ecotoxicology and Environmental Safety. 170:25-32.
Nagasaka, A., and Hidaka, H. (1976). Effect of antithyroid agents 6-propyl-2-thiouracil and 1-mehtyl-2-mercaptoimidazole on human thyroid iodine peroxidase. J. Clin. Endocrinol. Metab. 43:152–158.
Nelson K, Schroeder A, Ankley G, Blackwell B, Blanksma C, Degitz S, Flynn K, Jensen K, Johnson R, Kahl M et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part i: Fathead minnow. Aquatic Toxicology. 173:192-203.
Opitz, R., Maquet, E., Zoenen, M., Dadhich, R., Costagliola, S., 2011. TSH Receptor Function Is Required for Normal Thyroid Differentiation in Zebrafish. Molecular Endocrinology 25, 1579-1599.
Power, D.M., Llewellyn, L., Faustino, M., Nowell, M.A., Bjornsson, B.T., Einarsdottir, I.E., Canario, A.V., Sweeney, G.E., 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol 130, 447-459.
Raldua, D., Babin, P.J., 2009. Simple, Rapid Zebrafish Larva Bioassay for Assessing the Potential of Chemical Pollutants and Drugs to Disrupt Thyroid Gland Function. Environmental Science & Technology 43, 6844-6850.
Rehberger, K., Baumann, L., Hecker, M., Braunbeck, T., 2018. Intrafollicular thyroid hormone staining in whole-mount zebrafish (Danio rerio) embryos for the detection of thyroid hormone synthesis disruption. Fish Physiology and Biochemistry 44, 997-1010.
Ruf, J., and Carayon, P. (2006). Structural and functional aspects of thyroid peroxidase. Archives of biochemistry and biophysics 445(2), 269-77, 10.1016/j.abb.2005.06.023.
Stinckens E, Vergauwen L, Blackwell BR, Anldey GT, Villeneuve DL, Knapen D. 2020. Effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology. 54(10):6213-6223.
Stinckens E, Vergauwen L, Schroeder A, Maho W, Blackwell B, Witters H, Blust R, Ankley G, Covaci A, Villeneuve D et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part ii: Zebrafish. Aquatic Toxicology. 173:204-217.
Taurog A. 2005. Hormone synthesis. In: Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text (Braverman LE, Utiger RD, eds). Philadelphia:Lippincott, Williams and Wilkins, 47–81.
Taurog, A., Dorris, M. L., and Doerge, D. R. (1996). Mechanism of simultaneous iodination and coupling catalyzed by thyroid peroxidase. Archives of biochemistry and biophysics 330(1), 24-32,
Thienpont, B., Tingaud-Sequeira, A., Prats, E., Barata, C., Babin, P.J., Raldua, D., 2011. Zebrafish Eleutheroembryos Provide a Suitable Vertebrate Model for Screening Chemicals that Impair Thyroid Hormone Synthesis. Environmental Science & Technology 45, 7525-7532.
Tietge JE, Butterworth BC, Haselman JT, Holcombe GW, Hornung MW, Korte JJ, Kosian PA, Wolfe M, Degitz SJ.   Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis.  Aquat Toxicol. 2010 98(1):44-50
van der Ven LTM, van den Brandhof EJ, Vos JH, Power DM, Wester PW. 2006. Effects of the antithyroid agent propylthiouracil in a partial life cycle assay with zebrafish. Environmental Science & Technology. 40(1):74-81.
Vickers AE, Heale J, Sinclair JR, Morris S, Rowe JM, Fisher RL. Thyroid organotypic rat and human cultures used to investigate drug effects on thyroid function, hormone synthesis and release pathways. Toxicol Appl Pharmacol. 2012 260(1):81-8.
Wabukebunoti MAN, Firling CE. 1983. The prehatching development of the thyroid-gland of the fathead minnow, pimephales-promelas (rafinesque). General and Comparative Endocrinology. 49(2):320-331.
Walter KM, Miller GW, Chen XP, Yaghoobi B, Puschner B, Lein PJ. 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (danio rerio). General and Comparative Endocrinology. 272:20-32.
Walter, K.M., Miller, G.W., Chen, X.P., Yaghoobi, B., Puschner, B., Lein, P.J., 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (Danio rerio). General and Comparative Endocrinology 272, 20-32.
Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347.
</div>
<div>
<h4><a href="/relationships/305">Relationship: 305: TH synthesis, Decreased leads to T4 in serum, Decreased</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/42">Inhibition of Thyroperoxidase and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/65">XX Inhibition of Sodium Iodide Symporter and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/134">Sodium Iodide Symporter (NIS) Inhibition and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/54">Inhibition of Na+/I- symporter (NIS) leads to learning and memory impairment </a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/159">Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Low</td>
</tr>
<tr>
<td><a href="/aops/175">Thyroperoxidase inhibition leading to altered amphibian metamorphosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/176">Sodium Iodide Symporter (NIS) Inhibition leading to altered amphibian metamorphosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/363">Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/364">Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/365">Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/119">Inhibition of thyroid peroxidase leading to follicular cell adenomas and carcinomas (in rat and mouse)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/110">Inhibition of iodide pump activity leading to follicular cell adenomas and carcinomas (in rat and mouse)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/128">Kidney dysfunction by decreased thyroid hormone</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/188">Iodotyrosine deiodinase (IYD) inhibition leading to altered amphibian metamorphosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/192">Pendrin inhibition leading to altered amphibian metamorphosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/193">Dual oxidase (DUOX) inhibition leading to altered amphibian metamorphosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/457">Succinate dehydrogenase inhibition leading to increased insulin resistance through reduction in circulating thyroxine</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/459">AhR activation in the thyroid leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>human</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Xenopus laevis</td>
<td>Xenopus laevis</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fathead minnow</td>
<td>Pimephales promelas</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
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<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
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</tbody>
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Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
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</thead>
<tbody class="tbody-striped">
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<td>Male</td>
<td>High</td>
</tr>
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<td>Female</td>
<td>High</td>
</tr>
</tbody>
</table>
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Taxonomic: This KER is plausibly applicable across vertebrates. While a majority of the empirical evidence comes from work with laboratory rodents, there is a large amount of supporting data from humans (with anti-hyperthyroidism drugs including propylthiouracil and methimazole), some amphibian species (e.g., frog), fish species (e.g., zebrafish and fathead minnow), and some avian species (e.g, chicken).  The following are samples from a large literature that supports this concept: Cooper et al. (1982; 1983); Hornung et al. (2010); Van Herck et al. (2013); Paul et al. (2013); Nelson et al. (2016); Alexander et al. (2017); Stinckens et al. (2020).
Life stage: Applicability to certain life stages may depend on the species and their dependence on maternally transferred thyroid hormones (TH) during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body TH      levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH system disruptors.
Sex: The KE is plausibly applicable to both sexes. Thyroid hormones are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of thyroid hormone levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in thyroid hormone levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.
<h4>Key Event Relationship Description</h4>
Thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) are synthesized by NIS and TPO in the thyroid gland as iodinated thyroglobulin (Tg) and stored in the colloid of thyroid follicles across vertebrates. Secretion from the follicle into serum is a multi-step process. The first involves thyroid stimulating hormone (TSH) stimulation of the separation of the peptide linkage between Tg and TH. The next steps involve endocytosis of colloid, fusion of the endosome with the basolateral membrane of the thyrocyte, and finally release of TH into blood. More detailed descriptions of this process can be found in reviews by Braverman and Utiger (2012) and Zoeller et al. (2007).
<h4>Evidence Supporting this KER</h4>
The weight of evidence linking these two KEs of decreased TH synthesis and decreased T4 in serum is strong. It is commonly accepted dogma that decreased synthesis in the thyroid gland will result in decreased circulating TH (serum T4).
Biological Plausibility
The biological relationship between two KEs in this KER is well understood and documented fact within the scientific community.
Empirical Evidence
It is widely accepted that TPO inhibition leads to declines in serum T4 levels in adult mammals. This is due to the fact that the sole source for circulating T4 derives from hormone synthesis in the thyroid gland. Indeed, it has been known for decades that insufficient dietary iodine will lead to decreased serum TH concentrations due to inadequate synthesis. Strong qualitative and quantitative relationships exist between reduced TH synthesis and reduced serum T4 (Ekerot et al., 2013; Degon et al., 2008; Cooper et al., 1982; 1983; Leonard et al., 2016; Zoeller and Tan, 2007).  There is more limited evidence supporting the relationship between decreased TH synthesis and lowered circulating hormone levels during development.  Lu and Anderson (1994) followed the time course of TH synthesis, measured as thyroxine secretion rate, in non-treated pregnant rats and correlated it with serum T4 levels. Modeling of TH in the rat fetus demonstrates the quantitative relationship between TH synthesis and serum T4 concentrations (Hassan et al., 2017, 2020; Handa et al., 2021). Furthermore, a wide variety of drugs and chemicals that inhibit TPO are known to result in decreased release of TH from the thyroid gland, as well as decreased circulating TH concentrations. This is evidenced by a very large number of studies that employed a wide variety of techniques, including thyroid gland explant cultures, tracing organification of 131-I and in vivo treatment of a variety of animal species with known TPO inhibitors (King and May,1984; Atterwill et al., 1990; Brown et al., 1986; Brucker-Davis, 1998; Haselman et al., 2020; Hornung et al., 2010; Hurley et al., 1998; Kohrle, 2008; Tietge et al., 2010).
Additionally, evidence is available from studies investigating responses to TPO inhibitors in fish. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. Several other studies have also shown that chemically induced Inhibition of TPO results in reduced TH synthesis in zebrafish (Van der Ven et al., 2006; Raldua and Babin, 2009; Liu et al., 2011; Thienpont et al., 2011; Rehberger et al., 2018). A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.
Temporal Evidence: In mammals, the temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005).  There are currently no studies that measured both TPO synthesis and TH production during development. However, the impact of decreased TH synthesis on serum hormones is similar across all ages in mammals. Good evidence for the temporal relationship comes from thyroid system modeling of the impacts of iodine deficiency and NIS inhibition (e.g., Degon et al., 2008; Fisher et al., 2013). In addition, recovery experiments have demonstrated that serum thyroid hormones recovered in athyroid mice following grafting of in-vitro derived follicles (Antonica et al., 2012). In Xenopus, it has been shown that depression of TH synthesis in the thyroid gland precedes depression of circulating TH within 7 days of exposure during pro-metamorphosis (Haselman et al., 2020).
Temporal Evidence: In mammals, the temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005).  There are currently no studies that measured both TPO synthesis and TH production during development. However, the impact of decreased TH synthesis on serum hormones is similar across all ages in mammals. Good evidence for the temporal relationship comes from thyroid system modeling of the impacts of iodine deficiency and NIS inhibition (e.g., Degon et al., 2008; Fisher et al., 2013). In addition, recovery experiments have demonstrated that serum thyroid hormones recovered in athyroid mice following grafting of in-vitro derived follicles (Antonica et al., 2012).
In <strong><em>Xenopus, it has been shown that depression of TH synthesis in the thyroid gland precedes depression of circulating TH within 7 days of exposure during pro-metamorphosis (Haselman et al., 2020).
In oviparous fish such as zebrafish and fathead minnow, the nature of this KER depends on the life stage since the earliest stages of embryonic development rely on maternal THs transferred to the eggs. Embryonic TH synthesis is activated later during embryo-larval development. (See Domain of applicability)
Dose-response Evidence: Dose-response data is lacking from studies that include concurrent measures of both TH synthesis and serum TH concentrations. However, data is available demonstrating correlations between thyroidal TH and serum TH concentrations during gestation and lactation during development (Gilbert et al., 2013). This data was used to develop a rat quantitative biologically-based dose-response model for iodine deficiency (Fisher et al., 2013). In Xenopus, dose-responses were demonstrated in both thyroidal T4 and circulating T4 following exposure to three TPO inhibitors (Haselman et al., 2020).
Uncertainties and Inconsistencies
There are no inconsistencies in this KER, but there are some uncertainties. The first uncertainty stems from the paucity of data for quantitative modeling of the relationship between the degree of synthesis decrease and resulting changes in circulating T4 concentrations. In addition, most of the data supporting this KER comes from inhibition of TPO, and there are a number of other processes (e.g., endocytosis, lysosomal fusion, basolateral fusion and release) that are not as well studied.
