AOP ID and Title:
Graphical Representation
Status
Author status | OECD status | OECD project | SAAOP status |
---|---|---|---|
Open for adoption | Under Development | 1.35 | Included in OECD Work Plan |
Abstract
This AOP describes the sequence of events leading from thyroperoxidase inhibition to increased mortality via reduced anterior swim bladder inflation. The enzyme thyroperoxidase (TPO) is essential for the synthesis of thyroxine (T4) and triiodothyronine (T3) in the thyroid follicles. Inhibition of TPO reduces thyroid hormone (TH) levels. Thyroid hormones are critical in regulating developmental processes and thyroid hormone disruption can interfere with normal development. Swim bladder inflation is known to be under TH control (Brown et al., 1988; Liu and Chan, 2002). Many fish species have a swim bladder which is a gas-filled organ that typically consists of two chambers (Robertson et al., 2007). The posterior chamber inflates during early development in the embryonic phase, while the anterior chamber inflates during late development in the larval phase. Both the posterior and the anterior chamber have an important role in regulating buoyancy, and the anterior chamber has an additional role in hearing (Robertson et al., 2017). This AOP describes how inhibition of TPO results in decreased synthesis of T4 in the thyroid follicles. This reduces the availability of T4 for conversion to the more biologically active T3. Reduced T3 levels prohibit normal inflation of the anterior swim bladder chamber. Due to its role in regulating buoyancy, this results in reduced swimming performance. Since reduced swimming performance resuls in a decreased ability to forage and avoid predators, this reduces chances of survival. The final adverse outcome is a decrease of the population trajectory. Since many AOPs eventually lead to this more general adverse outcome at the population level, the more specific and informative adverse outcome at the organismal level, increased mortality, is used in the AOP title. Support for this AOP is mainly based on chemical exposures in zebrafish and fathead minnows (Nelson et al., 2016; Godfrey et al., 2017; Stinckens et al., 2016, 2020). Additional evidence of a link between reduced TH synthesis and reduced anterior chamber inflation is available from a study where a mutation was inserted in the gene coding for dual oxidase, an other enzyme that is important for TH synthesis (Chopra et al., 2019). This AOP is part of a larger AOP network describing how decreased synthesis and/or decreased biological activation of THs leads to incomplete or improper inflation of the swim bladder, leading to reduced swimming performance, increased mortality and decreased population trajectory (Knapen et al., 2018; Knapen et al., 2020; Villeneuve et al., 2018). An AOP initiated by TPO inhibition and leading to posterior chamber inflation has not been developed so far, since available evidence shows that TPO inhibition is less likely to cause effects on inflation of the posterior chamber during early embryonic development. This important aspect of life stage specificity is explained in more detail below (see Domain of applicability).
Background
The larger AOP network describing the effect of deiodinase and thyroperoxidase inhibition on swim bladder inflation consists of 5 AOPs:
- Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation: https://aopwiki.org/aops/155
- Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation: https://aopwiki.org/aops/156
- Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation : https://aopwiki.org/aops/157
- Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation : https://aopwiki.org/aops/158
- Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation: https://aopwiki.org/aops/159
Summary of the AOP
Events
Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)
Sequence | Type | Event ID | Title | Short name |
---|---|---|---|---|
1 | MIE | 279 | Thyroperoxidase, Inhibition | Thyroperoxidase, Inhibition |
2 | KE | 277 | Thyroid hormone synthesis, Decreased | TH synthesis, Decreased |
