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 thyroperoxidase and resulting changes in hormone 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) in serum"), 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 reponse, 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 eyes.

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 developemnt 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: "fish", "eye development", "retina", "thyroid/hormone disorders", "visual behaviour", "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 these structured studies were analysed in terms of experimental design and information on different biological levels. Most of the data is on 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, 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) in serum decreased) is plausibly applicable across vertebrates, too, variation can be expecteddue to feedback/compensatory mechanisms that can also differ across species. In zebrafish and fathead minnow, several studies reported the evidence for a relationship between circulating T4 and T3 levels (Nelson et al., 2016; Stinckens et al., 2020, Wang et al., 2020).

The linkage between the MIE, decreased T3 in serum, and the KE of altered retinal layer structure, is evidental 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 of RPE, photoreceptors, neurons and choroid. It is plausible to assume that TH levels are important for healthy eye development in all vertebrates.

Thyroid hormone 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. 2021)). 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 embryolarval life stage. In order to more specifically evaluate the life stage applicability of the impact on thyroperoxidase 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.

In zebrafish, 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 continues until well beyond 84 hpf (Raymond and others 1995). The first thyroid follicle appears around 55 hpf and endogenous T4 production has been observed at 72 hpf (Walter and others 2019). Since thyroperoxidase 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 thyroperoxidase 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 thyroperoxidase inhibition in zebrafish and fathead minnow (Nelson and others 2016; Stinckens and others 2016). Thyroperoxidase expression has however additionally 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.

In summary, in zebrafish the formation of the retinal layers occurs before the activation of thyroidal TH synthesis but further differentiation and maturation continues until well after the onset of thyroidal TH synthesis. There is ample evidence of the impact of thyroperoxidase inhibition on retinal layer structure at the age of 5 dpf, and there is some evidence showing early effects at 48, 66 and 72 hpf (Komoike and others 2013; Reider and Connaughton 2014) suggesting the importance of local TH synthesis in the eyes. Additional mechanisms (e.g., deiodinase inhibition) could also play a role. Currently there is insufficient evidence to clearly evaluate the importance of inhibition of local TPO in the eyes versus thyroidal TPO for the development of proper retinal layer structure.

Mammals on the other hand continuously receive maternal THs via the placenta. 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 (REF).

Taken together, there is strong support for applicability of the current AOP to embryolarval/embryofoetal stages of vertebrates. Since the term 'eleutheroembryo' (stage starting at hatching and ending with free-feeding fish) is not available, 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[LB1]  (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 where 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)). Furthermore, disrupted retinal layers are often observed using a semi-quantitative classification system rather than quantitative measurements.

However, the combinations of some studies show some correlations: 
For example, the study by Rehberger et al. (2018) shows a tendency for T3 and T4 to decrease with increasing PTU concentration, Baumann et al. (2016) found both a disturbed retina and behavioural abnormalities due to impaired visual performance in larvae at these concentrations (and at much higher concentrations). Baumann et al. (2016) also shows a correlation between increased TPO expression (measured as a fold change) and decreased RPE diameter with increasing PTU exposure.

There are 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 concentration 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.

References

 

Antonica, F., Kasprzyk, D. F., Opitz, R., Iacovino, M., Liao, X. H., Dumitrescu, A. M., Refetoff, S., Peremans, K., Manto, M., Kyba, M., & Costagliola, S. (2012). Generation of functional thyroid from embryonic stem cells. Nature, 491(7422), 66–71. https://doi.org/10.1038/nature11525

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

Crofton, K. M., Kodavanti, P. R. S., Derr-Yellin, E. C., Casey, A. C., & Kehn, L. S. (2000). PCBs, thyroid hormones, and ototoxicity in rats: Cross-fostering experiments demonstrate the impact of postnatal lactation exposure. Toxicological Sciences, 57(1), 131–140. https://doi.org/10.1093/toxsci/57.1.131

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.

Li Z, Ptak D, Zhang L, Walls EK, Zhong W, Leung YF. 2012. Phenylthiourea Specifically Reduces Zebrafish Eye Size. Plos One 7(6).

Maack G, Segner H. 2003. Morphological development of the gonads in zebrafish. Journal of Fish Biology 62(4):895-906.

Morris AC, Fadool JM. 2005. Studying rod photoreceptor development in zebrafish. Physiology & Behavior 86(3):306-313.

Opitz, R., Antonica, F., & Costagliola, S. (2013). New Model Systems to Illuminate Thyroid Organogenesis. Part I: An Update on the Zebrafish Toolbox. European Thyroid Journal, 2(4), 229–242. https://doi.org/10.1159/000357079

Paul, K. B., Hedge, J. M., Macherla, C., Filer, D. L., Burgess, E., Simmons, S. O., Crofton, K. M., & Hornung, M. W. (2013). Cross-species analysis of thyroperoxidase inhibition by xenobiotics demonstrates conservation of response between pig and rat. Toxicology, 312(1), 97–107. https://doi.org/10.1016/j.tox.2013.08.006

Taurog, A. (1999). Molecular evolution of thyroid peroxidase. Biochimie, 81(5), 557–562. https://doi.org/10.1016/S0300-9084(99)80110-2

Vickers, A. E. M., Heale, J., Sinclair, J. R., Morris, S., Rowe, J. M., & Fisher, R. L. (2012). Thyroid organotypic rat and human cultures used to investigate drug effects on thyroid function, hormone synthesis and release pathways. Toxicology and Applied Pharmacology, 260(1), 81–88. https://doi.org/10.1016/j.taap.2012.01.029

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.

Racheva K, Totev T, Natchev E, Bocheva N, Beirne R, Zlatkova M. 2020. Color discrimination assessment in patients with hypothyroidism using the farnsworth-munsell 100 hue test. Journal of the Optical Society of America a-Optics Image Science and Vision. 37(4):A18-A25.

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 VP. 2014. Effects of Low-Dose Embryonic Thyroid Disruption and Rearing Temperature on the Development of the Eye and Retina in Zebrafish. Birth Defects Research Part B-Developmental and Reproductive Toxicology 101(5):347-354.

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.

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.

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.