For example, Kim et al. (2015) investigated the adverse effects of Triphenyl phosphate (TPP), a substance that disrupts the thyroid system. Therefore, Rat pituitary (GH3) and thyroid follicular cell lines (FRTL-5) were studied. In the GH3 cells, TPP led to an upregulation of the expression of important thyroid genes (tsh, tr alpha and tr beta) while T3, a positive control, downregulated the expression of these genes. In FRTL-5 cells, the expression of nis and tpo genes was significantly upregulated, suggesting that TPP stimulates TH synthesis in the thyroid gland.
In zebrafish larvae at the age of 7 days post-fertilisation (dpf), TPP exposure resulted in a significant increase in T3 and T4 concentrations and the expression of genes involved in thyroid hormone synthesis. Exposure to TPP also significantly regulated the expression of genes involved in the metabolism (dio1), transport (ttr) and excretion (ugt1ab) of THs. The down-regulation of the crh and tsh genes in the zebrafish larvae suggests the activation of a central regulatory feedback mechanism that is triggered by the increased T3 levels in vivo. Taken together, these observations indicate that TPP increases TH concentrations in early life stages of zebrafish by disrupting central regulatory and hormone synthesis pathways.
<h4>Quantitative Understanding of the Linkage</h4>
In rats, Hassan et al. (2020) demonstrated in vitro: ex vivo correlations of TPO inhibition using PTU and MMI and constructed a quantitative model relating level of TPO inhibition with changes in circulating T4 levels. They determined that 30% inhibition of TPO was sufficient to decrease circulating T4 levels by 20%. This is further supported by studies of Hassan et al. (2017) and Handa et al. (2021)
In Xenopus, Haselman et al. (2020) collected temporal and dose-response data for both thyroidal and circulating T4 which showed strong qualitative concordance of the response-response relationship. A quantitative relationship exists there in, but is yet to be demonstrated mathematically in this species.
Response-response relationship
Fisher et al. (2013) published a quantitative biologically-based dose-response model for iodine deficiency in the rat. This model provides quantitative relationships for thyroidal T4 synthesis (iodine organification) and predictions of serum T4 concentrations in developing rats. There are other computational models that include thyroid hormone synthesis. Ekerot et al. (2012) modeled TPO, T3, T4 and TSH in dogs and humans based on exposure to myeloperoxidase inhibitors that also inhibit TPO.  This model was recently adapted for rats(Leonard et al., 2016) and Hassan et al (2017) have extended it to include the pregnant rat dam in response to TPO inhibition induced by PTU. While the original model predicted serum TH and TSH levels as a function of oral dose, it was not used to explicitly predict the relationship between serum hormones and TPO inhibition, or TH synthesis. Leonard et al. (2016) recently incorporated TPO inhibition into the model. Degon et al (2008) developed a human thyroid model that includes TPO, but does not make quantitative prediction of organification changes due to inhibition of the TPO enzyme. Further empirical support for the response-response relationship has been demonstrated in the amphibian model, Xenopus laevis, exposed to TPO inhibitors during pro-metamorphosis (Haselman et al., 2020) wherein temporal profiles were measured for both thyroidal and circulating T4.
Time-scale
Given that the thyroid gland contains follicular lumen space filled with stored thyroglobulin/T4, complete inhibition of thyroid hormone synthesis at a given point in time will not result in an instantaneous decrease in circulating T4. The system will be capable of maintaining sufficient circulating T4 levels until the gland stores are depleted. The time it takes to deplete stored hormone will greatly depend on species, developmental status and numerous other factors.
In Xenopus, Haselman et al. (2020) demonstrated an approximately 5 day difference between a significant decrease in thyroidal T4 preceding a significant decrease in circulating T4 while exposed to a potent TPO inhibitor (MMI) continuously during pro-metamorphosis.
Known modulating factors
During Xenopus metamorphosis, circulating T4 steadily increases to peak levels at metamorphic climax. Therefore, during Xenopus metamorphosis, this KER is operable at an increased rate as compared to a system that is maintaining steady circulating T4 levels through homeostatic control. In this case, developmental status is a modulating factor for the rates and trajectories of these KEs.
Known Feedforward/Feedback loops influencing this KER
This KER is entirely influenced by the feedback loop between circulating T4 originating from the thyroid gland and circulating TSH originating from the pituitary. Intermediate biochemical processes exist within the hypothalamus to affirm feedback and coordinately release TSH from the pituitary. However, quantitative representations of these feedback processes are limited to models discussed previously.
In Xenopus, circulating levels of T4 increase through pro-metamorphosis indicating a "release" of feedback to allow circulating levels of T4 to increase and drive metamorphic changes (Sternberg et al., 2011). This provides evidence that homeostatic control of feedback can be developmentally dependent, and likely species dependent.
<h4>References</h4>
Alexander EK, Pearce EN, Brent GA, Brown RS, Chen H, Dosiou C, Grobman WA, Laurberg P, Lazarus JH, Mandel SJ, Peeters RP, Sullivan S.  2017 Guidelines of the American Thyroid Association for the Diagnosis and Management of Thyroid Disease During Pregnancy and the Postpartum. Thyroid. 2017 Mar;27(3):315-389.
Antonica F, Kasprzyk DF, Opitz R, Iacovino M, Liao XH, Dumitrescu AM, Refetoff S, Peremans K, Manto M, Kyba M, Costagliola S.  Generation of functional thyroid from embryonic stem cells.  Nature. 2012 491(7422):66-71.
Atterwill CK, Fowler KF. A comparison of cultured rat FRTL-5 and porcine thyroid cells for predicting the thyroid toxicity of xenobiotics. Toxicol In Vitro. 1990. 4(4-5):369-74.
Braverman, L.E. and Utiger, R.D. (2012). Werner and Ingbar's The Thyroid: A Fundamental and Clinical Text (10 ed.). Philadelphia, PA: Lippincott Williams & Wilkins. pp. 775-786. ISBN 978-1451120639.
Brown CG, Fowler KL, Nicholls PJ, Atterwill C. Assessment of thyrotoxicity using in vitro cell culture systems. Food Chem Toxicol. 1986 24(6-7):557-62.
Brucker-Davis F. Effects of environmental synthetic chemicals on thyroid function. Thyroid. 1998 8(9):827-56.
Chang J, Wang M, Gui W, Zhao Y, Yu L, Zhu G. 2012. Changes in thyroid hormone levels during zebrafish development. Zoological Science. 29(3):181-184.
Cooper DS, Kieffer JD, Halpern R, Saxe V, Mover H, Maloof F, Ridgway EC (1983) Propylthiouracil (PTU) pharmacology in the rat. II. Effects of PTU on thyroid function. Endocrinology 113:921-928.
Cooper DS, Saxe VC, Meskell M, Maloof F, Ridgway EC.Acute effects of propylthiouracil (PTU) on thyroidal iodide organification and peripheral iodothyronine deiodination: correlation with serum PTU levels measured by radioimmunoassay. J Clin Endocrinol Metab. 1982 54(1):101-7.
Crane HM, Pickford DB, Hutchinson TH, Brown JA. 2006. The effects of methimazole on development of the fathead minnow, pimephales promelas, from embryo to adult. Toxicological Sciences. 93(2):278-285.
Degon, M., Chipkin, S.R., Hollot, C.V., Zoeller, R.T., and Chait, Y. (2008). A computational model of the human thyroid. Mathematical Biosciences 212, 22–53
Ekerot P, Ferguson D, Glämsta EL, Nilsson LB, Andersson H, Rosqvist S, Visser SA. Systems pharmacology modeling of drug-induced modulation of thyroid hormones in dogs and translation to human. Pharm Res. 2013 30(6):1513-24.
Fisher JW, Li S, Crofton K, Zoeller RT, McLanahan ED, Lumen A, Gilbert ME.  Evaluation of iodide deficiency in the lactating rat and pup using a biologically based dose-response model. Toxicol Sci. 2013 132(1):75-86.
Gilbert ME, Hedge JM, Valentín-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett JM, Fisher JW.  An animal model of marginal iodine deficiency during development: the thyroid axis and neurodevelopmental outcome.  Toxicol Sci. 2013 132(1):177-95.
Handa S, Hassan I, Gilbert M, El-Masri H. 2021. Mechanistic Computational Model for Extrapolating In Vitro Thyroid Peroxidase (TPO) Inhibition Data to Predict Serum Thyroid Hormone Levels in Rats. Toxicological Sciences 183(1):36-48.
Haselman, J.T., Olker, J.H., Kosian, P.A., Korte, J.J., Swintek, J.A., Denny, J.S., Nichols, J.W., Tietge, J.E., Hornung, M.W. and Degitz, S.J., 2020. Targeted pathway-based in vivo testing using thyroperoxidase inhibition to evaluate plasma thyroxine as a surrogate metric of metamorphic success in model amphibian Xenopus laevis. Toxicological Sciences, 175(2), pp.236-250.
Hassan, I, El-Masri, H., Kosian, PA, Ford, J, Degitz, SJ and Gilbert, ME. Quantitative Adverse Outcome Pathway for Neurodevelopmental Effects of Thyroid Peroxidase-Induced Thyroid Hormone Synthesis Inhibition. Toxicol Sci. 2017 Nov 1;160(1):57-73
Hassan, I., El-Masri, H., Ford, J., Brennan, A., Handa, S., Paul Friedman, K. and Gilbert, M.E., 2020. Extrapolating in vitro screening assay data for thyroperoxidase inhibition to predict serum thyroid hormones in the rat. Toxicological Sciences, 173(2), pp.280-292.
Hernandez-Mariano JA, Torres-Sanchez L, Bassol-Mayagoitia S, Escamilla-Nunez M, Cebrian ME, Villeda-Gutierrez EA, Lopez-Rodriguez G, Felix-Arellano EE, Blanco-Munoz J. 2017. Effect of exposure to p,p '-dde during the first half of pregnancy in the maternal thyroid profile of female residents in a mexican floriculture area. Environmental Research. 156:597-604.
Hornung MW, Degitz SJ, Korte LM, Olson JM, Kosian PA, Linnum AL, Tietge JE. Inhibition of thyroid hormone release from cultured amphibian thyroid glands by methimazole, 6-propylthiouracil, and perchlorate. Toxicol Sci. 2010 118(1):42-51.
Hurley PM. Mode of carcinogenic action of pesticides inducing thyroid follicular cell tumors in rodents. Environ Health Perspect. 1998 106(8):437-45.
Kim, S., Jung, J., Lee, I., Jung, D., Youn, H., & Choi, K. (2015). Thyroid disruption by triphenyl phosphate, an organophosphate flame retardant, in zebrafish (Danio rerio) embryos/larvae, and in GH3 and FRTL-5 cell lines. Aquatic Toxicology, 160, 188–196. https://doi.org/10.1016/j.aquatox.2015.01.016
King DB, May JD. Thyroidal influence on body growth. J Exp Zool. 1984 Dec;232(3):453-60.
Köhrle J. Environment and endocrinology: the case of thyroidology. Ann Endocrinol (Paris). 2008 69(2):116-22.
Leonard JA, Tan YM, Gilbert M, Isaacs K, El-Masri H. Estimating margin of exposure to thyroid peroxidase inhibitors using high-throughput in vitro data, high-throughput exposure modeling, and physiologically based pharmacokinetic/pharmacodynamic modeling. Toxicol Sci. 2016 151(1):57-70.
Liu CS, Zhang XW, Deng J, Hecker M, Al-Khedhairy A, Giesy JP, Zhou BS. 2011. Effects of prochloraz or propylthiouracil on the cross-talk between the hpg, hpa, and hpt axes in zebrafish. Environmental Science & Technology. 45(2):769-775.
Liu XS, Cai Y, Wang Y, Xu SH, Ji K, Choi K. 2019. Effects of tris(1,3-dichloro-2-propyl) phosphate (tdcpp) and triphenyl phosphate (tpp) on sex-dependent alterations of thyroid hormones in adult zebrafish. Ecotoxicology and Environmental Safety. 170:25-32.
Lu, M-H, and Anderson, RR. Thyroxine secretion rats during pregnancy in the rat.  Endo Res. 1994. 20(4):343-364.
Nelson K, Schroeder A, Ankley G, Blackwell B, Blanksma C, Degitz S, Flynn K, Jensen K, Johnson R, Kahl M et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part i: Fathead minnow. Aquatic Toxicology. 173:192-203.
Opitz R, Maquet E, Zoenen M, Dadhich R, Costagliola S. 2011. Tsh receptor function is required for normal thyroid differentiation in zebrafish. Molecular Endocrinology. 25(9):1579-1599.
Paul KB, Hedge JM, Macherla C, Filer DL, Burgess E, Simmons SO, Crofton KM, Hornung MW. Cross-species analysis of thyroperoxidase inhibition by xenobiotics demonstrates conservation of response between pig and rat. Toxicology. 2013. 312:97-107.