3 | KE | 281 | Thyroxine (T4) in serum, Decreased | T4 in serum, Decreased |
4 | KE | 1003 | Decreased, Triiodothyronine (T3) in serum | Decreased, Triiodothyronine (T3) in serum |
5 | KE | 1007 | Reduced, Anterior swim bladder inflation | Reduced, Anterior swim bladder inflation |
6 | KE | 1005 | Reduced, Swimming performance | Reduced, Swimming performance |
7 | AO | 351 | Increased Mortality | Increased Mortality |
8 | AO | 360 | Decrease, Population trajectory | Decrease, Population trajectory |
Key Event Relationships
Upstream Event | Relationship Type | Downstream Event | Evidence | Quantitative Understanding |
---|---|---|---|---|
Thyroperoxidase, Inhibition | adjacent | Thyroid hormone synthesis, Decreased | Moderate | Low |
Thyroid hormone synthesis, Decreased | adjacent | Thyroxine (T4) in serum, Decreased | Moderate | Low |
Thyroxine (T4) in serum, Decreased | adjacent | Decreased, Triiodothyronine (T3) in serum | Moderate | Moderate |
Decreased, Triiodothyronine (T3) in serum | adjacent | Reduced, Anterior swim bladder inflation | Moderate | Moderate |
Reduced, Anterior swim bladder inflation | adjacent | Reduced, Swimming performance | Moderate | Low |
Reduced, Swimming performance | adjacent | Increased Mortality | Moderate | Low |
Increased Mortality | adjacent | Decrease, Population trajectory | High | Moderate |
Thyroperoxidase, Inhibition | non-adjacent | Thyroxine (T4) in serum, Decreased | High | Low |
Thyroxine (T4) in serum, Decreased | non-adjacent | Reduced, Anterior swim bladder inflation | Moderate | Moderate |
Stressors
Name | Evidence |
---|---|
Methimazole | High |
Mercaptobenzothiazole | High |
Propylthiouracil | High |
Overall Assessment of the AOP
The attached document includes:
- Support for biological plausibility of KERs
- Support for essentiality of KEs
- Empirical support for KERs
- Dose and temporal concordance table covering the larger AOP network
Overall, the weight of evidence for the sequence of key events laid out in the AOP is moderate to high. Nonetheless, the exact underlying mechanism of TH disruption leading to impaired swim bladder inflation is not understood.
Domain of Applicability
Life Stage ApplicabilityLife Stage | Evidence |
---|---|
larvae | High |
Term | Scientific Term | Evidence | Links |
---|---|---|---|
zebrafish | Danio rerio | High | NCBI |
fathead minnow | Pimephales promelas | High | NCBI |
Sex | Evidence |
---|---|
Unspecific | High |
Taxonomic: Organogenesis of the swim bladder begins with an evagination from the gut. In physostomous fish, a connection between the swim bladder and the gut is retained. In physoclystous fish, once initial inflation by gulping atmospheric air at the water surface has occurred, the swim bladder is closed off from the digestive tract and swim bladder volume is regulated by gas secretion into the swim bladder (Wooley and Qin, 2010). This AOP is currently mainly based on experimental evidence from studies on zebrafish and fathead minnows, physostomous fish with a two-chambered swim bladder. This AOP is not applicable to fish that do not have a second swim bladder chamber that inflates during larval development, e.g., the Japanese rice fish (Oryzias latipes).
Life stage: The current AOP is applicable to the larval life stage, the period in which the anterior chamber of the swim bladder inflates (21 days post fertilization in zebrafish). While evidence supports a link between reduced serum T4 levels and reduced anterior swim bladder inflation in late larvae, experimental evidence suggests that inhibition of T4 synthesis is less likely to result in reduced posterior swim bladder inflation in embryos. The reduced likelihood of effects on posterior chamber inflation is probably due to maternal transfer of T4 into the eggs limiting the importance of TH synthesis by TPO in the earliest embryonic stages. These maternally derived THs are depleted at 21 days post fertilization (dpf) and this is probably why 21 day old larvae are sensitive to TPO inhibtion, resulting in impaired anterior chamber inflation.