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 thyroid 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 rat compared to human (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. Similary, 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 thyroid hormones during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf 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 thyroid hormone 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 thyroid hormone synthesis, including thyroperoxidase, are identical in both sexes. Therefore inhibition of thyroperoxidase 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 and 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 inhibiton 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 below 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, TH 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 thyroid hormone system is highly conserved across vertebrates, there are some taxon-specific considerations.

Zebrafish and fathead minnows are oviparous fish species in which maternal thyroid hormones are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic thyroid hormone synthesis is initiated. Maternal transfer of thyroid hormones 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 thyroperoxidase can only occur after activation of embryonic TH synthesis mediated by thyroperoxidase. Endogenous transcription profiles of thyroid-related genes in zebrafish and fathead minnow showed that mRNA coding for thyroid peroxidase 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 thyroid hormone synthesis in vitro.  

In fish, increases of TPO mRNA levels are often used as indirect evidence of TPO inhibition in in vivo experiments (Baumann et al., 2016; Nelson et al., 2016; Wang et al., 2020).

References

Baumann L, Ros A, Rehberger K, Neuhauss SCF, Segner H. 2016. Thyroid disruption in zebrafish (danio rerio) larvae: Different molecular response patterns lead to impaired eye development and visual functions. Aquatic Toxicology. 172:44-55.

Brown RS, Shalhoub V, Coulter S, Alex S, Joris I, De Vito W, Lian J, Stein GS.  Developmental regulation of thyrotropin receptor gene expression in the fetal and neonatal rat thyroid: relation to thyroid morphology and to thyroid-specific gene expression.  Endocrinology. 2000 Jan;141(1):340-5.

Brucker-Davis F. 1998. Effects of environmental synthetic chemicals on thyroid function. Thyroid 8:827-856.

Campinho MA, Saraiva J, Florindo C, Power DM. 2014. Maternal thyroid hormones are essential for neural development in zebrafish. Molecular Endocrinology. 28(7):1136-1149.

Chang, H. C. and D. R. Doerge (2000) Dietary genistein inactivates rat thyroid peroxidase in vivo without an apparent hypothyroid effect. Toxicol Appl Pharmacol. 168:244-252.

Chang J, Wang M, Gui W, Zhao Y, Yu L, Zhu G. 2012. Changes in thyroid hormone levels during zebrafish development. Zoological Science. 29(3):181-184.

Crane HM, Pickford DB, Hutchinson TH, Brown JA. 2004. Developmental changes of thyroid hormones in the fathead minnow, pimephales promelas. General and Comparative Endocrinology. 139(1):55-60.

Divi, R. L., & Doerge, D. R. (1994). Mechanism-based inactivation of lactoperoxidase and thyroid peroxidase by resorcinol derivatives. Biochemistry 33(32), 9668–9674.

Divi, R. L., Chang, H. C., & Doerge, D. R. (1997). Anti-Thyroid Isoflavones from Soybean. Biochem. Pharmacol.  54(10), 1087–1096.

Doerge DR, Chang HC. Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo. J Chromatogr B Analy Technol Biomed Life Sci. 2002 Sep 25;777(1-2):269-79.

Ealey PA, Henderson B, Loveridge N.A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland. Histochem J. 1984 Feb;16(2):111-22.

Fukiishi Y, Harauchi T, Yoshizaki T, Hasegawa Y, Eguchi Y.  Ontogeny of thyroid peroxidase activity in perinatal rats. Acta Endocrinol (Copenh). 1982 101(3):397-402.

Holzer G, Morishita Y, Fini JB, Lorin T, Gillet B, Hughes S, Tohmé M, Deléage G, Demeneix B, Arvan P, Laudet V. Thyroglobulin Represents a Novel Molecular Architecture of Vertebrates. J Biol Chem. 2016 Jun 16.

Hornung, M. W., Degitz, S. J., Korte, L. M., Olson, J. M., Kosian, P. a, Linnum, A. L., & Tietge, J. E. (2010). Inhibition of thyroid hormone release from cultured amphibian thyroid glands by methimazole, 6-propylthiouracil, and perchlorate. Toxicol Sci 118(1), 42–51.

Hurley PM. 1998. Mode of carcinogenic action of pesticides inducing thyroid follicular cell tumors in rodents. Environ Health Perspect 106:437-445.

Kaczur, V., Vereb, G., Molnár, I., Krajczár, G., Kiss, E., Farid, N. R., & Balázs, C. (1997). Effect of anti-thyroid peroxidase (TPO) antibodies on TPO activity measured by chemiluminescence assay. Clin. Chem 43(8 Pt 1), 1392–6.

Kessler, J., Obinger, C., Eales, G., 2008. Factors influencing the study of peroxidase- generated iodine species and implications for thyroglobulin synthesis. Thyroid 18, 769–774.

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.

OECD (2017) New Scoping Document on in vitro and ex vivo Assays for the Identification of Modulators of Thyroid Hormone Signalling. Series on Testing and Assessment. No. 207.  ISSN: 20777876 (online) http://dx.doi.org/10.1787/20777876

Opitz R, Maquet E, Zoenen M, Dadhich R, Costagliola S. 2011. Tsh receptor function is required for normal thyroid differentiation in zebrafish. Molecular Endocrinology. 25(9):1579-1599.

Paul KB, Hedge JM, Macherla C, Filer DL, Burgess E, Simmons SO, Crofton KM, Hornung MW. Cross-species analysis of thyroperoxidase inhibition by xenobiotics demonstrates conservation of response between pig and rat. Toxicology. 2013. 312:97-107

Paul, K.B., Hedge, J.M., Rotroff, D.M., Hornung, M.W., Crofton, K.M., Simmons, S.O. 2014. Development of a thyroperoxidase inhibition assay for high-throughput screening. Chem.  Res. Toxicol. 27(3), 387-399.

Paul-Friedman K, Watt ED, Hornung MW, Hedge JM, Judson RS, Crofton KM, Houck KA, Simmons SO. 2016. Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors Within the ToxCast Phase I and II Chemical Libraries.  Toxicol Sci. 151:160-80.