Power DM, Llewellyn L, Faustino M, Nowell MA, Bjornsson BT, Einarsdottir IE, Canario AV, Sweeney GE. 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. 130(4):447-459.
Raldua D, Babin PJ. 2009. Simple, rapid zebrafish larva bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function. Environmental Science & Technology. 43(17):6844-6850.
Rehberger K, Baumann L, Hecker M, Braunbeck T. 2018. Intrafollicular thyroid hormone staining in whole-mount zebrafish (danio rerio) embryos for the detection of thyroid hormone synthesis disruption. Fish Physiology and Biochemistry. 44(3):997-1010.
Sternberg, R.M., Thoemke, K.R., Korte, J.J., Moen, S.M., Olson, J.M., Korte, L., Tietge, J.E. and Degitz Jr, S.J., 2011. Control of pituitary thyroid-stimulating hormone synthesis and secretion by thyroid hormones during Xenopus metamorphosis. General and comparative endocrinology, 173(3), pp.428-437.
Stinckens E, Vergauwen L, Blackwell BR, Anldey GT, Villeneuve DL, Knapen D. 2020. Effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology. 54(10):6213-6223.
Stinckens E, Vergauwen L, Schroeder A, Maho W, Blackwell B, Witters H, Blust R, Ankley G, Covaci A, Villeneuve D et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part ii: Zebrafish. Aquatic Toxicology. 173:204-217.
Thienpont B, Tingaud-Sequeira A, Prats E, Barata C, Babin PJ, Raldua D. 2011. Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis. Environmental Science & Technology. 45(17):7525-7532.
Tietge, J.E., Butterworth, B.C., Haselman, J.T., Holcombe, G.W., Hornung, M.W., Korte, J.J., Kosian, P.A., Wolfe, M. and Degitz, S.J., 2010. Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis. Aquatic Toxicology, 98(1), pp.44-50.
van der Ven LTM, van den Brandhof EJ, Vos JH, Power DM, Wester PW. 2006. Effects of the antithyroid agent propylthiouracil in a partial life cycle assay with zebrafish. Environmental Science & Technology. 40(1):74-81.
Van Herck SL, Geysens S, Delbaere J, Darras VM.  Regulators of thyroid hormone availability and action in embryonic chicken brain development. Gen Comp Endocrinol. 2013. 190:96-104.
Wabukebunoti MAN, Firling CE. 1983. The prehatching development of the thyroid-gland of the fathead minnow, pimephales-promelas (rafinesque). General and Comparative Endocrinology. 49(2):320-331.
Walter KM, Miller GW, Chen XP, Yaghoobi B, Puschner B, Lein PJ. 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (danio rerio). General and Comparative Endocrinology. 272:20-32.
Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347.
Zoeller, R. T., Tan, S. W., and Tyl, R. W. (2007). General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical reviews in toxicology 37(1-2), 11-53.
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<div>
<h4><a href="/relationships/2038">Relationship: 2038: T4 in serum, Decreased leads to Decreased, Triiodothyronine (T3)</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/159">Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/363">Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/364">Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/365">Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fathead minnow</td>
<td>Pimephales promelas</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Juvenile</td>
<td>Moderate</td>
</tr>
<tr>
<td>Larvae</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic: Thyroid follicles mainly produce T4 and to a lesser extent T3 across vertebrates. When serum T4 levels are decreased, less T4 is available for conversion to the more biologically active T3. This key event relationship is not always evident. This could be due to feedback/compensatory mechanisms that in some cases seem to be able to maintain T3 levels even though T4 levels are reduced, for example through increased conversion of T4 to T3 by deiodinases. These feedback mechanisms can also differ across species. Therefore, although this KER is plausibly applicable across vertebrates, variation can be expected. In zebrafish and fathead minnow, several studies reported evidence for a relationship between whole body T4 and T3 levels (Nelson et al., 2016; Stinckens et al., 2020, Wang et al., 2020).
Life stage: This key event relationship is applicable to late larvae and juveniles rather than to embryos, because of the presence of maternal TH in embryos.
Uncertainties during embryonic life stage:
<ul>
<li>A decrease in whole body T4 was observed in fathead minnows exposed to 1 mg/L 2-mercaptobenzothiazole (MBT), a TPO inhibitor, until 6 dpf (Nelson et al., 2016). In contrast, there was no observed effect on T3 in fathead minnows exposed to MBT until 6 dpf. Comparably, zebrafish exposed to 0.4 or 0.7 mg/L MBT thruntilough 120 hpf showed decreased whole body T4 but not T3 (Stinckens et al., 2016). During this early larval life stage, T3 may have been derived from maternal T4. In addition, it could be produced from further depletion of any T4 still produced by the thyroid gland (as TPO may not have been fully inhibited at the tested exposure concentrations).</li>
<li>Since exposure to PFAS did result in decreased whole-body T4 and T3 in 5 day old zebrafish, the life-stage specificity possibly depends on the mechanism that lies at the basis of the TH changes (Wang et al., 2020). The exact mechanisms by which PFAS disrupt the TH system remain uncertain. Compounds that directly reduce T3 levels (e.g., deiodinase inhibitors) in addition to reducing T4 levels via another mechanism can be expected to result in decreased T4 and T3 levels.</li>
</ul>
Sex: The KE is plausibly applicable to both sexes. Thyroid hormones are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of thyroid hormone levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in thyroid hormone levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.
<h4>Key Event Relationship Description</h4>
When serum thyroxine (T4) levels are decreased, less T4 is available for conversion to the more biologically active triiodothyronine (T3). While some thyroid hormone (TH) disrupting mechanisms can immediately affect T3 levels, including deiodinase inhibition, other mechanisms reduce T4 levels, for example through inhibition of TH synthesis, leading to decreased T3 levels.
Since in fish early life stages TH are typically measured on a whole body level, it is currently uncertain whether TH levels changes occur at the serum and/or tissue level. Pending more dedicated studies, whole body TH levels are often considered a proxy for serum TH levels.
Since in fish early life stages TH are typically measured on a whole-body level, it is currently uncertain whether TH levels changes occur at the serum and/or tissue level. Pending more dedicated studies, whole-body TH levels are often considered a proxy for serum TH levels.
This key event relationship is not always evident. This could be due to feedback/compensatory mechanisms that in some cases seem to be able to maintain T3 levels even though T4 levels are reduced, for example through increased conversion of T4 to T3 by deiodinases.
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
When serum thyroxine (T4) levels are decreased, less T4 is available for conversion to the more biologically active triiodothyronine (T3). It is plausible to assume that while some thyroid hormone (TH) disrupting mechanisms can immediately affect T3 levels, including deiodinase inhibition, other mechanisms reduce T4 levels, for example through inhibition of TH synthesis, leading to decreased T3 levels.
Empirical Evidence
<ul>
<li>A decrease in whole-body T4 and T3 was observed in zebrafish exposed to methimazole from fertilization until the age of 21 and 32 days and to propylthiouracil until the age of 14, 21 and 32 days (Stinckens et al., 2020). Additionally, a strong correlation was observed between T4 and T3 levels. Both compounds are TPO inhibitors expected to inhibit TH synthesis.</li>
<li>A dose-dependent decrease in whole-body T4 and T3 was observed in zebrafish exposed to perfluorooctanoic acid and perfluoropolyether carboxylic acids from fertilization until the age of 5 days (Wang et al., 2020). The exact mechanisms by which PFAS disrupt the TH system remain uncertain.</li>
<li>While T4 measurements could not be acquired in fathead minnows exposed to 1 mg/L 2-mercaptobenzothiazole, a TPO inhibitor, for 14 days, a significant decrease in T3 was observed (Nelson et al., 2016). The decreased T3 levels were likely the result of reduced T4 synthesis.</li>
<li>Besson et al. (2020) showed both decreased T4 levels and decreased T3 levels in metamorphosing convict surgeonfish exposed to chlorpyrifos.</li>
</ul>
Uncertainties and Inconsistencies
<ul>
<li>Since in fish early life stages THs are typically measured on a whole body level, it is currently uncertain whether TH level changes occur at the serum and/or tissue level. Pending more dedicated studies, whole body TH levels are considered a proxy for serum TH levels.</li>
<li>This key event relationship is not always evident. This could be due to feedback/compensatory mechanisms that in some cases seem to be able to maintain T3 levels even though T4 levels are reduced, for example through increased conversion of T4 to T3 by deiodinases. Examples of studies showing reduced T4 levels in the absence of reduced T3 levels:
<ul style="list-style-type:circle">
<li>Zebrafish exposed to 0.35 mg/L 2-mercaptobenzothiazole, a TPO inhibitor, through 32 dpf showed decreased whole-body T4, but T3 levels showed particularly large variation and overall were not significantly decreased (Stinckens et al., 2016).</li>
<li>Although T4 content of 28 dpf larval fathead minnows exposed to 32 or 100 µg/l methimazole, a TPO inhibitor, was reduced, these fish showed no change in whole body T3 content (Crane et al., 2006). Significantly higher T3/T4 ratios in fish held in 100 µg/l methimazole suggest an increased conversion of T4 to T3 or reduced degradation and conjugation during continued exposure to methimazole</li>
</ul>
</li>
</ul>
<h4>Quantitative Understanding of the Linkage</h4>
Stinckens et al. (2020, supplementary information) showed a significant linear relationship between whole body T3 and T4 concentrations at 21 and 32 days post fertilization after continuous exposure of zebrafish to methimazole and propylthiouracil, two inhibitors of TH synthesis.
Known Feedforward/Feedback loops influencing this KER
This key event relationship is not always evident. This could be due to feedback/compensatory mechanisms that in some cases seem to be able to maintain T3 levels even though T4 levels are reduced, for example through increased conversion of T4 to T3 by deiodinases. Examples of studies showing reduced T4 levels in the absence of reduced T3 levels:
<ul>
<li>Zebrafish exposed to 0.35 mg/L 2-mercaptobenzothiazole, a TPO inhibitor, through 32 dpf showed decreased whole-body T4, but T3 levels showed particularly large variation and overall were not significantly decreased (Stinckens et al., 2016).</li>
<li>Although T4 content of 28 dpf larval fathead minnows exposed to 32 or 100 µg/l methimazole, a TPO inhibitor, was reduced, these fish showed no change in whole body T3 content (Crane et al., 2006). Significantly higher T3/T4 ratios in fish held in 100 µg/l methimazole suggest an increased conversion of T4 to T3 or reduced degradation and conjugation during continued exposure to methimazole</li>
</ul>
This relationship depends on the MIE that is causing the decrease in T3. For example, deiodinase inhibition results in reduced activation of T4 to T3 and thus in reduced T3 levels; increased T4 levels have been observed, probably as a compensatory mechanism in response to the lower T3 levels. For example, Cavallin et al. (2017) exposed fathead minnows to iopanoic acid, a deiodinase inhibitor, and observed T4 increases together with T3 decreases.
<h4>References</h4>
Besson, M., Feeney, W. E., Moniz, I., François, L., Brooker, R. M., Holzer, G., Metian, M., Roux, N., Laudet, V., & Lecchini, D. (2020). Anthropogenic stressors impact fish sensory development and survival via thyroid disruption. Nature Communications, 11(1). https://doi.org/10.1038/s41467-020-17450-8
Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.
Crane, H.M., Pickford, D.B., Hutchinson, T.H., Brown, J.A., 2006. The effects of methimazole on development of the fathead minnow, pimephales promelas, from embryo to adult. Toxicological Sciences 93, 278-285.
Hernandez-Mariano JA, Torres-Sanchez L, Bassol-Mayagoitia S, Escamilla-Nunez M, Cebrian ME, Villeda-Gutierrez EA, Lopez-Rodriguez G, Felix-Arellano EE, Blanco-Munoz J. 2017. Effect of exposure to p,p '-dde during the first half of pregnancy in the maternal thyroid profile of female residents in a mexican floriculture area. Environmental Research. 156:597-604.
Liu XS, Cai Y, Wang Y, Xu SH, Ji K, Choi K. 2019. Effects of tris(1,3-dichloro-2-propyl) phosphate (tdcpp) and triphenyl phosphate (tpp) on sex-dependent alterations of thyroid hormones in adult zebrafish. Ecotoxicology and Environmental Safety. 170:25-32.
Nelson, K., Schroeder, A., Ankley, G., Blackwell, B., Blanksma, C., Degitz, S., Flynn, K., Jensen, K., Johnson, R., Kahl, M., Knapen, D., Kosian, P., Milsk, R., Randolph, E., Saari, T., Stinckens, E., Vergauwen, L., Villeneuve, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquatic Toxicology 173, 192-203.
Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.
Stinckens, E., Vergauwen, L., Schroeder, A., Maho, W., Blackwell, B., Witters, H., Blust, R., Ankley, G., Covaci, A., Villeneuve, D., Knapen, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish. Aquatic Toxicology 173, 204-217.
Wang, J.X., Shi, G.H., Yao, J.Z., Sheng, N., Cui, R.N., Su, Z.B., Guo, Y., Dai, J.Y., 2020. Perfluoropolyether carboxylic acids (novel alternatives to PFOA) impair zebrafish posterior swim bladder development via thyroid hormone disruption. Environment International 134.
Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347.
</div>
<div>
<h4><a href="/relationships/2373">Relationship: 2373: Decreased, Triiodothyronine (T3) leads to Altered, retinal layer structure</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/363">Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Low</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Embryo</td>
<td>High</td>
</tr>
<tr>
<td>Larvae</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
</div>
Life-stage applicability: Most studies on TH-regulated retinal structure are performed during vertebrate development. There is evidence of the impact of reduced T3 (caused by inhibition of thyroperoxidase) on retinal layer structure at 48, 66, 72, 96 and 120 hpf during zebrafish embryo-eleutheroembryo development (Baumann and others 2016; Komoike and others 2013; Reider and Connaughton 2014).
Taxonomic applicability: The visual system of the zebrafish follows the typical organisation of vertebrates and is often used as a model to study human eye diseases. Although there are some differences in eye structure between fish and mammals, it is plausible to assume that TH levels are important for healthy eye development across all vertebrates.
Sex applicability: Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Effects on retinal layers resulting from TH level changes during early development are therefore expected to be independent of sex.
<h4>Key Event Relationship Description</h4>
Although the exact mechanisms need further investigation, studies show that thyroid hormones (THs) are required for healthy eye development in vertebrates (Wester et al. 1990, Suliman & Novales Flamarique 2013, Deveau et al., 2019) and it has been described that retinal development, photoreceptor differentiation and colour vision are directly regulated by THs. Not only in zebrafish (Bertrand et al. 2007), but also in mice (Ng et al. 2010) and chickens (Trimarchi et al. 2008), THs are directly linked to the transcription of essential visual opsins and the differentiation of retinal cells, as well as the overall structure of the retina, which is essential for proper visual functioning. Therefore, decreased triidothyronine (T3) levels during eye development are likely to lead to structural and morphological alterations of the retina. The site of decreased T3 in this case is the retinal layers.
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
THs, TH receptors, and deiodinase (DIO) enzymes are important for eye and retinal development in vertebrates. Dio enzymes activate and inactivate THs, consequently playing a central role in regulating TH levels in target tissues. In zebrafish, TH receptors and dio enzymes have been localized in the retina from 24 hpf onwards, probably regulating the differentiation of retinal structures and photoreceptors (Gan et al. (2010), Duval, M. G., & Allison, W. T. (2018)). It is known from amphibians that when TH levels start to rise at the beginning of metamorphosis, the morphology of the eyes starts changing (Fini et al. 2012). In chicken, the developing eye shows a dynamic expression pattern of Deiodinase 2 (DIO2) and Deiodinase 3 (DIO3), probably regulating photoreceptor differentiation and cornea development (reviewed by Darras 2015).
Empirical Evidence
There is ample evidence that reduced THs have an influence on development of the retinal layer structure in fish and other vertebrates. Although the assumed site of T3 decrease is assumed to be in the retinal layers itself, most fish early life stage studies only quantify whole body T3 levels which does not allow for making the distinction between systemic and local T3 levels.
Evidence from exposure to PTU, 6-n-propylthouracil, a classic positive control for inhibition of TPO responsible for TH synthesis:
<ul>
<li>Reduced whole body T4 and T3 levels at 14, 21 and 32 days post fertilization (dpf) were observed after exposure of zebrafish to 111 mg/L PTU (Stinckens et al., 2020). Exposure to 37 mg/L PTU reduced T4 levels at 14, 21 and 32 dpf and significantly reduced T3 levels at 32 dpf, while the more limited decrease of T3 levels at 14 and 21 dpf was not statistically significant (Stinckens et al., 2020). Schmidt and Braunbeck (2011) also showed reduced T4 levels in juvenile zebrafish exposed to PTU for 5 weeks. PTU was also shown to reduce T4 levels already at 72 and 120 hours post fertilization (Walter et al., 2019). T3 levels tended to decrease at 72 and 120 hpf but these changes were not significant. Exposures were always continuous and started immediately after fertilization.</li>
<li>Baumann et al. (2016) described alterations in retinal structure, pigmentation and eye size in 5 day old zebrafish embryos after exposure to PTU. Exposures to 100 and 250 mg/L PTU reduced retinal pigment epithelial diameter and exposure to 250 mg/L increased the grey value of the pigment layer which is a measure of decreased pigmentation.</li>
<li>Gan et al. (2010) showed that thyroid hormones accelerate opsin expression in differentiating cones and induce the opsin switch, a shift from expression of UV opsin to blue opsin, in differentiated single cones in salmonids. Using in situ hybridization, they characterized the spatiotemporal dynamics of opsin expression and switching in embryos treated with exogenous TH or PTU. The results show that PTU repressed the opsin switch. THs are required for opsin switching in the retina of salmonid fishes.</li>
</ul>
Evidence from exposure to methimazole, a model thyroperoxidase inhibitor:
<ul>
<li>Methimazole was shown to reduce whole body T4 and T3 levels at 14, 21 and 32 days post fertilization after exposure of zebrafish to 50 and 100 mg/L (Stinckens et al., 2020). Exposures were always continuous and started immediately after fertilization.</li>
<li>Komoike et al. (2013) exposed zebrafish embryos to 10 mM methimazole and observed moderately disrupted retinal structure with apoptosis of retinal      cells already at 48 hpf and more severely disrupted retinal structure at 72 hpf. Major gaps and malformations of the retinal structure occurred at 72 hpf. The observed retinal anomalous morphologies have a direct analogy to the congenital anomalies observed in children exposed to methimazole in utero.</li>
<li>Reider and Connaughton (2014) exposed zebrafish embryos to methimazole until 66, 70 or 72 hpf and analysed the retina at 72 hpf. The thickness of the ganglion cell layer (GCL) was decreased in embryos exposed to MMI until 66 hpf compared to controls. An increase in GCL thickness was observed in embryos exposed until 70 hpf, and normal thickness was observed in embryos exposed until 72 hpf. Although the impact of the exposure windows cannot be entirely explained, this confirms the relation between reduced T3 and altered retinal structure.</li>
</ul>
Evidence from other chemical exposures:
<ul>
<li>Baumann et al. (2016): After exposure to 200 and 300 μg/L TBBPA, a compound with several mechanisms including a direct interaction with the TH     receptor and binding to the TH binding protein transthyretin, grey values were increased at 5 dpf indicative of reduced pigmentation in the eyes. There were no significant effects on the retinal pigment epithelium diameter. Experiments from Zhu et al. (2018) and Yu et al. (2021) confirm a reduction in T3 levels in both the larvae and embryos (whole body) after exposure to 300 μg/L TBBPA and the locomotor activity of larval offspring was significantly reduced.</li>
<li>Besson et al. (2020) used treatment with NH3 to highlight the role that THs play in retinal development in metamorphosing convict surgeonfish. They analysed different cell segments, types, and layers of the retina, such as (i) the densities of photoreceptor external segments (perceiving light signals), (ii) photoreceptor nuclei, (iii) bipolar cells (which integrate the synaptic signals originating from the photoreceptors), and (iv) ganglion cells (which integrate signals from bipolar cells and create action potential toward the optic nerve). They investigated the role of THs in the development of these sensory structures by injecting fish daily from d0 to d5 with NH3 (10−6 M), a TH antagonist, to achieve TH signal disruption. NH3 prevents the binding of TH such as T3 to TR, therefore impairing the binding of transcriptional coactivators to TR, which therefore remain in an inactive and repressive conformation.The NH3 treatment was thus applied to repress TH signaling by disrupting the TH pathway leading to an adverse outcome on retinal layer level. Repressed retinal development at both d2 and d5 with a 10- 25 % decrease of bipolar cell density was detected.</li>
<li>Besson et al. (2020) further showed that treatment with chlorpyrifos reduced T3 levels and reduced bipolar cell density by 10%.</li>
<li>Bhumika et al. (2014) found that lowering T3 signaling through exposure to different chemicals accelerates optic tectum reinnervation following optic      nerve crush in zebrafish and that this is accompanied by a more rapid resolution of the inflammatory response. Unlike in mammals, full recovery of the damaged CNS is possible in adult fish and amphibians and, for instance, the optic nerve of fish can regenerate completely after injury. Adult zebrafish were exposed to 10 μM of iopanoic acid (IOP), which lowered intracellular T3 availability, or to 7 μM of the TH receptor β antagonist methylsulfonylnitrobenzoate (C1). Both treatments accelerated optic tectum (OT) reinnervation. At 7 days post injury (7 dpi) there was a clear increase in the biocytin labeled area in the OT following anterograde tracing as well as an increased immunostaining of Gap43, a protein expressed in outgrowing axons. This effect was attenuated by T3 supplementation to IOP-treated fish. ON crush induced limited cell death and proliferation at the level of the retina in control, IOP- and C1-treated fish.</li>
</ul>
Evidence from genetic knockdown and knockout studies:
<ul>
<li>Houbrechts (2016) performed deiodinase (DIO) knockdown in zebrafish embryos and observed reduced eye size, disturbed retinal lamination and strong reduction in rods and all four cone types. DIO 1 and 2 are both responsible for converting T4 to the more active T3. Combined knockdown of DIO 1 and 2, leading to reduced T3 levels, altered the structure of the ganglion cell layer (GCL), making it wider and less dense. DIO3 deactivates T3 and defects were more prominent and persistent in D3-deficient fish with observations of marked disorganization across all retinal layers.</li>
<li>Using genetic zebrafish experiments Duval and Allison (2018) investigated the role of the thyroid hormone receptor thrb in cone differentiation at different time points. Disrupting thrb activity via expression of a dominant negative thrb (dnthrb) at either early or late retinal development had differential outcomes on red cones (reduced abundance), versus UV and blue cones (increased abundance). The effects of thrb change through photoreceptor development, first promoting red cones and restricting UV cones, and later restricting UV and blue cones. Knockdown of thrb causes near-complete absence of red cones and an increase in UV cone abundance (by approximately 35%), whereas expression of dnthrb via heat shock at 52 hpf leads to increased UV (by 27%) and blue cone abundance (by 36%) relative to heat shocked nontransgenic siblings. Inducing dnthrb expression at other time points, including 24 hpf, 30 hpf, and 36 hpf, did not alter cone abundances as dramatically relative to controls (<20% change). This revealed an effect of thrb that is limited to later photoreceptor development: the endogenous receptor negatively regulates blue cone determination. In contrast, disrupting Thrb activity either early (with morpholino knock down) or late leads to more UV cones.</li>
<li>Ng et al. (2010) showed in mice that knockout of the thyroid receptor, THRb2, results in important changes in the numbers of specific cone types in the retina and M opsins do not even appear at all. Knockout of a thyroid hormone receptor conceptually corresponds to decreased activation of the thyroid hormone receptor due to decreased T3 levels.</li>
</ul>
Other models of hypothyroidism:
<ul>
<li>Gamborino (2000) analysed eye development in a rat model of congenital-neonatal hypothyroidism (HG), induced by combined chemical-surgical thyroidectomy. Histopathological analyses of the eyes of TH-deficient animals revealed decrease in photoreceptor and ganglion cell layer thickness, a delay in photoreceptor outer segment morphogenesis and significantly lower values for ganglion cell nuclear volumes and nuclear pore density.</li>
</ul>
indirect evidence:
Trimarchi et al. (2008) observed three waves of expression of components of the HPT-axis in specific locations in the retina in progenitor cells and photoreceptor cells during development of the chicken, indicating that thyroid hormones are required for normal retinal development and photoreceptor differentiation
Uncertainties and Inconsistencies
Several studies have shown molecular responses to hypothyroidism that are related to eye development (Bagci et al., 2015; Houbrechts et al., 2016; Baumann et al., 2019) but the exact molecular processes linking lower TH level to disturbances of the layers in the retina is not yet fully understood.
Both decreased as well as increased TH action has been shown to impact retinal development.
