AOP ID and Title:

Short Title: TPOi retinal layer structure

Authors

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

Status

Abstract

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.

Background

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.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Overall Assessment of the AOP

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

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).

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.

Essentiality of the Key Events

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.

 

Weight of Evidence Summary

1. Biological plausibility:

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. 

2. Empirical support is moderate for most KERs in the AOP and low for the most upstream KERs.
3. 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.

Quantitative Consideration

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.  

Considerations for Potential Applications of the AOP (optional)

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.

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Appendix 1

List of MIEs in this AOP

Event: 279: Thyroperoxidase, Inhibition

Short Name: Thyroperoxidase, Inhibition

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

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).

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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.

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.

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.

Key Event Description

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.

Figure 1      illustrates the enzymatic and nonenzymatic reactions mediated by TPO that result in the synthesis of thyroxine (T4) .

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).

The ontogeny of TPO has been determined using both direct and indirect evidence in mammals.  Available evidence suggests the 11^th to 12^th 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 5^th 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).

How it is Measured or Detected

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).

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).

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List of Key Events in the AOP

Event: 277: Thyroid hormone synthesis, Decreased

Short Name: TH synthesis, Decreased

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Evidence for Perturbation by Stressor

Propylthiouracil

6-n-proylthiouracil is a common positive control for inhibition of TPO

Methimazole

Methimazole is a very common positve control for inhibition of TPO

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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).

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.

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.

 

Key Event Description

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 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.

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.

How it is Measured or Detected

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).

References

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.

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.

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.

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Event: 281: Thyroxine (T4) in serum, Decreased

Short Name: T4 in serum, Decreased

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Organ term

Evidence for Perturbation by Stressor

Propylthiouracil

6-n-propylthouracil is a classic positive control for inhibition of TPO

Methimazole

Methimazole is a classic positive control for inhibition of TPO.

Perchlorate

Perchlorate ion (ClO− ₄) is a classic positive control for inhibition of NIS

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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.

Key Event Description

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). 

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.

How it is Measured or Detected

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).

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.

References

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Event: 1003: Decreased, Triiodothyronine (T3)

Short Name: Decreased, Triiodothyronine (T3)

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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.

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.

 

Key Event Description

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.

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.

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).

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.    

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 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).

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).

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).

How it is Measured or Detected

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).

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.

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).

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Event: 1877: Altered, retinal layer structure

Short Name: Altered, retinal layer structure

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Organ term

Evidence for Perturbation by Stressor

Propylthiouracil

6-n-propylthouracil is a classic positive control for inhibition of TPO.

Methimazole

Methimazole is a classic positive control for inhibition of TPO.

Perchlorate

Perchlorate ion (ClO− ₄) is a classic positive control for inhibition of NIS.

 

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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.

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).

 

Key Event Description

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).

 

How it is Measured or Detected

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)

References

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. https://doi.org/10.1002/cne.21164

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. https://doi.org/10.1016/j.aquatox.2015.12.015

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. https://doi.org/10.3390/ijms20071543

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. https://doi.org/10.1167/iovs.18-24461

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. https://doi.org/10.1002/dvdy.22392

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. https://doi.org/10.1371/journal.pone.0040132

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. https://doi.org/10.1242/jeb.030866

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. https://doi.org/10.1002/bdrb.21118

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.https://doi.org/10.1073/pnas.1303551110

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.

 

Event: 1643: Altered, Visual function

Short Name: Altered, Visual function

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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).

 

Key Event Description

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).

 

How it is Measured or Detected

Measurements of visual function can be performed at the level of neuronal activity:

• Electroretinography (Chrispell et al., 2015)
• 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).
• 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.

Other measurements are performed at the level of the eyes:

• 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.      

Yet other studies use assessment of vision-related behaviours: 

• 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)).
• Light-dark transition or vision startle response: reaction to change in light intensity (light sensitivity) (Brastrom et al., 2019)
• Black-white preference test (Baumann et al., 2016)

• 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).

References

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

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List of Adverse Outcomes in this AOP

Event: 351: Increased Mortality

Short Name: Increased Mortality

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

All living things are susceptible to mortality.

Key Event Description

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.

How it is Measured or Detected

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:

• in the lab by recording mortality during exposure experiments
• in dedicated setups simulating a realistic situation such as mesocosms or drainable ponds for aquatic species
• 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.

Regulatory Significance of the AO

Increased mortality is one of the most common regulatory assessment endpoints, along with reduced growth and reduced reproduction.

Event: 360: Decrease, Population growth rate

Short Name: Decrease, Population growth rate

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Consideration of population size and changes in population size over time is potentially relevant to all living organisms.

Key Event Description

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.

 

How it is Measured or Detected

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, N_t=0 (population size at time t=0), and the population size at the end of the interval, N_t=1 (population size at time t = 1), and then subsequently dividing by the initial population size. 

Equation 2:  r = (N_t=1 - N_t=0) / N_t=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).

 

Regulatory Significance of the AO

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.

References

• 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.
• Bookhout TA. 1994. Research and management techniques for wildlife and habitats. The Wildlife Society, Bethesda, Maryland. 740 pp.
• Caswell H. 2001. Matrix Population Models. Sinauer Associates, Inc., Sunderland, MA, USA
• 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.
• 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.
• Etterson MA, Ankley GT.  2021.  Endogenous Lifecycle Models for Chemical Risk Assessment. Environ. Sci. Technol. 55:  15596-15608. 
• Gotelli NJ, 2008. A Primer of Ecology. Sinauer Associates, Inc., Sunderland, MA, USA.
• 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.
• 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.
• 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. 
• 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.
• 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.
• 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.
• 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.
• 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.
• Murray DL, Sandercock BK (editors).  2020.  Population ecology in practice.  Wiley-Blackwell, Oxford UK, 448 pp.
• 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.
• Nisbet RM, Muller EB, Lika K, Kooijman SALM. 2000. From molecules to ecosystems through dynamic energy budgets. J Anim Ecol 69:  913–926.
• 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. 
• Vandermeer JH, Goldberg DE. 2003.  Population ecology: first principles.  Princeton University Press, Princeton NJ, 304 pp.
• 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
• 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.

Appendix 2

List of Key Event Relationships in the AOP