Pintar, J.E. (2000) Normal development of the hypothalamic-pituitary-thyroid axis. In. Werner & Ingbar’s The Thyroid. (8^th ed), Braverman. L.E. and Utiger, R.D. (eds) Lippincott Williams and Wilkins, Philadelphia.

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.

Remy L, Michel-Bechet M, Athouel-Haon AM, Magre S. Critical study of endogenous peroxidase activity: its role in the morphofunctional setting of the thyroid follicle in the rat fetus. Acta Histochem. 1980;67(2):159-72.

Ruf, J., & Carayon, P. (2006). Structural and functional aspects of thyroid peroxidase. Archives of Biochemistry and Biophysics, 445(2), 269–77.

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.

Schmutzler, C., Bacinski, A., Gotthardt, I., Huhne, K., Ambrugger, P., Klammer, H., Schlecht, C., Hoang-Vu, C., Gruters, A., Wuttke, W., Jarry, H., Kohrle, J., 2007a. The ultraviolet filter benzophenone 2 interferes with the thyroid hormone axis in rats and is a potent in vitro inhibitor of human recombinant thyroid peroxidase. Endocrinology 148, 2835–2844.

Stinckens E, Vergauwen L, Blackwell BR, Anldey GT, Villeneuve DL, Knapen D. 2020. Effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology. 54(10):6213-6223.

Stinckens E, Vergauwen L, Schroeder A, Maho W, Blackwell B, Witters H, Blust R, Ankley G, Covaci A, Villeneuve D et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part ii: Zebrafish. Aquatic Toxicology. 173:204-217.

Taurog A. 2005. Hormone synthesis. In: Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text (Braverman LE, Utiger RD, eds). Philadelphia:Lippincott, Williams and Wilkins, 47–81

Taurog, a, Dorris, M. L., & Doerge, D. R. (1996). Mechanism of simultaneous iodination and coupling catalyzed by thyroid peroxidase. Archives of Biochemistry and Biophysics, Taurog A. Molecular evolution of thyroid peroxidase. Biochimie. 1999 May;81(5):557-62

Takayama S, Aihara K, Onodera T, Akimoto T. Antithyroid effects of propylthiouracil and sulfamonomethoxine in rats and monkeys. Toxicol Appl Pharmacol. 1986 Feb;82(2):191-9.

Thorne N, Auld DS, Inglese J.  Apparent activity in high-throughput screening: origins of compound-dependent assay interference. Curr Opin Chem Biol. 2010 Jun;14(3):315-24.

Thorpe-Beeston JG, Nicolaides KH, McGregor AM. Fetal thyroid function. Thyroid. 1992 Fall;2(3):207-17. Review.

Tietge JE, Degitz SJ, Haselman JT, Butterworth BC, Korte JJ, Kosian PA, Lindberg-Livingston AJ, Burgess EM, Blackshear PE, Hornung MW. 2013. Inhibition of the thyroid hormone pathway in xenopus laevis by 2-mercaptobenzothiazole. Aquatic Toxicology. 126:128-136.

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.

Vickers AE, Heale J, Sinclair JR, Morris S, Rowe JM, Fisher RL. Thyroid organotypic rat and human cultures used to investigate drug effects on thyroid function, hormone synthesis and release pathways. Toxicol Appl Pharmacol. 2012 Apr 1;260(1):81-8.

Wabukebunoti MAN, Firling CE. 1983. The prehatching development of the thyroid-gland of the fathead minnow, pimephales-promelas (rafinesque). General and Comparative Endocrinology. 49(2):320-331.

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.

Wang JX, Shi GH, Yao JZ, Sheng N, Cui RN, Su ZB, Guo Y, Dai JY. 2020. Perfluoropolyether carboxylic acids (novel alternatives to pfoa) impair zebrafish posterior swim bladder development via thyroid hormone disruption. Environment International. 134.

Zoeller, R. T., Tan, S. W., & Tyl, R. W. (2007). General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical Reviews in Toxicology, 37(1-2), 11–53.

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 specis, 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 medical 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 thyroid hormones 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 thyroid hormone 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 thyroid hormone production is initiated in zebrafish.

Sex: The KE is plausibly applicable to both sexes. Thyroid hormones are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of thyroid hormone levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in thyroid hormone levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.

 

Key Event Description

The thyroid hormones (TH), triiodothyronine (T3) and thyroxine (T4) are thyrosine based hormones. Synthesis of TH 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 concentration 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 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 concentations rise continuously until birth. At term, fetal T4 is similar to maternal levels, but T3 remains 2-3 fold lower than maternal levels.  In rats, THs can be detected in the fetus as early as the second gestational week, but fetal synthesis does not start until gestational day 17 with birth at gestational day 22-23. Maternal THs continue to supplement fetal production until parturition. (see Howdeshell, 2002; Santisteban and Bernal, 2005 for review). The ontogeny of TPO inhibition during development by environmental chemicals is 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 thyroid 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 lifestages store different amounts of TH precursor 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 thyroid hormone system is highly conserved across vertebrates, there are some taxon-specific considerations.

Zebrafish and fathead minnows are oviparous fish species in which maternal thyroid hormones are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic thyroid hormone synthesis is initiated. Maternal transfer of thyroid hormones 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 thyroid hormone 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 radiolabel 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 thyroid hormone 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 inbibitors), 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 thyroid hormone level measurements in fish early life stages are used as indirect evidence of decreased thyroid hormone synthesis (Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020). Analytical determination of thyroid hormone 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 thyroid hormone synthesis (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.

Zoeller RT.  Interspecies differences in susceptibility to perturbation of thyroid hormone homeostasis requires a definition of "sensitivity" that is informative for risk analysis. Regul Toxicol Pharmacol. 2004 Dec;40(3):380.

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 existence and importance has also been described in many different animal and plant kingdoms (Eales, 1997; Heyland and Moroz, 2005), while 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 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 thyroid hormone 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 disruptors.

Sex: The KE is plausibly applicable to both sexes. Thyroid hormones are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of thyroid hormone levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in thyroid hormone levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.