<ul>
<li>For example, Ng et al. (2010) showed altered cone appearance in the retina following both DIO3 knockout (leading to hyperthyroidism) and THRb2 knockout (corresponding to hypothyroidism).</li>
<li>Besson et al. (2020) used pharmacological treatments (T3 + iopanoic acid (IOP), NH3) to not only disrupt but also activate the TH signaling pathway. They used 10−6M T3 + (iopanoic acid) (T3 treatment) to achieve TH signal activation. Here, IOP was used as an inhibitor of deiodinase enzymes, following comparable work in mammals and amphibians, and as routinely used in fish to prevent the immediate degradation of injected T3. The combined treatment thus causes elevated T3 levels. Detected effects on retinal layers were elevated densities of bipolar cells at day 2 in surgeonfish.</li>
<li>Suppressing TH signaling in retina dystrophy mouse models (a mouse model of retinal degeneration) seems to protect cone viability (Ma et al., 2014; 2016). The authors suggested that the impact of TH on cone survival is independent of its impact on cone opsin expression. The mechanism underlying the effect on cone viability has not been elucidated. </li>
<li>Bhumika et al. (2014) showed accelerated reinnervation of the optic tectum after optic nerve crush in zebrafish that had been treated with IOP or a TR antagonist. B     oth treatments cause hypothyroidism. Supplementation of T3 reduced the rate of reinnervation.</li>
</ul>
Another uncertainty lies in the systemic versus local changes in T3 levels. Although the assumed site of T3 decrease is assumed to be in the retinal layers itself, most fish early life stage studies only quantify whole body T3 levels which does not allow for making the distinction between systemic and local T3 levels.
Most knowledge comes from effects observed in developing organisms. There are some gaps in our knowledge about how TH levels affect the eyes of already fully developed organisms and/or whether they have similarly serious effects on the retinal layers. It can be assumed that the effects, if any, are weaker. Studies (Reider et al. 2014) found that layer thickness varied across ages suggesting that these retinal layers are differentially sensitive to for example MMI and/or that there are different critical periods of sensitivity of the retinal tissue.
<h4>Quantitative Understanding of the Linkage</h4>
There is no direct quantitative relation available at this point.
Known Feedforward/Feedback loops influencing this KER
<ul>
<li>One feedback loop mechanism could be triggered by iodine deficiency or inhibition of iodine uptake. It appears probably that the inhibition increases the secretion of Thyroid stimulating hormone, which could stimulate the expression of the NIS-transporter. This increase in TSH could shift the ratio in favour of T3.</li>
</ul>
<h4>References</h4>
Baumann L, Ros A, Rehberger K, Neuhauss SCF, Segner H. 2016. Thyroid disruption in zebrafish (Danio rerio) larvae: Different molecular response patterns lead to impaired eye development and visual functions. Aquatic Toxicology 172:44-55.
Bagci E, Heijlen M, Vergauwen L, Hagenaars A, Houbrechts AM, Esguerra CV, Blust R, Darras VM, Knapen D. 2015. Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction. PLOS One 10:e0123285.
Baumann L, Segner H, Ros A, Knapen D, Vergauwen L. 2019. Thyroid Hormone Disruptors Interfere with Molecular Pathways of Eye Development and Function in Zebrafish. International Journal of Molecular Sciences 20(7).
Besson, M., Feeney, W. E., Moniz, I., François, L., Brooker, R. M., Holzer, G., Metian, M., Roux, N., Laudet, V., & Lecchini, D. (2020). Anthropogenic stressors impact fish sensory development and survival via thyroid disruption. Nature Communications, 11(1). https://doi.org/10.1038/s41467-020-17450-8
Bhumika, S., & Darras, V. M. (2014). Role of thyroid hormones in different aspects of nervous system regeneration in vertebrates. General and Comparative Endocrinology, 203, 86–94. https://doi.org/10.1016/j.ygcen.2014.03.017
Duval, M. G., & Allison, W. T. (2018). Photoreceptor progenitors depend upon coordination of gdf6a, thrβ, and tbx2b to generate precise populations of cone photoreceptor subtypes. Investigative Ophthalmology and Visual Science, 59(15), 6089–6101. https://doi.org/10.1167/iovs.18-24461
Darras VM, Houbrechts AM, Van Herck SLJ. Intracellular thyroid hormone metabolism as a local regulator of nuclear thyroid hormone receptor-mediated impact on vertebrate development. Biochim Biophys Acta - Gene Regul Mech. 2015;1849(2):130-141. doi:10.1016/j.bbagrm.2014.05.004
Fini, J. B., le Mével, S., Palmier, K., Darras, V. M., Punzon, I., Richardson, S. J., Clerget-Froidevaux, M. S., & Demeneix, B. A. (2012). Thyroid hormone signaling in the Xenopus laevis embryo is functional and susceptible to endocrine disruption. Endocrinology, 153(10), 5068–5081. <a href="https://doi.org/10.1210/en.2012-1463">https://doi.org/10.1210/en.2012-1463</a>
Gan, K. J., & Flamarique, I. N. (2010). Thyroid hormone accelerates opsin expression during early photoreceptor differentiation and induces opsin switching in differentiated TRα-expressing cones of the salmonid retina. Developmental Dynamics, 239(10), 2700–2713. https://doi.org/10.1002/dvdy.22392
Houbrechts AM, Delarue J, Gabriëls IJ, Sourbron J, Darras VM. Permanent deiodinase type 2 Deficiency strongly perturbs zebrafish development, growth, and fertility. Endocrinology. 2016;157(9):3668-3681. doi:10.1210/en.2016-1077
Komoike Y, Matsuoka M, Kosaki K. 2013. Potential Teratogenicity of Methimazole: Exposure of Zebrafish Embryos to Methimazole Causes Similar Developmental Anomalies to Human Methimazole Embryopathy. Birth Defects Research Part B-Developmental and Reproductive Toxicology 98(3):222-229.
Gamborino MJ, Sevilla-Romero E, Muñoz A, Hernández-Yago J, Renau-Piqueras J, Pinazo-Durán MD. Role of thyroid hormone in craniofacial and eye development using a rat model. Ophthalmic Res. 2001;33(5):283-291. doi:10.1159/000055682
Ma HW, Ding XQ. 2016. Thyroid hormone signaling and cone photoreceptor viability. Retinal Degenerative Diseases: Mechanisms and Experimental Therapy. 854:613-618.
Ma HW, Thapa A, Morris L, Redmond TM, Baehr W, Ding XQ. 2014. Suppressing thyroid hormone signaling preserves cone photoreceptors in mouse models of retinal degeneration. Proceedings of the National Academy of Sciences of the United States of America. 111(9):3602-3607.
Ng L, Lyubarsky A, Nikonov SS, Ma M, Srinivas M, Kefas B, St Germain DL, Hernandez A, Pugh EN, Jr., Forrest D. 2010. Type 3 deiodinase, a thyroid-hormone-inactivating enzyme, controls survival and maturation of cone photoreceptors. J Neurosci. 30(9):3347-3357.
Reider M, Connaughton VP. 2014. Effects of Low-Dose Embryonic Thyroid Disruption and Rearing Temperature on the Development of the Eye and Retina in Zebrafish. Birth Defects Research Part B-Developmental and Reproductive Toxicology 101(5):347-354.
Quesada-García A, Encinas P, Valdehita A, et al. Thyroid active agents T3 and PTU differentially affect immune gene transcripts in the head kidney of rainbow trout (Oncorynchus mykiss). Aquat Toxicol. 2016;174:159-168. doi:10.1016/j.aquatox.2016.02.016
Schmidt F and Braunbeck T. Alterations along the Hypothalamic-Pituitary-Thyroid Axis of the Zebrafish (Danio rerio ) after Exposure to Propylthiouracil. 2011. Journal of Thyroid Research. Volume 2011, Article ID 376243, 17 pages. doi:10.4061/2011/376243
Stinckens E, Vergauwen L, Blackwell BR, Anldey GT, Villeneuve DL, Knapen D. 2020. Effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology. 54(10):6213-6223.
Wang, W. Der, Hsu, H. J., Li, Y. F., & Wu, C. Y. (2017). Retinoic acid protects and rescues the development of zebrafish embryonic retinal photoreceptor cells from exposure to paclobutrazol. International Journal of Molecular Sciences, 18(1). https://doi.org/10.3390/ijms18010130
Walter KM, Miller GW, Chen XP, Yaghoobi B, Puschner B, Lein PJ. 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (danio rerio). General and Comparative Endocrinology. 272:20-32.
Yu, Y., Hou, Y., Dang, Y., Zhu, X., Li, Z., Chen, H., Xiang, M., Li, Z., & Hu, G. (2021). Exposure of adult zebrafish (Danio rerio) to Tetrabromobisphenol A causes neurotoxicity in larval offspring, an adverse transgenerational effect. Journal of Hazardous Materials, 414, 125408. https://doi.org/https://doi.org/10.1016/j.jhazmat.2021.125408
Zhu, B., Zhao, G., Yang, L., & Zhou, B. (2018). Tetrabromobisphenol A caused neurodevelopmental toxicity via disrupting thyroid hormones in zebrafish larvae. Chemosphere, 197, 353–361. https://doi.org/10.1016/j.chemosphere.2018.01.080
</div>
<div>
<h4><a href="/relationships/2374">Relationship: 2374: Altered, retinal layer structure leads to Altered, Visual function</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/363">Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>adjacent</td>
<td>High</td>
<td>Low</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Embryo</td>
<td>High</td>
</tr>
<tr>
<td>Adult</td>
<td>Moderate</td>
</tr>
<tr>
<td>Juvenile</td>
<td>Moderate</td>
</tr>
<tr>
<td>Larvae</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic applicability: The visual system of the zebrafish follows the typical organisation of vertebrates and is often used as a model to study human eye diseases. Although there are some differences in eye structure between zebrafish and humans, it is plausible to assume that a functioning eye structure is important for visual function across all vertebrates and invertebrates that have eyes.
Life stage applicability: The first visual responses based on retinal functionality appear around 70 hpf in zebrafish (Schmitt and Dowling 1999). It is plausible to assume that alterations of the eye structure would result in altered visual function across all life stages, but such alterations are most likely to occur during the development of the normal eye structure, which occurs in the embryo-eleutheroembryo phase.
Sex applicability: Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Effects on visual function resulting from altered eye structure during early development are therefore expected to be independent of sex.
<h4>Key Event Relationship Description</h4>
The structure of the vertebrate retina is well conserved and consists of the following layers: The retinal pigment epithelium (RPE), the photoreceptor layer (PRL), the outer plexiform layer (OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL) and the ganglion cell layer (GCL). Each of these layers has a specific function for the physiology of the visual system. The RPE serves to protect and maintain the photoreceptors and absorbs excess light. The photoreceptors in the PRL consist of a light-receiving outer segment (OS) and the inner segment (IS), which contains the cell bodies. They send their signals to the bipolar cells in the INL, which transmit the signal to the ganglion cells. These form the optic nerve and are responsible for transmitting signals to the optic nerves. In both plexiform layers, the retinal neurons form their synaptic connections (Bibliowicz et al. 2011).
To study the eye, the zebrafish (Danio rerio) is at the forefront of many studies as a model organism. In zebrafish, eye development begins around 12 hpf (Houbrechts et al., 2016b) and by 72 hpf the layers of the retina are well developed (Malicki et al., 2016). Functional vision is established by 4-5 dpf (Brockerhoff, 2006; Chhetri et al., 2014).
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
It should be emphasised that all layers of the retina are interdependent. The RPE plays an essential role in the retinoid cycle for the photoreceptors (PRL), which perceive the light stimulus and transmit it via the bipolar cells to the ganglia (IPL), which form the optic nerve and transmit the signal to the optic nerve (Connaughton 2005). If these key sites of the phototransduction pathway are disrupted by, for example, endocrine disruptors, it stands to reason that there would be a significant impact on the optical sense and it is plausible that disorders of the eye structure can lead to visual disorders.