Maternal thyroid hormone levels in embryos have been demonstrated in zebrafish, fathead minnow, brown trout, striped bass, tilapia, rabbitfish, conger eel, sea bream and different species of salmon (Ruuskanen and Hsu, 2018; Walpita et al., 2007; Chang et al., 2012; Hsu et al., 2014; Power et al., 2001; Brown et al., 1987, 1988). Campinho et al. (2014) confirmed that maternal thyroid hormones are essential for normal brain development in zebrafish by knocking down MCT8, responsable for transporting thyroid hormones into the cells. Alt et al. (2006) found a first differentiated thyroid follicle in zebrafish at 55 hours post fertilization. Elsalini et al. (2003) used immunohistochemistry to show the development of the first thyroid follicles producing thyroid hormone at 72 hours post fertilization (hpf) in zebrafish. Walter et al. (2019) showed decreased levels of T4 in 72 hpf embryos exposed to propylthiouracil (PTU), a TPO inhibitor, confirming embryonic TH synthesis at 72 hpf. At 24 hpf PTU had no effect on TH levels, confirming that at least at the whole body level, there is no detectable TH synthesis at 24 hpf. Time points between 24 and 72 hpf have not been investigated. Therefore, it is still uncertain when exactly embryonic TH synthesis is activated. Therefore early developmental processes (before thyroid activation) that are dependent on T4, such as posterior swim bladder inflation, may be less sensitive to chemicals reducing T4 synthesis. Nelson et al. (2016) and Stinckens et al. (2016) indeed found that 2-mercaptobenzothiazole (a thyroperoxidase inhibitor) decreased T4 levels in both zebrafish (5 days post fertilization) and fathead minnow (6 days post fertilization), which is after activation of the thyroid gland for both species, while it did not affect posterior chamber inflation. Godfrey et al. (2017) also reported normal posterior chamber inflation after exposure of zebrafish embryos to methimazole, a TPO inhibitor. In the latter study, only perfluorooctanoic acid (a deiodinase inhibitor in pig, Stinckens et al., 2018) affected posterior chamber inflation. Stinckens et al. (2018) also showed that sodium perchlorate, an inhibitor of the sodium iodide symporter (NIS), did not impact posterior chamber inflation. Since this symporter is essential for providing iodide for TH synthesis, this is indirect evidence for this hypothesis of life stage specificity. It is important to note that posterior chamber inflation should be observed until at least 6 dpf in zebrafish to exclude delayed swim bladder inflation (which may be due to a more general developmental delay, rather than a specific effect on the swim bladder).
Sex: Sex differences are typically not investigated in tests using early life stages of fish and it is currently unclear whether sex-related differences are important in this AOP. 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. Since the anterior chamber inflates around 21 days post fertilization, when sex differentiation is still in its early stages, sex differences are expected to play a minor role in the current AOP.
Essentiality of the Key Events
Overall, the confidence in the supporting data for essentiality of KEs within the AOP is moderate. There is indirect evidence that reduced thyroid hormone synthesis causes reduced anterior swim bladder inflation from a study where a similar MIE was targeted: Chopra et al. (2019) showed that knockdown of dual oxidase, important for thyroid hormone synthesis, reduced anterior swim bladder inflation. Additionally, there is indirect evidence from deiodinase knockdowns supporting the downstream part of the AOP linking decreased T3 levels to reduced swim bladder inflation (targeted at posterior chamber inflation, not specifically at anterior chamber inflation).
Weight of Evidence Summary
Biological plausibility: see Table. Overall, the weight of evidence for the biological plausibility of the KERs in the AOP is moderate since there is empirical support for an association between the sets of KEs and the KERs are plausible based on analogy to accepted biological relationships, but scientific understanding is not completely established.
Empirical support: see Table. Overall, the empirical support for the KERs in the AOP is moderate since dependent changes in sets of KEs following exposure to several specific stressors has been demonstrated, with limited evidence for dose and temporal concordance and some uncertainties.
Quantitative Consideration
There is some level of quantitative understanding that can form the basis for development of a quantitative AOP. Quantitative relationships between reduced T4 and reduced T3, and between reduced T3 and reduced anterior chamber inflation were established. The latter is particularly critical for linking impaired swim bladder inflation to TH disruption.