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 thyroid hormones 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 thyroid hormone 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 thyroid hormone synthesis comes from measurements of whole body thyroid hormone levels using LC-MS techniques (Hornung et al., 2015) are increasingly used to accurately quantify whole body thyroid hormone levels as a proxy for serum thyroid hormone levels (Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020). It is important to note that thyroid hormones concentrations can be influenced by a number of intrinsic and extrinsic factors (e.g., circadian rhythms, stress, food intake, housing, noise) (see for example, Döhler et al., 1979).

Any of these measurements should be evaluated for the relationship to the actual endpoint of interest, repeatability, reproducibility, and lower limits of quantification using a fit-for-purpose approach (i.e., different regulatory needs will require different levels of confidence in the AOP). This is of particular significance when assessing the very low levels of TH present in fetal serum. Detection limits of the assay must be compatible with the levels in the biological sample.  All three of the methods summarized above would be fit-for-purpose, depending on the number of samples to be evaluated and the associated costs of each method. Both RIA and ELISA measure THs by an indirect methodology, whereas analytical determination is the most direct measurement available. All these methods, particularly RIA, are repeatable and reproducible.

 

References

Axelrad DA, Baetcke K, Dockins C, Griffiths CW, Hill RN, Murphy PA, Owens N, Simon NB, Teuschler LK. Risk assessment for benefits analysis: framework for analysis of a thyroid-disrupting chemical. J Toxicol Environ Health A. 2005 68(11-12):837-55.

Baret A. and Fert V.  T4 and ultrasensitive TSH immunoassays using luminescent enhanced xanthine oxidase assay. J Biolumin Chemilumin. 1989. 4(1):149-153

Bartalena L, Robbins J. Thyroid hormone transport proteins. Clin Lab Med. 1993 Sep;13(3):583-98. Bassett JH, Harvey CB, Williams GR. (2003). Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol Cell Endocrinol. 213:1-11.

Capen CC. Mechanistic data and risk assessment of selected toxic end points of the thyroid gland. Toxicol Pathol. 1997 25(1):39-48.

Carr JA, Patino R. 2011. The hypothalamus-pituitary-thyroid axis in teleosts and amphibians: Endocrine disruption and its consequences to natural populations. General and Comparative Endocrinology. 170(2):299-312.

Chang J, Wang M, Gui W, Zhao Y, Yu L, Zhu G. 2012. Changes in thyroid hormone levels during zebrafish development. Zoological Science. 29(3):181-184.

Cope RB, Kacew S, Dourson M. A reproductive, developmental and neurobehavioral study following oral exposure of tetrabromobisphenol A on Sprague-Dawley rats. Toxicology. 2015 329:49-59.

Corcoran JM, Eastman CJ, Carter JN, Lazarus L. (1977). Circulating thyroid hormone levels in children. Arch Dis Child. 52: 716-720.

Crane HM, Pickford DB, Hutchinson TH, Brown JA. 2006. The effects of methimazole on development of the fathead minnow, pimephales promelas, from embryo to adult. Toxicological Sciences. 93(2):278-285.

Crofton KM. Developmental disruption of thyroid hormone: correlations with hearing dysfunction in rats. Risk Anal. 2004 Dec;24(6):1665-71.

DeVito M, Biegel L, Brouwer A, Brown S, Brucker-Davis F, Cheek AO, Christensen R, Colborn T, Cooke P, Crissman J, Crofton K, Doerge D, Gray E, Hauser P, Hurley P, Kohn M, Lazar J, McMaster S, McClain M, McConnell E, Meier C, Miller R, Tietge J, Tyl R. (1999). Screening methods for thyroid hormone disruptors. Environ Health Perspect. 107:407-415.

Döhler KD, Wong CC, von zur Mühlen A (1979).   The rat as model for the study of drug effects on thyroid function: consideration of methodological problems.  Pharmacol Ther B. 5:305-18.

Eales JG. (1997). Iodine metabolism and thyroid related functions in organisms lacking thyroid follicles: Are thyroid hormones also vitaminsProc Soc Exp Biol Med. 214:302-317.

Furlow JD, Neff ES. (2006). A developmental switch induced by thyroid hormone: Xenopus laevis metamorphosis. Trends Endocrinol Metab. 17:40–47.

Goldey ES, Crofton KM. Thyroxine replacement attenuates hypothyroxinemia, hearing loss, and motor deficits following developmental exposure to Aroclor 1254 in rats. Toxicol Sci. 1998 45(1):94-10

Goldey ES, Kehn LS, Lau C, Rehnberg GL, Crofton KM.  Developmental exposure to polychlorinated biphenyls (Aroclor 1254) reduces circulating thyroid hormone concentrations and causes hearing deficits in rats. Tox Appl Pharmacol. 1995 135(1):77-88.

Harris AR, Fang SL, Prosky J, Braverman LE, Vagenakis AG.  Decreased outer ring monodeiodination of thyroxine and reverse triiodothyronine in the fetal and neonatal rat.  Endocrinology. 1978 Dec;103(6):2216-22

Hernandez-Mariano JA, Torres-Sanchez L, Bassol-Mayagoitia S, Escamilla-Nunez M, Cebrian ME, Villeda-Gutierrez EA, Lopez-Rodriguez G, Felix-Arellano EE, Blanco-Munoz J. 2017. Effect of exposure to p,p '-dde during the first half of pregnancy in the maternal thyroid profile of female residents in a mexican floriculture area. Environmental Research. 156:597-604.

Heyland A, Hodin J. (2004). Heterochronic developmental shift caused by thyroid hormone in larval sand dollars and its implications for phenotypic plasticity and the evolution of non-feeding development. Evolution. 58: 524-538.

Heyland A, Moroz LL. (2005). Cross-kingdom hormonal signaling: an insight from thyroid hormone functions in marine larvae. J Exp Biol. 208:4355-4361.

Hill RN, Crisp TM, Hurley PM, Rosenthal SL, Singh DV. Risk assessment of thyroid follicular cell tumors.  Environ Health Perspect. 1998 Aug;106(8):447-57.