Empirical Evidence
<ul>
<li>Baumann et al., 2016 used propylthiouracil (PTU) and tetrabromobisphenol A (TBBPA) to disrupt the thyroid hormone system in zebrafish larvae. This exposure induced different molecular response patterns leading to impaired eye development (reduction of RPE cell diameter, pigmentation and eye size). Behavioural analyses showed that these larvae were also disrupted in their visual capacities, such as decrease in optokinetic response and increase in light preference of PTU-treated larvae.</li>
<li>Avallone et al. (2015) studied the effects of cadmium exposure on the vision of adult zebrafish. The morpho-cytological changes of the retina (Nerve fiber layer clearly thickened and vacuolated, presence of compact pycnotic nuclei, empty area, change in the thickness of pigmented retinal epithelium and at the level of cones inner segments, extended folds of treated retinas, presence of cell debris and/of blood cells in vitreal chamber) were investigated by light and electron microscopy, while the functionality of the cadmium-exposed retinas was assessed by re-illumination behavioural tests with white or coloured light. Cadmium toxicity was shown to cause significant cell degeneration and loss of organisation at both macroscopic and microscopic levels. These changes were directly related to functional responses, particularly by increasing light sensitivity of exposed fish. Avoidance of bright light had increased in exposed fish.</li>
<li>Houbrechts et al. (2016) used a knockdown of deiodinase 1 and 2 genes in zebrafish embryos to induce transient hypothyroidism and observed a wider and less dense ganglion cell layer at 3 dpf together with a reduced response (increase of swimming activity) to light at 4 dpf. By 7 dpf both the change in the ganglion cell layer as well as the altered response to light had recovered and resembled those of the untreated larvae. </li>
<li>Flamarique et al. (2013) used thyroid hormone treatment to transform the UV cones of young rainbow trout into blue cones and showed that this reduced the distances and angles at which prey were located (variables that are known indicators of foraging performance). Using optical measurements and photon-catch calculations, the study showed that control rainbow trouts perceived prey (Daphnia) with greater contrast compared to thyroid-hormone-treated fish, demonstrating that the presence of UV cones enhances foraging performance of young rainbow trout.</li>
<li>Walter et al. (2019) found out that developmental exposure to either T4 or T3 in zebrafish embryos altered photomotor behavior. The response to a sudden transition from light to dark differed from that in untreated fish.</li>
<li>Heijlen et al. (2014) showed that knockdown of Type 3 Iodothyronine Deiodinase, known to disrupt retinal layer structure (Houbrechts et al. (2016), caused embryos to spend significantly less time moving, and perturbed the escape response after a tactile stimulus. It is unclear to what extent this relationship is determined by alterations in muscle development or other factors contributing to these types of behaviour.</li>
<li>Houbrechts et al. (2016b) showed that permanent deiodinase 2 deficiency in zebrafish resulted in a reduction of the number of R/G cones and rods that persisted through 7 dpf together with a reduced response to light (observed at 6 dpf).</li>
<li>Chawla et al. (2018) investigated the role of Retinoic Acid (RA) in embryonic development of craniofacial structures in zebrafish. An increase in RA caused morphological changes of the eyes: a decrease of both cellular density of the corneal epithelium and cellularity of the inner segment. Inhibition of RA synthesis with 4-diethylamino- benzaldehyde (DEAB) resulted in structural changes of the retina, including the obliteration of photoreceptors and ganglion cell layer, and decreased cellularity of the outer and inner nuclear layers. Treated fish showed strong impairment of the optokinetic reflex.</li>
</ul>
Uncertainties and Inconsistencies
Often, high variances occur in the results of behavioural studies that may be due to a variety of factors including genetic differences, variation in feeding status, etc. It is also difficult to compare data from different laboratories in such experiments. Similarly, extrapolating data from fish to mammalian data is particularly difficult for behavioural studies.
<h4>Quantitative Understanding of the Linkage</h4>
Quantitative understanding of this linkage is currently limited.
Time-scale
Temporal evidence is supported by the studies of Houbrechts et al. (2016) and Van Camp et al. (2018) in genetic knockdown and knockout zebrafish respectively. Houbrechts et al. (2016) used a DIO 1 and 2 knockdown, which causes transient hypothyroidism. At 3 dpf they showed altered retinal layer structure and at 4 dpf they showed an altered response to light. By 7 dpf both the retinal layer structure and the response to light had returned to normal. Van Camp et al. (2018) used a DIO2 knockout model causing permanent hypothyroidism. They did shown both altered numbers of rods and cones in the retina and an altered response to light at 7 dpf.
<h4>References</h4>
Avallone B, Crispino R, Cerciello R, Simoniello P, Panzuto R, Motta CM. Cadmium effects on the retina of adult Danio rerio. CR Biologies. 2015;338:40–7.
Baumann, L., Ros, A., Rehberger, K., Neuhauss, S. C. F., & Segner, H. (2016). Thyroid disruption in zebrafish (Danio rerio) larvae: Different molecular response patterns lead to impaired eye development and visual functions. Aquatic Toxicology, 172, 44–55. https://doi.org/10.1016/j.aquatox.2015.12.015
Bibliowicz, J., Tittle, R. K., & Gross, J. M. (2011). Toward a better understanding of human eye disease: Insights from the zebrafish, Danio rerio. In Progress in Molecular Biology and Translational Science(Vol. 100, Issue Table 1). https://doi.org/10.1016/B978-0-12-384878-9.00007-8
Brockerhoff, S. E. (2006). Measuring the optokinetic response of zebrafish larvae. Nature Protocols, 1(5), 2448–2451. https://doi.org/10.1038/nprot.2006.255
Chawla, B., Swain, W., Williams, A. L., & Bohnsack, B. L. (2018). Retinoic acid maintains function of neural crest–derived ocular and craniofacial structures in adult zebrafish. Investigative Ophthalmology and Visual Science, 59(5), 1924–1935. https://doi.org/10.1167/iovs.17-22845
Chhetri, J., Jacobson, G., & Gueven, N. (2014). Zebrafish-on the move towards ophthalmological research. Eye (Basingstoke), 28(4), 367–380. https://doi.org/10.1038/eye.2014.19
Crowley-Perry, M., Barberio, A. J., Zeino, J., Winston, E. R., & Connaughton, V. P. (2021). Zebrafish optomotor response and morphology are altered by transient, developmental exposure to bisphenol-a. Journal of Developmental Biology,9(2). https://doi.org/10.3390/jdb9020014
Connaughton, V. P., Graham, D., & Nelson, R. (2004). Identification and morphological classification of horizontal, bipolar, and amacrine cells within the zebrafish retina. Journal of Comparative Neurology, 477(4), 371–385. https://doi.org/10.1002/cne.20261
Flamarique, I. N. (2013). Opsin switch reveals function of the ultraviolet cone in fish foraging. Proceedings of the Royal Society B: Biological Sciences, 280(1752). https://doi.org/10.1098/rspb.2012.2490
Frau, S., Novales Flamarique, I., Keeley, P. W., Reese, B. E., & Muñoz-Cueto, J. A. (2020). Straying from the flatfish retinal plan: Cone photoreceptor patterning in the common sole (Solea solea) and the Senegalese sole (Solea senegalensis). Journal of Comparative Neurology, 528(14), 2283–2307. https://doi.org/10.1002/cne.24893
Heijlen, M., Houbrechts, A. M., Bagci, E., Van Herck, S. L. J., Kersseboom, S., Esguerra, C. V., Blust, R., Visser, T. J., Knapen, D., & Darras, V. M. (2014). Knockdown of type 3 iodothyronine deiodinase severely perturbs both embryonic and early larval developme
Houbrechts, A. M., Vergauwen, L., Bagci, E., Van houcke, J., Heijlen, M., Kulemeka, B., Hyde, D. R., Knapen, D., & Darras, V. M. (2016). Deiodinase knockdown affects zebrafish eye development at the level of gene expression, morphology and function. Molecular and Cellular Endocrinology, 424, 81–93.      https://doi.org/10.1016/j.mce.2016.01.018
Houbrechts, A. M., Delarue, J., Gabriëls, I. J., Sourbron, J., & Darras, V. M. (2016b). Permanent deiodinase type 2 Deficiency strongly perturbs zebrafish development, growth, and fertility. Endocrinology, 157(9), 3668–3681. https://doi.org/10.1210/en.2016-1077
Malicki, J., Pooranachandran, N., Nikolaev, A., Fang, X., & Avanesov, A. (2016). Analysis of the retina in the zebrafish model. In Methods in Cell Biology(Vol. 134). Elsevier Ltd. https://doi.org/10.1016/bs.mcb.2016.04.017
Masuda, T., Shimazawa, M., Hara, H., 2017. Retinal diseases associated with oxidative stress and the effects of a free radical scavenger (edaravone). Oxid. Med. Cell. Longev. 2017, 9208489.
Moreno-Marmol T, Cavodeassi F, Bovolenta P. 2018. Setting Eyes on the Retinal Pigment Epithelium. Frontiers in Cell and Developmental Biology 6.
Morris AC, Fadool JM. 2005. Studying rod photoreceptor development in zebrafish. Physiology & Behavior 86(3):306-313.
Naujokas, M.F., Anderson, B., Ahsan, H., et al., 2013. The broad scope of health effects from chronic arsenic exposure: update on a worldwide public health problem. Environ. Health Perspect. 121 (3), 295e302.
Raymond PA, Barthel LK, Curran GA. 1995. DEVELOPMENTAL PATTERNING OF ROD AND CONE PHOTORECEPTORS IN EMBRYONIC ZEBRAFISH. Journal of Comparative Neurology 359(4):537-550.
Schmitt EA, Dowling JE. 1999. Early retinal development in the zebrafish, Danio rerio: Light and electron microscopic analyses. Journal of Comparative Neurology 404(4):515-536.
Strauss O. The retinal pigment epithelium in visual function. Physiol Rev. 2005 Jul;85(3):845-81. doi: 10.1152/physrev.00021.2004. PMID: 15987797.
Vancamp P, Houbrechts AM, Darras VM. Insights from zebrafish deficiency models to understand the impact of local thyroid hormone regulator action on early development. Gen Comp Endocrinol. 2019 Aug 1;279:45-52. doi: 10.1016/j.ygcen.2018.09.011. Epub 2018 Sep 19. PMID: 30244055.
Walter, K. M., Miller, G. W., Chen, X., Harvey, D. J., Puschner, B., & Lein, P. J. (2019). Changes in thyroid hormone activity disrupt photomotor behavior of larval zebrafish. Neurotoxicology, 74, 47–57. https://doi.org/10.1016/j.neuro.2019.05.008
</div>
<div>
<h4><a href="/relationships/2375">Relationship: 2375: Altered, Visual function leads to Increased Mortality</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/399">Inhibition of Fyna leading to increased mortality via decreased eye size (Microphthalmos)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/363">Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Low</td>
</tr>
<tr>
<td><a href="/aops/364">Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/365">Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic applicability: The visual system of the fish (e.g., zebrafish) follows the typical organisation of vertebrates and is often used as a model to study human eye diseases. Although there are some differences, it is plausible to assume that visual function is important for survival across all vertebrates and invertebrates that have eyes.
Sex applicability: Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Effects on mortality resulting from altered visual function are therefore expected to be independent of sex.
Life stage applicability: It is plausible to assume that altered visual function of the eye would result in a higher mortality across all life stages. This could be especially true for the embryonic stages, the most sensitive stage of life. Vision plays a crucial role (in the early life stages) of most species, as eye development and establishment of functional vision is essential for perception of food or avoidance of predators for example (Carvalho et al., 2002).
<h4>Key Event Relationship Description</h4>
In animals, whatever the taxa, visual abilities are strongly linked to their lifestyle (feeding, avoidance of predators, movement, protection....). When these capacities are impaired, they lead to reduced fitness and are therefore strongly linked to a decrease in survival, particularly in the early stages of life.
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
Decreases in visual functions can have a strong impact on behavior, leading to changes in individual response and abilities in the environment, including, for example, perception of food or avoidance of predators. Variation in the visual system can also influence learning tasks when visual stimuli are used (Corral-López et al., 2017).
Sensory drive has been implicated in speciation in various taxa, largely based on phenotype-environment correlations and signatures of selection in sensory genes, including vision (Maan et al, 2017).
It is biologically plausible that an animal which has difficulties in finding food and avoiding predators will have lower survival chances in wildlife.
Empirical Evidence
Only very few studies are available in which it was demonstrated that decreased visual capacities lead to reduced survival of the organism. In general, mortality is rarely assessed but survival-reducing factors (feeding, predation) are mainly investigated. Here we consider the work about different toxicants that disrupt complex fish behaviors, such as predator avoidance, reproductive, and social behaviors. Toxicant exposure often completely eliminates the performance of behaviors that are essential to fitness and survival in natural ecosystems, frequently after exposures of lesser magnitude than those causing significant mortality (Brown et al., 2004).