Considerations for Potential Applications of the AOP (optional)
A growing number of environmental pollutants are known to adversely affect the thyroid hormone system, and major gaps have been identified in the tools available for the identification, and the hazard and risk assessment of these thyroid hormone disrupting chemicals. Villeneuve et al. (2014) discussed the relevance of swim bladder inflation as a potential key event and endpoint of interest in fish tests. Knapen et al. (2020) provide an example of how the adverse outcome pathway (AOP) framework and associated data generation can address current testing challenges in the context of fish early-life stage tests, and fish tests in general. A suite of assays covering all the essential biological processes involved in the underlying toxicological pathways can be implemented in a tiered screening and testing approach for thyroid hormone disruption, using the levels of assessment of the OECD’s Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals as a guide. Specifically, for this AOP, thyroperoxidase inhibition can be assessed using an in chemico assay, measurements of T4 and T3 levels could be added to the Fish Embryo Acute Toxicity
(FET) test (OECD TG 236) and to the Fish Early Life Stage Toxicity (FELS) Test (OECD TG210), and assessments of anterior chamber inflation and swimming performance could be added to the FELS Test.
References
Alt, B., Reibe, S., Feitosa, N.M., Elsalini, O.A., Wendl, T., Rohr, K.B., 2006. Analysis of origin and growth of the thyroid gland in zebrafish. Dev. Dyn. 235, 1872–1883, http://dx.doi.org/10.1002/dvdy.20831.
Brown, C.L., Sullivan, C.V., Bern, H.A. and Dickhoff, W.W. 1987. Occurrence of thyroid hormones in early developmental stages of teleost fish. Trans. Am. Fish. Soc. Symp. 2: 144–150.
Brown, C.L., Doroshov, S.I., Nunez, J.M., Hadley, C., Vaneenennaam, J., Nishioka, R.S., Bern, H.A., 1988. MATERNAL TRIIODOTHYRONINE INJECTIONS CAUSE INCREASES IN SWIMBLADDER INFLATION AND SURVIVAL RATES IN LARVAL STRIPED BASS, MORONE-SAXATILIS. Journal of Experimental Zoology 248, 168-176.
Campinho, M.A., Saraiva, J., Florindo, C., Power, D.M., 2014. Maternal Thyroid Hormones Are Essential for Neural Development in Zebrafish. Molecular Endocrinology 28, 1136-1149.
Chang, J., Wang, M., Gui, W., Zhao, Y., Yu, L., Zhu, G., 2012. Changes in thyroid hormone levels during zebrafish development. Zool. Sci. 29, 181–184, http:// dx.doi.org/10.2108/zsj.29.181.
Chopra, K., Ishibashi, S., Amaya, E., 2019. Zebrafish duox mutations provide a model for human congenital hypothyroidism. Biology Open 8.
Elsalini, O.A., Rohr, K.B., 2003. Phenylthiourea disrupts thyroid function in developing zebrafish. Dev. Genes Evol. 212, 593–598, http://dx.doi.org/10. 1007/s00427-002-0279-3.
Godfrey, A., Hooser, B., Abdelmoneim, A., Horzmann, K.A., Freemanc, J.L., Sepulveda, M.S., 2017. Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish. Aquatic Toxicology 193, 228-235.
Hsu, C.W., Tsai, S.C., Shen, S.C., Wu, S.M., 2014. Profiles of thyrotropin, thyroid hormones, follicular cells and type I deiodinase gene expression during ontogenetic development of tilapia larvae and juveniles. Fish Physiology and Biochemistry 40, 1587-1599.
Knapen, D., Angrish, M.M., Fortin, M.C., Katsiadaki, I., Leonard, M., Margiotta-Casaluci, L., Munn, S., O'Brien, J.M., Pollesch, N., Smith, L.C., Zhang, X.W., Villeneuve, D.L., 2018. Adverse outcome pathway networks I: Development and applications. Environmental Toxicology and Chemistry 37, 1723-1733.