Hornung MW, Kosian P, Haselman J, Korte J, Challis K, Macherla C, Nevalainen E, Degitz S (2015) In vitro, ex vivo and in vivo determination of thyroid hormone modulating activity of benzothiazoles. Toxicol Sci 146:254-264.

Hulbert AJ. Thyroid hormones and their effects: a new perspective. Biol Rev Camb Philos Soc. 2000 Nov;75(4):519-631. Review.

Kapelari K, Kirchlechner C, Högler W, Schweitzer K, Virgolini I, Moncayo R. 2008. Pediatric reference intervals for thyroid hormone levels from birth to adulthood: a retrospective study. BMC Endocr Disord. 8: 15.

Lau C, Thibodeaux JR, Hanson RG, Rogers JM, Grey BE, Stanton ME, Butenhoff JL, Stevenson LA.  Exposure to perfluorooctane sulfonate during pregnancy in rat and mouse. II: postnatal evaluation.  Toxicol Sci. 2003 Aug;74(2):382-92.

Leloup, J., and M. Buscaglia. La triiodothyronine: hormone de la métamorphose des amphibiens. CR Acad Sci 284 (1977): 2261-2263.

Liu J, Liu Y, Barter RA, Klaassen CD.: Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: a dose-response study. J Pharmacol Exp Ther 273, 977-85, 1994

Liu XS, Cai Y, Wang Y, Xu SH, Ji K, Choi K. 2019. Effects of tris(1,3-dichloro-2-propyl) phosphate (tdcpp) and triphenyl phosphate (tpp) on sex-dependent alterations of thyroid hormones in adult zebrafish. Ecotoxicology and Environmental Safety. 170:25-32.

Liu YW, Chan WK. 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation. 70(1):36-45.

Manzon RG, Youson JH. (1997). The effects of exogenous thyroxine (T4) or triiodothyronine (T3), in the presence and absence of potassium perchlorate, on the incidence of metamorphosis and on serum T4 and T3 concentrations in larval sea lampreys (Petromyzon marinus L.). Gen Comp Endocrinol. 106:211-220. 

McClain RM. Mechanistic considerations for the relevance of animal data on thyroid neoplasia to human risk assessment. Mutat Res. 1995 Dec;333(1-2):131-42

Miller MD, Crofton KM, Rice DC, Zoeller RT.  Thyroid-disrupting chemicals: interpreting upstream biomarkers of adverse outcomes. Environ Health Perspect. 2009 117(7):1033-41

Morse DC, Wehler EK, Wesseling W, Koeman JH, Brouwer A. Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to polychlorinated biphenyls (Aroclor 1254). Toxicol Appl Pharmacol. 1996 Feb;136(2):269-79.

Nelson K, Schroeder A, Ankley G, Blackwell B, Blanksma C, Degitz S, Flynn K, Jensen K, Johnson R, Kahl M et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part i: Fathead minnow. Aquatic Toxicology. 173:192-203.

NTP National Toxicology Program.: NTP toxicology and carcinogenesis studies of 3,3'-dimethylbenzidine dihydrochloride (CAS no. 612-82-8) in F344/N rats (drinking water studies). Natl Toxicol Program Tech Rep Ser 390, 1-238, 1991.

O'Connor, J. C., J. C. Cook, et al. (1998). "An ongoing validation of a Tier I screening battery for detecting endocrine-active compounds (EACs)." Toxicol Sci 46(1): 45-60.

O'Connor, J. C., L. G. Davis, et al. (2000). "Detection of dopaminergic modulators in a tier I screening battery for identifying endocrine-active compounds (EACs)." Reprod Toxicol 14(3): 193-205.

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.

Rathmann D, Rijntjes E, Lietzow J, Köhrle J. (2015) Quantitative Analysis of Thyroid Hormone Metabolites in Cell Culture Samples Using LC-MS/MS. Eur Thyroid J. Sep;4(Suppl 1):51-8.

Rouaze-Romet M, Savu L, Vranckx R, Bleiberg-Daniel F, Le Moullac B, Gouache P, Nunez EA. 1992. Reexpression of thyroxine-binding globulin in postweaning rats during protein or energy malnutrition. Acta Endocrinol (Copenh).127:441-448.

Samanidou VF, Kourti PV. (2009) Rapid HPLC method for the simultaneous monitoring of duloxetine, venlaflaxine, fluoxetine and paroxetine in biofluids. Bioanalysis. 2009 Aug;1(5):905-17.

Savu L, Vranckx R, Maya M, Gripois D, Blouquit MF, Nunez EA. 1989. Thyroxine-binding globulin and thyroxinebinding prealbumin in hypothyroid and hyperthyroid developing rats. BiochimBiophys Acta. 992:379-384.

Schneider S, Kaufmann W, Strauss V, van Ravenzwaay B.    Vinclozolin: a feasibility and sensitivity study of the ILSI-HESI F1-extended one-generation rat reproduction protocol. Regul Toxicol Pharmacol. 2011 Feb;59(1):91-100.

Schussler, G.C. (2000). The thyroxine-binding proteins. Thyroid 10:141–149.

Spencer, CA. (2013). Assay of thyroid hormone and related substances. In De Groot, LJ et al. (Eds). Endotext. South Dartmouth, MA

Sternberg RM, Thoemke KR, Korte JJ, Moen SM, Olson JM, Korte L, Tietge JE, Degitz SJ Jr. Control of pituitary thyroid-stimulating hormone synthesis and secretion by thyroid hormones during Xenopus metamorphosis. Gen Comp Endocrinol. 2011. 173(3):428-37

Stinckens E, Vergauwen L, Blackwell BR, Anldey GT, Villeneuve DL, Knapen D. 2020. Effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology. 54(10):6213-6223.

Stinckens E, Vergauwen L, Schroeder A, Maho W, Blackwell B, Witters H, Blust R, Ankley G, Covaci A, Villeneuve D et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part ii: Zebrafish. Aquatic Toxicology. 173:204-217.

Taurog A. 2005. Hormone synthesis. In: Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text (Braverman LE, Utiger RD, eds). Philadelphia:Lippincott, Williams and Wilkins, 47–81Walker P, Dubois JD, Dussault JH.  Free thyroid hormone concentrations during postnatal development in the rat.  Pediatr Res. 1980 Mar;14(3):247-9.