<ul>
<li>Fuiman et al. (2006) specifically investigated the importance of several putative survival skills for escaping a predator. They first analysed routine swimming, acoustic startle stimulus and visual startle stimulus of red drum larvae and subsequently performed a predation experiment using the same larvae in the presence of a live predator. The authors found that the effectiveness of escape responses was almost 100% and thus responsiveness determined survival under predation. Of the different putative survival skills, only visual responsiveness was significantly correlated to escape potential, while others such as acoustic responsiveness were not significantly contributing to escape potential. Further investigation showed that only visual responsiveness differed significantly between poorly responding larvae and better responders.</li>
<li>Dehnert et al., 2019: In zebrafish, 2, 4-Dichlorophenoxyacetic acid exposure during eye development impaired visual behavior, i.e. reduced prey capture. Additionally, exposed fish showed reduced neural activity within the optic tectum following prey exposure.</li>
<li>Besson et al., 2020 exposed metamorphosing convict surgeonfish to pharmacological treatments. They performed a 10−6 M NH3 treatment (a TH antagonist) to achieve TH signal disruption and they observed an adverse outcome on retinal layer level. Repressed retinal development at both day 2 and day 5 with a 10-25 % decrease of bipolar cell density was detected. They followed up with a behavior test at day 2 with blacktail snapper as a predator and got the following results: </li>
</ul>
1. In the test using chemical cues of the predator the NH3-treated fish did not discriminate between water sources, while control fish clearly avoided predator cues.
2. In the visual cues test the NH3-treated fish showed no preference and spent 25 % more time in visual stimulus compared to controls.
3. In a survival predation test in an in situ arena they observed that day 2 NH3 treated fish exhibited 30% lower survival than d2 control fish.
<ul>
<li>Furthermore Besson et al., 2020 conducted a Chlorpyrifos (CPF) treatment 30 μg L−1 and observed a significant reduction (25%) in T4 levels at day 2 in CPF30 fish, as well as significantly reduced T3 levels in CPF30 fish (28%) compared with control fish. CPF30 fish also exhibited reduced densities of bipolar cell (10%) of retinal layer and CPF30 fish experienced lower survival.</li>
<li>Flamarique et al. (2013) showed that thyroid hormone treatment impacted the development of the visual system in rainbow trout and reduced the distances and angles at which prey were located (variables that are known indicators of foraging performance). Using optical measurements and photon-catch calculations, the study showed that control rainbow trouts perceived prey (Daphnia) with greater contrast compared to thyroid-hormone-treated fish. Reduced foraging performance is likely to reduce survival in the wild.</li>
<li>Heijlen et al. (2014) showed that knockdown of Type 3 Iodothyronine Deiodinase, known to disrupt eye development (Houbrechts et al. (2016), causes embryos to spend significantly less time moving, and perturbs the escape response after a tactile stimulus. An inability to escape predators likely reduces survival in the wild.</li>
</ul>
Uncertainties and Inconsistencies
It is obvious that impaired vision leads to higher mortality, as the sense of sight is important for survival, and if it is impaired, feeding or escape becomes more difficult. However, the number of studies investigating this connection is limited. It is often unclear to what extent this relationship is determined by altered visual function versus other pathways such as alterations in muscle development or other factors contributing to these types of behaviour. Also, the natural conditions, which depend on many variables, are difficult to reproduce in the laboratory or to compare between different laboratories.
<h4>Quantitative Understanding of the Linkage</h4>
Known modulating factors
Increase according to global health of the population (e.g on trout (Post and Parkinson, 2001)
<h4>References</h4>
Babkiewicz, E., Bazała, M., Urban, P., Maszczyk, P., Markowska, M., & Maciej Gliwicz, Z. (2020). The effects of temperature on the proxies of visual detection of Danio rerio larvae: observations from the optic tectum. Biology Open, 9(7). https://doi.org/10.1242/BIO.047779
Besson, M., Feeney, W. E., Moniz, I., François, L., Brooker, R. M., Holzer, G., Metian, M., Roux, N., Laudet, V., & Lecchini, D. (2020). Anthropogenic stressors impact fish sensory development and survival via thyroid disruption. Nature Communications, 11(1). https://doi.org/10.1038/s41467-020-17450-8
Brown, S. B., Adams, B. A., Cyr, D. G., & Eales, J. G. (2004). Contaminant effects on the teleost fish thyroid. Environmental Toxicology and Chemistry, 23(7), 1680–1701. https://doi.org/10.1897/03-242
Carvalho, P. S. M., Noltie, D. B., & Tillitt, D. E. (2002). Ontogenetic improvement of visual function in the medaka Oryzias latipes based on an optomotor testing system for larval and adult fish. Animal Behaviour, 64(1), 1–10. https://doi.org/10.1006/anbe.2002.3028
Corral-López, A., Garate-Olaizola, M., Buechel, S. D., Kolm, N., & Kotrschal, A. (2017). On the role of body size, brain size, and eye size in visual acuity. Behavioral Ecology and Sociobiology, 71(12). https://doi.org/10.1007/s00265-017-2408-z
Dehnert, G. K., Karasov, W. H., & Wolman, M. A. (2019). 2,4-Dichlorophenoxyacetic acid containing herbicide impairs essential visually guided behaviors of larval fish. Aquatic Toxicology, 209(October 2018), 1–12. https://doi.org/10.1016/j.aquatox.2019.01.015
Flamarique IN. 2013. Opsin switch reveals function of the ultraviolet cone in fish foraging. Proceedings of the Royal Society B-Biological Sciences 280(1752).
Fuiman LA, Rose KA, Cowan JH, Smith EP. 2006. Survival skills required for predator evasion by fish larvae and their relation to laboratory measures of performance. Animal Behaviour 71:1389-1399.
Heijlen M, Houbrechts A, Bagci E, Van Herck S, Kersseboom S, Esguerra C, Blust R, Visser T, Knapen D, Darras V. 2014. Knockdown of type 3 iodothyronine deiodinase severely perturbs both
Houbrechts AM, Delarue J, Gabriels IJ, Sourbron J, Darras VM. 2016. Permanent Deiodinase Type 2 Deficiency Strongly Perturbs Zebrafish Development, Growth, and Fertility. Endocrinology 157(9):3668-3681.
</div>
<div>
<h4><a href="/relationships/2013">Relationship: 2013: Increased Mortality leads to Decrease, Population growth rate</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/312">Acetylcholinesterase Inhibition leading to Acute Mortality via Impaired Coordination & Movement</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/16">Acetylcholinesterase inhibition leading to acute mortality</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/155">Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/156">Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/157">Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation </a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/158">Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/159">Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/363">Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/364">Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/365">Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/399">Inhibition of Fyna leading to increased mortality via decreased eye size (Microphthalmos)</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/410">GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/536">Estrogen receptor agonism leading to reduced survival and population growth due to renal failure</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/564">DBDPE-induced inhibition of mitochondrial complex Ⅰ leading to population decline via neurotoxicity and metabotoxicity.</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fathead minnow</td>
<td>Pimephales promelas</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic: All organisms must survive to reproductive age in order to reproduce and sustain populations. The additional considerations related to survival made above are applicable to other fish species in addition to zebrafish and fathead minnows with the same reproductive strategy (r-strategist as described in the theory of MaxArthur and Wilson (1967). The impact of reduced survival on population size is even greater for k-strategists that invest more energy in a lower number of offspring.
Life stage: Density dependent effects start to play a role in the larval stage of fish when free-feeding starts (Hazlerigg et al., 2014).
Sex: This linkage is independent of sex.
<h4>Key Event Relationship Description</h4>
Increased mortality in the reproductive population may lead to a declining population. This depends on the excess mortality due to the applied stressor and the environmental parameters such as food availability and predation rate. Most fish species are r-strategist, meaning they produce a lot of offspring instead of investing in parental care. This results in natural high larval mortality causing only a small percentage of the larvae to survive to maturity. If the excess larval mortality due to a stressor is small, the population dynamics might result in constant population size. Should the larval excess be more significant, or last on the long-term, this will affect the population. To calculate the long-term persistence of the population, population dynamic models should be used.
<h4>Evidence Supporting this KER</h4>
Survival rate is an obvious determinant of population size and is therefore included in population modeling (e.g., Miller et al., 2020).
Biological Plausibility
<ul>
<li>Survival to reproductive maturity is a parameter of demographic significance. Assuming resource availability (i.e., food, habitat, etc.) is not limiting to the extant population, sufficient mortality in the reproductive population may ultimately lead to declining population trajectories.</li>
<li>Under some conditions, reduced larval survival may be compensated by reduced predation and increased food availability, and therefore not result in population decline (Stige et al., 2019).</li>
</ul>
Empirical Evidence
<ul>
<li>According to empirical data, combined with population dynamic models, feeding larvae are the crucial life stage in zebrafish (and other r-strategists) for the regulation of the population. (Schäfers et al., 1993)</li>
<li>In fathead minnow, natural survival of early life stages has been found to be highly variable and influential on population growth (Miller and Ankley, 2004)</li>
<li>Rearick et al. (2018) used data from behavioural assays linked to survival trials and applied a modelling approach to quantify changes in antipredator escape performance of larval fathead minnows in order to predict changes in population abundance. This work was done in the context of exposure to an environmental oestrogen. Exposed fish had delayed response times and slower escape speeds, and were more susceptible to predation. Population modelling showed that this can result in population decline.</li>
<li>In the context of fishing and fisheries, ample evidence of a link between increased mortality and a decrease of population size has been given. Important insights can result from the investigation of optimum modes of fishing that allow for maintaining a population (Alekseeva and Rudenko, 2018). Jacobsen and Essington (2018) showed the impact of varying predation mortality on forage fish populations.</li>
<li>Boreman (1997) reviewed methods for comparing the population-level effects of mortality in fish populations induced by pollution or fishing.</li>
</ul>
Uncertainties and Inconsistencies
<ul>
<li>The extent to which larval mortality affects population size could depend on the fraction of surplus mortality compared to a natural situation.</li>
<li>There are scenarios in which individual mortality may not lead to declining population size. These include instances where populations are limited by the availability of habitat and food resources, which can be replenished through immigration. Effects of mortality in the larvae can be compensated by reduced competition for resources (Stige et al., 2019).</li>
<li>The direct impact of pesticides on migration behavior can be difficult to track in the field, and documentation of mortality during migration is likely underestimated (Eng 2017).</li>
</ul>
<h4>References</h4>
Alekseeva SM, Rudenko AI. 2018. Modeling of optimum fishing population. Marine Intellectual Technologies. 3(4):142-146.
Beaudouin, R., Goussen, B., Piccini, B., Augustine, S., Devillers, J., Brion, F., Pery, A.R., 2015. An individual-based model of zebrafish population dynamics accounting for energy dynamics. PloS one 10, e0125841.
Boreman J. 1997. Methods for comparing the impacts of pollution and fishing on fish populations. Transactions of the American Fisheries Society. 126(3):506-513.
Caswell, H., 2000. Matrix population models. Sinauer Sunderland, MA, USA.
Eng, M.L., Stutchbury, B.J.M. & Morrissey, C.A. Imidacloprid and chlorpyrifos insecticides impair migratory ability in a seed-eating songbird. Sci Rep 7, 15176 (2017)
Hazlerigg, C.R., Lorenzen, K., Thorbek, P., Wheeler, J.R., Tyler, C.R., 2012. Density-dependent processes in the life history of fishes: evidence from laboratory populations of zebrafish Danio rerio. PLoS One 7, e37550.
Jacobsen NS, Essington TE. 2018. Natural mortality augments population fluctuations of forage fish. Fish and Fisheries. 19(5):791-797.
MacArthur, R., Wilson, E., 1967. The Theory of Island Biogeography. Princeton: Princeton Univ. Press. 203 p.
Miller, D.H., Ankley, G.T., 2004. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17β-trenbolone as a case study. Ecotoxicology and Environmental Safety 59, 1-9.
Miller, D.H., Clark, B.W., Nacci, D.E. 2020. A multidimensional density dependent matrix population model for assessing risk of stressors to fish populations. Ecotoxicology and environmental safety 201, 110786
Pinceel, T., Vanschoenwinkel, B., Brendonck, L., Buschke, F., 2016. Modelling the sensitivity of life history traits to climate change in a temporary pool crustacean. Scientific reports 6, 29451.
Rearick, D.C., Ward, J., Venturelli, P., Schoenfuss, H., 2018. Environmental oestrogens cause predation-induced population decline in a freshwater fish. Royal Society open science 5, 181065.
Schäfers, C., Oertel, D., Nagel, R., 1993. Effects of 3, 4-dichloroaniline on fish populations with differing strategies of reproduction. In: Braunbeck, T. , Hanke, W and Segner, H. (eds) Ecotoxicology and Ecophysiology, VCH, Weinheim, 133-146.