Knapen, D., Stinckens, E., Cavallin, J.E., Ankley, G.T., Holbech, H., Villeneuve, D.L., Vergauwen, L., 2020. Toward an AOP Network-Based Tiered Testing Strategy for the Assessment of Thyroid Hormone Disruption. Environmental Science & Technology 54, 8491-8499.
Liu, Y.W., Chan, W.K., 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 70, 36-45.
Maack, G., Segner, H., 2003. Morphological development of the gonads in zebrafish. Journal of Fish Biology 62, 895-906.
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.
Power, D.M., Llewellyn, L., Faustino, M., Nowell, M.A., Bjornsson, B.T., Einarsdottir, I.E., Canario, A.V., Sweeney, G.E., 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol 130, 447-459.
Robertson, G.N., McGee, C.A.S., Dumbarton, T.C., Croll, R.P., Smith, F.M., 2007. Development of the swimbladder and its innervation in the zebrafish, Danio rerio. Journal of Morphology 268, 967-985.
Ruuskanen, S., Hsu, B.Y., 2018. Maternal Thyroid Hormones: An Unexplored Mechanism Underlying Maternal Effects in an Ecological Framework. Physiological and Biochemical Zoology 91, 904-916.
Stinckens, E., Vergauwen, L., Ankley, G.T., Blust, R., Darras, V.M., Villeneuve, D.L., Witters, H., Volz, D.C., Knapen, D., 2018. An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish. Aquatic Toxicology 200, 1-12.
Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.
Stinckens, E., Vergauwen, L., Schroeder, A., Maho, W., Blackwell, B., Witters, H., Blust, R., Ankley, G., Covaci, A., Villeneuve, D., Knapen, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish. Aquatic Toxicology 173, 204-217.
Villeneuve, D., Angrish, M., Fortin, M., Katsiadaki, I., Leonard, M., Margiotta-Casaluci, L., Munn, S., O'Brien, J., Pollesch, N., Smith, L., Zhang, X., Knapen, D., 2018. Adverse Outcome Pathway Networks II: Network Analytics. Environ Toxicol Chem doi: 10.1002/etc.4124.
Villeneuve, D., Volz, D.C., Embry, M.R., Ankley, G.T., Belanger, S.E., Leonard, M., Schirmer, K., Tanguay, R., Truong, L., Wehmas, L., 2014. Investigating alternatives to the fish early-life stage test: a strategy for discovering and annotating adverse outcome pathways for early fish development. Environmental Toxicology and Chemistry 33, 158-169.
Walpita, C.N., Van der Geyten, S., Rurangwa, E., Darras, V.M., 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, 206–214, http://dx.doi.org/ 10.1016/j.ygcen.2007.02.020.
Walter, K.M., Miller, G.W., Chen, X.P., Yaghoobi, B., Puschner, B., Lein, P.J., 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (Danio rerio). General and Comparative Endocrinology 272, 20-32.
Woolley, L.D., Qin, J.G., 2010. Swimbladder inflation and its implication to the culture of marine finfish larvae. Reviews in Aquaculture 2, 181-190.