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 Sep 1;45(17):7525-32.

Wabukebunoti MAN, Firling CE. 1983. The prehatching development of the thyroid-gland of the fathead minnow, pimephales-promelas (rafinesque). General and Comparative Endocrinology. 49(2):320-331.

Walter KM, Miller GW, Chen XP, Yaghoobi B, Puschner B, Lein PJ. 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (danio rerio). General and Comparative Endocrinology. 272:20-32.

Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347.

Yamauchi K1, Ishihara A. Evolutionary changes to transthyretin: developmentally regulated and tissue-specific gene expression. FEBS J. 2009. 276(19):5357-66.

Yaoita Y, Brown DD. (1990). A correlation of thyroid hormone receptor gene expression with amphibian metamorphosis. Genes Dev. 4:1917-1924.

Yen PM. (2001). Physiological and molecular basis of thyroid hormone action. Physiol Rev. 81:1097-1142.

Zoeller RT, Tan SW, Tyl RW. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Crit Rev Toxicol. 2007 Jan-Feb;37(1-2):11-53

Zoeller, R. T., R. Bansal, et al. (2005). "Bisphenol-A, an environmental contaminant that acts as a thyroid hormone receptor antagonist in vitro, increases serum thyroxine, and alters RC3/neurogranin expression in the developing rat brain." Endocrinology 146(2): 607-612.

Event: 1003: Decreased, Triiodothyronine (T3) in serum

Short Name: Decreased, Triiodothyronine (T3) in serum

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

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 circulating T3 and T4 that are bound to transport proteins in blood. 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 in serum come from rat studies, and 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, wherease 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 serum THs is still regarded as a measurable key event causatively linked to downstream adverse outcomes.

THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in amphibian and larbean 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 existence and importance has been also described in many differrent animal and plant kingdoms (Eales, 1997; Heyland and Moroz, 2005), while 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: Thyroid hormones are essential in all life stages, but decreases of circulating levels are associated with specific developmental events. 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. Thyroid hormones are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of thyroid hormone levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in thyroid hormone levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.

 

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 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 serum 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 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 store which ensure 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). Until recently, 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), but the specific pathways that are activated in this regard need to be elucidated.

The production of THs in the thyroid gland and the circulation levels in the bloodstream are self-controlled by an efficiently regulated feedback mechanism across the Hypothalamus-Pituitary-Thyroid (HPT) axis. TH levels are regulated, not only in the plasma level, but also in the individual cell level, to maintain homeostasis. This is succeeded by the efficient regulatory mechanism of the thyroid hormone axis which consists of the following: (1) the hypothalamic secretion of the thyrotropin-releasing hormone (TRH), (2) the thyroid-stimulating hormone (TSH) secretion from the anterior pituitary, (3) hormonal transport by the plasma binding proteins, (4) cellular uptake mechanisms in the cell level, (5) intracellular control of TH concentration by the deiodinating mechanism (6) transcriptional function of the nuclear thyroid hormone receptor and (7) in the fetus, the transplacental passage of T4 and T3 (Cheng et al., 2010).

In regards to the brain, the TH concentration involves also an additional level of regulation, namely the hormonal transport through the Blood Brain Barrier (BBB) (Williams, 2008). The TRH and the TSH are actually regulating the production of pro-hormone T4 and in a lesser extent of T3, which is the biologically active TH. 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 up to date but the monocarboxylate transporters (Mct8, Mct10) and the anion-transporting polypeptide (OATP1c1) show the highest degree of affinity towards TH (Jansen et al., 2005).

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 thyroid hormone system as well as thyroid hormone synthesis and action are highly conserved across vertebrates, there are some taxon-specific considerations.

Although the HPT axis is highly conserved, there are some differences between fish and mammals (Blanton and Specker, 2007; Deal and Volkoff, 2020). For example, in fish, corticotropin releasing hormone (CRH) often plays a more important role in regulating thyrotropin (TSH) secretion by the pituitary and thus thyroid hormone synthesis compared to TSH-releasing hormone (TRH). TTRs from fish have low sequence identity with human TTR, for example seabream TTR has 54% sequence identity with human TTR but the only amino acid difference within the thyroxine-binding site is the conservative substitution of Ser117 in human TTR to Thr117 in seabream TTR (Santos and Power, 1999; Yamauchi et al., 1999; Eneqvist et al., 2004). In vitro binding experiments showed that TH 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 minnows are oviparous fish species in which maternal thyroid hormones are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic thyroid hormone synthesis is initiated. Maternal transfer of thyroid hormones, both T4 and T3, to the eggs has been demonstrated in zebrafish (Walpita et al., 2007; Chang et al., 2012) and fathead minnows (Crane et al., 2004; Nelson et al., 2016).

Several studies have reported evidence of T3 decreases after exposure to TPO inhibitors and deiodinase inhibitors in early life stages of zebrafish (Stinckens et al., 2016; Stinckens et al., 2020; Wang et al., 2020) and fathead minnow (Nelson et al., 2016; Cavallin et al., 2017).

How it is Measured or Detected

T3 and T4 can be measured as free (unbound) or total (bound + unbound). Free hormone 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 a 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 a an indirect methodology, whereas analytical determination is the most direct measurement available. All of these methods, particularly RIA, are repeatable and reproducible.

In fish early life stages most evidence for the ontogeny of thyroid hormone synthesis comes from measurements of whole body thyroid hormone levels and using LC-MS techniques (Hornung et al., 2015) are increasingly used to accurately quantify whole body thyroid hormone levels as a proxy for serum thyroid hormone levels (Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020).

References

Bartalena L, Robbins J.Thyroid hormone transport proteins.Clin Lab Med. 1993 Sep;13(3):583-98.

Bassett JH, Harvey CB, Williams GR. (2003). Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol Cell Endocrinol. 213:1-11.

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.

Brent GA. (2012). Mechanisms of thyroid hormone action. J Clin Invest. 122: 3035-3043.

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.