Stige, L.C., Rogers, L.A., Neuheimer, A.B., Hunsicker, M.E., Yaragina, N.A., Ottersen, G., Ciannelli, L., Langangen, Ø., Durant, J.M., 2019. Density‐and size‐dependent mortality in fish early life stages. Fish and Fisheries 20, 962-976.Hazlerigg, C.R.E., Tyler, C.R., Lorenzen, K., Wheeler, J.R., Thorbek, P., 2014. Population relevance of toxicant mediated changes in sex ratio in fish: An assessment using an individual-based zebrafish (Danio rerio) model. Ecological Modelling 280, 76-88.
Stige, L.C., Rogers, L.A., Neuheimer, A.B., Hunsicker, M.E., Yaragina, N.A., Ottersen, G., Ciannelli, L., Langangen, O., Durant, J.M., 2019. Density- and size-dependent mortality in fish early life stages. Fish and Fisheries 20, 962-976.
</div>
<h3>List of Non Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/366">Relationship: 366: Thyroperoxidase, Inhibition leads to T4 in serum, Decreased</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/42">Inhibition of Thyroperoxidase and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>non-adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/159">Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>non-adjacent</td>
<td>High</td>
<td>Low</td>
</tr>
<tr>
<td><a href="/aops/175">Thyroperoxidase inhibition leading to altered amphibian metamorphosis</a></td>
<td>non-adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/363">Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>non-adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Xenopus laevis</td>
<td>Xenopus laevis</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>chicken</td>
<td>Gallus gallus</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>human</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fathead minnow</td>
<td>Pimephales promelas</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Amphibia</td>
<td>Amphibia</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8292" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fish</td>
<td>fish</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
<tr>
<td>Female</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic: Use of TPO inhibitors as anti-hyperthyroidism drugs in humans and pets (Emiliano et al., 2010; Trepanier, 2006) and effects of these drugs on serum TH concentrations in rats (US EPA, 2005), amphibian, fish and avian species (Coady et al., 2010; Grommen et al., 2011; Nelson et al., 2016; Rosebrough et al., 2006; Stinckens et al.; 2020; Tietge et al., 2012), strongly supports a causative linkage between inhibition of TPO and decreased serum T4 across species. Therefore, this KER is plausibly applicable across vertebrate species. Therefore, this KER is plausibly applicable across vertebrates.
Taxonomic: Use of TPO inhibitors as anti-hyperthyroidism drugs in humans and pets (Emiliano et al., 2010; Trepanier, 2006) and effects of these drugs on serum TH concentrations in rats (US EPA, 2005), amphibian, fish and avian species (Coady et al., 2010; Grommen et al., 2011; Nelson et al., 2016; Rosebrough et al., 2006; Stinckens et al.; 2020; Tietge et al., 2012), strongly supports a causative linkage between inhibition of TPO and decreased serum T4 across species. Therefore, this KER is plausibly applicable across vertebrate species. 
According to the evaluation of the empirical taxonomic domain of applicability (tDOA) of an adverse outcome pathway network for thyroid hormone system disruption (THSD) by Haigis et al., 2023, the level of confidence for a linkage between TPO inhibition and reduced thyroid hormone (TH) levels was considered high for mammals and fish (Dong et al., 2020, Fang et al., 2022, Hassan et al., 2020, Hornung et al., 2015, Jomaa et al., 2015, Nelson et al., 2016, O’Shaughnessy et al., 2018, Paul et al., 2013, Raldua and Babin, 2009, Ramhøj et al., 2021, 2022, Schmutzler et al., 2007, Stinckens et al., 2016, 2020, Thienpont et al., 2011, Tietge et al., 2013, Vickers et al., 2012, Wu et al., 2016) and moderate for amphibians (Haselman et al., 2020, Tietge et al., 2010, 2013). This was supported by structural protein conservation analysis by Lalone et al., 2018 and Haigis et al., 2023. Structural protein conservation of mammalian, fish, amphibian, reptilian and avian TPO was found compared to the human (Homo sapiens) protein target using the U.S. Environmental Protection Agency’s Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS v6.0; seqapass.epa.gov/seqapass/) tool, while acknowledging the potential existence of interspecies differences in conservation. No empirical evidence linking TPO inhibition to THSD was found for reptiles and birds.
Life stage: Applicability to certain life stages may depend on the species and their dependence on maternally transferred thyroid hormones during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). Therefore, iIt is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH system disruptors.
Sex: The KE is plausibly applicable to both sexes. THs are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of TH levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in TH levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.
<h4>Key Event Relationship Description</h4>
Thyroperoxidase (TPO) is the enzyme that catalyzes iodine organification of thyroglobulin to produce thyroglobulin (Tg)-bound T3 and T4 in the lumen of thyroid follicles. Tg-bound THs are endocytosed across the apical lumen-follicular cell membrane, undergo thyroglobulin proteolysis, followed by hormone section into the blood stream (see Taurog, 2005 for review). This indirect KER describes the relationship of TPO inhibition to reduced circulating levels of thyroid hormone (TH) in the serum.
<h4>Evidence Supporting this KER</h4>
The weight of evidence linking thyroperoxidase inhibition to reductions in circulating serum TH is strong. Many studies support this basic linkage. There is no inconsistent data.
Biological Plausibility
It is a well-accepted fact that inhibition of the only enzyme capable of synthesizing THs, TPO, results in subsequent decrease in serum TH concentrations.  A large amount of evidence from clinical and animal studies clearly support the commonly accepted dogma that inhibition of TPO leads to decreased serum THs.
Empirical Evidence
The majority of research in support of this KER involves exposure to known TPO inhibitors and measurement of serum hormones. There are many in vivo studies that link decreases in serum TH concentrations with exposure to xenobiotics that inhibit thyroperoxidase (TPO) in mammals (Brucker-Davis, 1998; Hurley, 1998; Boas et al., 2006; Crofton, 2008; Kohrle, 2008; Pearce and Braverman, 2009; Murk et al., 2013).
While these studies support the connection between exposure to a known TPO inhibitor and decreased TH, many of these studies do not empirically measure TPO inhibition or decreased TH synthesis. Thus, many studies support the indirect linkage between TPO inhibition (for chemicals identified as TPO inhibitors in in vivo or ex vivo studies) and decreased TH, with the well accepted theory that these proceed via decreased TH synthesis. That exposure to TPO inhibitors leads to decreased serum TH concentrations, via decreased TH synthesis is strongly supported by decades of mechanistic research in a variety of species.
This indirect relationship is also evidenced by the use of clinically-relevant anti-hyperthyroidism drugs, MMI and PTU (Laurberg & Anderson, 2014; Sundaresh et al., 2013). These drugs are both recognized TPO inhibitors and are part of a standard drug-based regimen of care for clinically hyperthyroid patients including those with Grave's disease. Serum THs are measured as the bioindicator of successful treatment with anti-hyperthyroidism drugs; the actual decrease in TH synthesis in the thyroid gland is implied in the efficacious use of these drugs (Trepanier, 2006).
In rats, MMI and PTU are often used as control chemicals to decrease serum THs to study biological phenomena related to disruption of TH homeostasis (many examples, including Zoeller and Crofton, 2005; Morreale de Escobar et al, 2004; Schwartz et al., 1997; Herwig et al., 2014; Wu et al., 2013; Pathak et al., 2011). Further, MMI is recommended as a positive control for use in the Amphibian Metamorphosis (Frog) Assay within Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program (US EPA, 2009; Coady et al., 2010), an assay used to evaluate the potential for chemicals to disrupt TH homeostasis. PTU has been suggested as positive control chemical in the guidance for the Comparative Developmental Thyroid Assay (US EPA, 2005), a non-guideline assay used to evaluate the potential for chemicals to disrupt TH homeostasis during gestation and early neonatal development.
Additionally, evidence is available from studies investigating responses to TPO inhibitors in fish. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.
Thus, an indirect key event relationship between TPO inhibition and decreased serum THs is strongly supported by a large database of clinical medicine and investigative research with whole animals (with a great deal of supporting evidence in rats and frogs).
Temporal Evidence: In mammals, the temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005). The qualitative impact of TPO inhibition on serum hormones is similar across all ages in mammals. The temporal nature of the impact on serum THs by TPO inhibitors in developmental exposure studies is evidenced by the duration of exposure and developmental age (Goldey et al., 1995; Ahmed et al., 2010; Tietge et al., 2010), as well as recovery after cessation of exposure (Cooke et al., 1993; Goldey et al., 1995; Sawin et al., 1998; Axelstad et al., 2008; Shibutani et al., 2009; Lasley and Gilbert, 2011). The temporal relationship between TPO inhibitor exposure duration and serum hormone decreases in adult organisms has been widely demonstrated (e.g., Hood et al., 1999; Mannisto et al., 1979). In addition, MMI and PTU induced decreases in serum T4 are alleviated by TH      replacement in both fetal and postnatal age rats (Calvo et al., 1990; Sack et al., 1995; Goldey and Crofton, 1998). Computational modeling of the thyroid also provides evidence for the indirect temporal relationship between these two KEs (e.g., Degon et al., 2008; Fisher et a., 2013).
In oviparous fish such as zebrafish and fathead minnow, the nature of this KER depends on the life stage since the earliest stages of embryonic development rely on maternal thyroid hormones transferred to the eggs. Embryonic thyroid hormone synthesis is activated later during embryo-larval development. (See Domain of applicability)
Dose-Response Evidence: Empirical data is available from enough studies in animals treated with TPO inhibitors during development to make it readily accepted dogma that a dose-response relationship exists between TPO inhibition and serum TH concentrations.  Again, these studies do not empirically measure TPO inhibition or decreased TH synthesis, but rely on the strong support of decades of mechanistic research in a variety of species of the causative relationship between these KEs.  Examples of dose-responsive changes in TH concentrations following developmental exposure to TPO inhibitors include studies a variety of species, including: rodents (Blake and Henning, 1985; Goldey et al., 1995; Sawin et al., 1998); frogs (Tietge et al., 2013); fish tissue levels (Elsalini and Rohr, 2003.); and, chickens (Wishe et al., 1979). Computational modeling of the thyroid also provides evidence for the indirect dose-response relationship between these two KEs (e.g., Leonard et al., 2016; Fisher et a., 2013).
Uncertainties and Inconsistencies
There are no inconsistencies in this KER, but there are some uncertainties. The predominant uncertainty regarding the indirect key event relationship between inhibition of TPO activity and decreased serum T4 is the quantitative nature of this relationship, i.e., to what degree must TPO be inhibited in order to decrease serum T4 by a certain magnitude. Many animal (rat) studies typically employ relatively high exposures of TPO-inhibiting chemicals that result in hypothyroidism (severe decrements in T4 and T3). Thus, a dose-response relationship between TPO inhibition and decreased serum T4 is not typically defined. However, there are numerous publications demonstrating clear dose- and duration- dependent relationships between TPO inhibitors dose and reduced serum T3 and T4 in rodent models (see for example: Cooper et al., 1983; Hood et al., 1999; Goldey et al., 2005; Gilbert, 2011). The relationship between maternal and fetal levels of hormone following chemically-induced TPO inhibiton has not been well characterized and may differ based on kinetics. Reductions in serum TH in the fetus, in rats and humans is derived from a chemical's effect on the maternal thyroid gland as well as the fetal thyroid gland.
<h4>Quantitative Understanding of the Linkage</h4>
Hassan et al. (2017, 2020) and Handa et al. (2021) worked out a quantitative model to predict serum T4 levels based on TPO inhibition in rats.
Response-response relationship
The indirect linkage between exposure to known TPO inhibitors and decreased serum TH has not been defined quantitatively. The two key event relationships that mediate this relationship (TPO inhibition leading to decreased TH synthesis, and decreased TH synthesis leading to decreased serum TH) have been incorporated into some quantitative models. A quantitative biologically-based dose-response model for iodine deficiency in the rat includes relationships between thyroidal T4 synthesis and serum T4 concentrations in developing rats Fisher et al. (2013). Ekerot et al. (2012) modeled TPO, T3, T4 and TSH in dogs and humans based on exposure to myeloperoxidase inhibitors that also inhibit TPO and was recently adapted for rat (Leonard et al., 2016). While the original model predicted serum TH and TSH levels as a function of oral dose, it was not used to explicitly predict the relationship between serum hormones and TPO inhibition, or TH synthesis. Leonard et al. (2016) recently incorporated TPO inhibition into the model. Degon et al (2008) developed a human thyroid model that includes TPO but does not make quantitative prediction of organification changes due to inhibition of the TPO enzyme.
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~~ ~~
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