Appendix 1
List of MIEs in this AOP
Event: 279: Thyroperoxidase, Inhibition
Short Name: Thyroperoxidase, Inhibition
Key Event Component
Process | Object | Action |
---|---|---|
iodide peroxidase activity | thyroid peroxidase | decreased |
AOPs Including This Key Event
AOP ID and Name | Event Type |
---|---|
Aop:42 - Inhibition of Thyroperoxidase and Subsequent Adverse Neurodevelopmental Outcomes in Mammals | MolecularInitiatingEvent |
Aop:119 - Inhibition of thyroid peroxidase leading to follicular cell adenomas and carcinomas (in rat and mouse) | MolecularInitiatingEvent |
Aop:159 - Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation | MolecularInitiatingEvent |
Aop:175 - Thyroperoxidase inhibition leading to altered amphibian metamorphosis | MolecularInitiatingEvent |
Aop:271 - Inhibition of thyroid peroxidase leading to impaired fertility in fish | MolecularInitiatingEvent |
Aop:363 - Thyroperoxidase inhibition leading to increased mortality via altered eye structure | MolecularInitiatingEvent |
Stressors
Name |
---|
2(3H)-Benzothiazolethione |
2-mercaptobenzothiazole |
Ethylene thiourea |
Mercaptobenzothiazole |
Methimazole |
Propylthiouracil |
Resorcinol |
Thiouracil |
Ethylenethiourea |
Amitrole |
131-55-5 |
2,2',4,4'-Tetrahydroxybenzophenone |
Daidzein |
Genistein |
4-Nonylphenol |
4-propoxyphenol |
Sulfamethazine |
Biological Context
Level of Biological Organization |
---|
Molecular |
Cell term
Cell term |
---|
thyroid follicular cell |
Organ term
Organ term |
---|
thyroid follicle |
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 ApplicabilityTerm | Scientific Term | Evidence | Links |
---|---|---|---|
rat | Rattus norvegicus | High | NCBI |
humans | Homo sapiens | High | NCBI |
pigs | Sus scrofa | High | NCBI |
Xenopus laevis | Xenopus laevis | High | NCBI |
chicken | Gallus gallus | High | NCBI |
zebrafish | Danio rerio | High | NCBI |
fathead minnow | Pimephales promelas | High | NCBI |
mouse | Mus musculus | NCBI |
Life Stage | Evidence |
---|---|
All life stages | High |
Sex | Evidence |
---|---|
Female | High |
Male | High |
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.
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., guicacol 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 assays 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. Available evidence suggests the 11th to 12th fetal week as the beginning of functional TPO in humans. In rodents, TPO function begins late in the second fetal week, with the first evidence of T4 secretion on gestational day 17 (Remy et al., 1980). Thyroid-specific genes appear in the thyroid gland according to a specific temporal pattern; thyroglobulin (Tg), TPO (Tpo), and TSH receptor (Tshr) genes are expressed by gestational day 14 in rats, and the sodium iodide symporter, NIS (Nis), is expressed by gestational day 16 in rats. Maturation to adult function is thought to occur within a few weeks after parturition in rats and mice, and within the first few months in neonatal humans (Santisteban and Bernal, 2005). Tg is first detected in human fetuses starting at 5th week of gestation and rises throughout gestation (Thorpe-Beeston et al., 1992), but iodine trapping and T4 production does not occur until around 10-12 weeks. Also, the dimerization of Tg, a characteristic of adult TH storage, is not found until much later in human gestation (Pintar, 2000). In rats, Tg immunoreactivity does not appear until day 15 of gestation (Fukiishi et al., 1982; Brown et al., 2000). The vast majority of research and knowledge on Tg is from mammals, although genomic orthologs are known for a variety of other species (Holzer et al., 2016). It is important to note that prior to the onset of fetal thyroid function, 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.
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). Limited information has been published using microsomes from human goiter samples (Vickers et al., 2012) and rat thyroid glands (Paul et al., 2013; 2014; Paul-Friedman et al., 2016).
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 of 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.
References
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.