Cavallin JE, Ankley GT, Blackwell BR, Blanksma CA, Fay KA, Jensen KM, Kahl MD, Knapen D, Kosian PA, Poole ST et al. 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry. 36(11):2942-2952.

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.

Cheng SY, Leonard JL, Davis PJ. (2010).Molecular aspects of thyroid hormone actions. Endocr Rev. 31:139–170.

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.

Davis PJ, Zhou M, Davis FB, Lansing L, Mousa SA, Lin HY. (2010). Mini-review: Cell surface receptor for thyroid hormone and nongenomic regulation of ion fluxes in excitable cells. Physiol Behav. 99:237–239.

Deal CK, Volkoff H. 2020. The role of the thyroid axis in fish. Frontiers in Endocrinology. 11.

DeVito M, Biegel L, Brouwer A, Brown S, Brucker-Davis F, Cheek AO, Christensen R, Colborn T, Cooke P, Crissman J, Crofton K, Doerge D, Gray E, Hauser P, Hurley P, Kohn M, Lazar J, McMaster S, McClain M, McConnell E, *Meier C, Miller R, Tietge J, Tyl R. (1999). Screening methods for thyroid hormone disruptors. Environ Health Perspect. 107:407-415.

Eales JG. (1997). Iodine metabolism and thyroid related functions in organisms lacking thyroid follicles: Are thyroid hormones also vitamins? Proc Soc Exp Biol Med. 214:302-317.

Eneqvist T, Lundberg E, Karlsson A, Huang SH, Santos CRA, Power DM, Sauer-Eriksson AE. 2004. High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine. Journal of Biological Chemistry. 279(25):26411-26416.

Friesema EC, Jansen J, Milici C, Visser TJ. (2005). Thyroid hormone transporters. Vitam Horm. 70: 137–167.

Furlow JD, Neff ES. (2006). A developmental switch induced by thyroid hormone: Xenopus laevis metamorphosis. Trends Endocrinol Metab. 17:40–47.

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.

Gilbert ME, Rovet J, Chen Z, Koibuchi N. (2012).Developmental thyroid hormone disruption: prevalence, environmental contaminants and neurodevelopmental consequences. Neurotoxicology. 33: 842-852.

Glinoer D. (2001).Potential consequences of maternal hypothyroidism on the offspring: evidence and implications. Horm Res. 55:109-114.

Hennemann G, Docter R, Friesema EC, de Jong M, Krenning EP, Visser TJ. (2001). Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocr Rev. 22:451-476.

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.

Heyland A, Hodin J. (2004). Heterochronic developmental shift caused by thyroid hormone in larval sand dollars and its implications for phenotypic plasticity and the evolution of non-feeding development. Evolution. 58: 524-538.

Heyland A, Moroz LL. (2005). Cross-kingdom hormonal signaling: an insight from thyroid hormone functions in marine larvae. J Exp Biol. 208:4355-4361.

Hornung, M.W., Kosian, P.A., Haselman, J.T., Korte, J.J., Challis, K., Macherla, C., Nevalainen, E., Degitz, S.J., 2015. In Vitro, Ex Vivo, and In Vivo Determination of Thyroid Hormone Modulating Activity of Benzothiazoles. Toxicological Sciences 146, 254-264.

Hulbert A J. (2000). Thyroid hormones and their effects: A new perspective. Biol Rev. 75: 519-631.

Jansen J, Friesema EC, Milici C, Visser TJ. (2005). Thyroid hormone transporters in health and disease. Thyroid. 15: 757-768.

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.

Liu YW, Chan WK. 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation. 70(1):36-45.

Manzon RG, Youson JH. (1997). The effects of exogenous thyroxine (T4) or triiodothyronine (T3), in the presence and absence of potassium perchlorate, on the incidence of metamorphosis and on serum T4 and T3 concentrations in larval sea lampreys (Petromyzon marinus L.). Gen Comp Endocrinol. 106:211-220.

Miller MD, Crofton KM, Rice DC, Zoeller RT. (2009).Thyroid-disrupting chemicals: interpreting upstream biomarkers of adverse outcomes. Environ Health Perspect. 117:1033-1041.

Moeller LC, Dumitrescu AM, Seo H, Refetoff S. (2006). Thyroid hormone mediated changes in gene expression can be initiated by cytosolic action of the thyroid hormone receptor β through the phosphatidylinositol 3-kinase pathway. NRS. 4:1-4.Nelson, K., Schroeder, A., Ankley, G., Blackwell, B., Blanksma, C., Degitz, S., Flynn, K., Jensen, K., Johnson, R., Kahl, M., Knapen, D., Kosian, P., Milsk, R., Randolph, E., Saari, T., Stinckens, E., Vergauwen, L., Villeneuve, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquatic Toxicology 173, 192-203.Obregon MJ, Mallol J, Escobar del Rey F, Morreale de Escobar G. (1981). Presence of l-thyroxine and 3,5,3-triiodo-l-thyronine in tissues from thyroidectomised rats. Endocrinology 109:908-913.

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.

Oetting A, Yen PM. (2007). New insights into thyroid hormone action. Best Pract Res Clin Endocrinol Metab. 21:193–208.

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.

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.

Santos CRA, Power DM. 1999. Identification of transthyretin in fish (sparus aurata): Cdna cloning and characterisation. Endocrinology. 140(5):2430-2433.

Schussler, G.C. (2000). The thyroxine-binding proteins. Thyroid 10:141–149.

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., Knapen, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish. Aquatic Toxicology 173, 204-217.

Thienpont B, Tingaud-Sequeira A, Prats E, Barata C, Babin PJ, Raldua D. 2011. Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis. Environmental Science & Technology. 45(17):7525-7532

Visser WE, Friesema EC, Jansen J, Visser TJ. (2008). Thyroid hormone transport in and out of cells. Trends Endocrinol Metab. 19:50-56.

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.

Wang JX, Shi GH, Yao JZ, Sheng N, Cui RN, Su ZB, Guo Y, Dai JY. 2020. Perfluoropolyether carboxylic acids (novel alternatives to pfoa) impair zebrafish posterior swim bladder development via thyroid hormone disruption. Environment International. 134.

Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347.

Williams GR. (2008). Neurodevelopmental and neurophysiological actions of thyroid hormone. J Neuroendocrinol. 20:784–794.

Yamauchi K, Nakajima J, Hayashi H, Hara A. 1999. Purification and characterization of thyroid-hormone-binding protein from masu salmon serum - a homolog of higher-vertebrate transthyretin. European Journal of Biochemistry. 265(3):944-949.

Yamauchi K1, Ishihara A. Evolutionary changes to transthyretin: developmentally regulated and tissue-specific gene expression.FEBS J. 2009 Oct;276(19):5357-66.

Yaoita Y, Brown DD. (1990). A correlation of thyroid hormone receptor gene expression with amphibian metamorphosis. Genes Dev. 4:1917-1924.

Yen PM. (2001). Physiological and molecular basis of thyroid hormone action. Physiol Rev. 81:1097-1142.

Zhang J, Grundstrom C, Brannstrom K, Iakovleva I, Lindberg M, Olofsson A, Andersson PL, Sauer-Eriksson AE. 2018. Interspecies variation between fish and human transthyretins in their binding of thyroid-disrupting chemicals. Environmental Science & Technology. 52(20):11865-11874.´

Zoeller RT, Tan SW, Tyl RW. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Crit Rev Toxicol. 2007 Jan-Feb;37(1-2):11-53

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 function.

Studies that detect and measure altered retinal layer structure after exposure to thyroid hormones 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 pigment epithelium (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).

 

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, 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.

(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 wide range of species including mammals, amphibians, fish and humans. Evaluation of these visual function modification 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 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. However, the main disadvantage of optokinetic tracking is that it is a subjective method in which the decision about whether the animal is performing the optokinetic tracking or not is made by an experimenter (Segura et al., 2018).

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

Babkiewicz, E., Bazała, M., Urban, P., Maszczyk, P., Markowska, M., & Maciej Gliwicz, Z. (2020). The effects of temperature on the proxies of visual detection of Danio rerio larvae: observations from the optic tectum. Biology Open, 9(7). https://doi.org/10.1242/BIO.047779

Brastrom, L.K., Scott, C.A., Dawson, D. V., Slusarski, D.C., 2019. A High-Throughput Assay for Congenital and Age-Related Eye Diseases in Zebrafish. Biomedicines 7, 28. https://doi.org/10.3390/biomedicines7020028

Carvalho, P.S.M., Noltie, D.B., Tillitt, D.E., 2002. Ontogenetic improvement of visual function in the medaka Oryzias latipes based on an optomotor testing system for larval and adult fish. Anim. Behav. 64, 1–10. https://doi.org/10.1006/anbe.2002.3028

Chrispell JD, Rebrik TI, Weiss ER. 2015. Electroretinogram Analysis of the Visual Response in Zebrafish Larvae. Jove-Journal of Visualized Experiments(97).

Corral-López, A., Garate-Olaizola, M., Buechel, S.D., Kolm, N., Kotrschal, A., 2017. On the role of body size, brain size, and eye size in visual acuity. Behav. Ecol. Sociobiol. 71. https://doi.org/10.1007/s00265-017-2408-z

Dehnert GK, Karasov WH, Wolman MA. 2019. 2,4-Dichlorophenoxyacetic acid containing herbicide impairs essential visually guided behaviors of larval fish. Aquatic Toxicology 209:1-12.

Gao, D., Wu, M., Wang, C., Wang, Y., Zuo, Z., 2015. Chronic exposure to low benzo[a]pyrene level causes neurodegenerative disease-like syndromes in zebrafish (Danio rerio). Aquat. Toxicol.

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

Raine, J. C., Lallemand, L., Pettem, C. M., & Janz, D. M. (2016). Effects of Chronic Dietary Selenomethionine Exposure on the Visual System of Adult and F1 Generation Zebrafish (Danio rerio). Bulletin of Environmental Contamination and Toxicology, 97(3), 331–336. https://doi.org/10.1007/s00128-016-1849-9

Randlett O, Wee CL, Naumann EA, Nnaemeka O, Schoppik D, Fitzgerald JE, Portugues R, Lacoste AMB, Riegler C, Engert F et al. . 2015. Whole-brain activity mapping onto a zebrafish brain atlas. Nature Methods 12(11):1039-1046.

Vancamp, P., Bourgeois, N. M. A., Houbrechts, A. M., & Darras, V. M. (2019). Knockdown of the thyroid hormone transporter MCT8 in chicken retinal precursor cells hampers early retinal development and results in a shift towards more UV/blue cones at the expense of green/red cones. Experimental Eye Research,178(September 2018), 135–147. https://doi.org/10.1016/j.exer.2018.09.018

Zhang, X., Hong, Q., Yang, L., Zhang, M., Guo, X., Chi, X., & Tong, M. (2015). PCB1254 exposure contributes to the abnormalities of optomotor responses and influence of the photoreceptor cell development in zebrafish larvae. Ecotoxicology and Environmental Safety, 118, 133–138. https://doi.org/10.1016/j.ecoenv.2015.04.026

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 trajectory

Short Name: Decrease, Population trajectory

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

Population ecology is the study of the sizes (and to some extent also the distribution) of plant and animal populations and of the processes, mainly biological in nature, that determine these sizes. As such, it provides an integrated measure of events occurring at lower levels of biological organization (biochemical, organismal, etc.). The population size in turn determines community and ecosystem structure. For fish, maintenance of sustainable fish and wildlife populations (i.e., adequate to ensure long-term delivery of valued ecosystem services) is an accepted regulatory goal upon which risk assessments and risk management decisions are based.

How it is Measured or Detected

Population trajectories, either hypothetical or site specific, can be estimated via population modeling based on measurements of vital rates or reasonable surrogates measured in laboratory studies. As an example, Miller and Ankley 2004 used measures of cumulative fecundity from laboratory studies with repeat spawning fish species to predict population-level consequences of continuous exposure.

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

• Miller DH, Ankley GT. 2004. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17ß-trenbolone as a case study. Ecotoxicology and Environmental Safety 59: 1-9.

Appendix 2

List of Key Event Relationships in the AOP