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List of Key Events in the AOP
Event: 277: Thyroid hormone synthesis, Decreased
Short Name: TH synthesis, Decreased
Key Event Component
Process | Object | Action |
---|---|---|
thyroid hormone generation | thyroid hormone | decreased |
AOPs Including This Key Event
Stressors
Name |
---|
Propylthiouracil |
Methimazole |
Biological Context
Level of Biological Organization |
---|
Cellular |
Cell term
Cell term |
---|
thyroid follicular cell |
Organ term
Organ term |
---|
thyroid gland |
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
not applicable as this KE is not an MIE
Propylthiouracil
6-n-proylthiouracil is a common positive control
Methimazole
Methimazole is a very common positve control
Domain of Applicability
Taxonomic ApplicabilityTerm | Scientific Term | Evidence | Links |
---|---|---|---|
rat | Rattus norvegicus | High | NCBI |
human | Homo sapiens | High | NCBI |
Pig | Pig | High | NCBI |
Xenopus laevis | Xenopus laevis | Moderate | NCBI |
zebrafish | Danio rerio | High | NCBI |
fathead minnow | Pimephales promelas | Moderate | NCBI |
Life Stage | Evidence |
---|---|
All life stages | High |
Sex | Evidence |
---|---|
Male | High |
Female | High |
Decreased TH synthesis resulting from TPO or NIS inhibition is conserved across 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).
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).
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.
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).
TIQDT (Thyroxine-immunofluorescence quantitative disruption test) is a method that provides an immunofluorescent based estimate of thyroxine in the gland of zebrafish (Thienpont et al 2011). This method has been used for ~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.
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.
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.
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.
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.
Huang CJ and Jap TS. 2015. A systematic review of genetic studies of thyroid disorders in Taiwan. J Chin Med Assoc. 78: 145-153.
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.
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.
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.
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.
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. 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.
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.
Event: 281: Thyroxine (T4) in serum, Decreased
Short Name: T4 in serum, Decreased
Key Event Component
Process | Object | Action |
---|---|---|
abnormal circulating thyroxine level | thyroxine | decreased |
AOPs Including This Key Event
Stressors
Name |
---|
Propylthiouracil |
Methimazole |
Biological Context
Level of Biological Organization |
---|
Tissue |
Organ term
Organ term |
---|
serum |
Evidence for Perturbation by Stressor
Propylthiouracil
6-n-propylthouracil is a classic positive control for inhibition of TPO
Perchlorate
Perchlorate ion (ClO− ₄) is a classic positive control for inhibition of NIS
Methimazole
Classic positive control
Domain of Applicability
Taxonomic ApplicabilityTerm | Scientific Term | Evidence | Links |
---|---|---|---|
human | Homo sapiens | High | NCBI |
rat | Rattus norvegicus | High | NCBI |
mouse | Mus musculus | High | NCBI |
chicken | Gallus gallus | Moderate | NCBI |
Xenopus laevis | Xenopus laevis | Moderate | NCBI |
Pig | Pig | High | NCBI |
zebrafish | Danio rerio | High | NCBI |
fathead minnow | Pimephales promelas | High | NCBI |
Life Stage | Evidence |
---|---|
All life stages | High |
Sex | Evidence |
---|---|
Female | High |
Male | High |
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 (Thienpont et al., 2011), 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 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.
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 inactive iodothyronines (rT3, 3,5-T2). T4 is the predominant TH in circulation, comprising approximately 80% of the TH excreted from the thyroid gland 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 result 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 (DeVito et al., 1999; Miller et al., 2009; Zoeller et al., 2007).
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).
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 a commonly used as a human clinical test method. Analytical determination of iodothyronines (T3, T4, rT3, T2) and their conjugates, though 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 ). 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
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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
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.
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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.
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Event: 1003: Decreased, Triiodothyronine (T3) in serum
Short Name: Decreased, Triiodothyronine (T3) in serum
Key Event Component
Process | Object | Action |
---|---|---|
abnormal circulating hormone level | 3,3',5'-triiodothyronine | decreased |
AOPs Including This Key Event
Biological Context
Level of Biological Organization |
---|
Tissue |
Organ term
Organ term |
---|
serum |
Domain of Applicability
Taxonomic ApplicabilityTerm | Scientific Term | Evidence | Links |
---|---|---|---|
zebrafish | Danio rerio | High | NCBI |
fathead minnow | Pimephales promelas | High | NCBI |
African clawed frog | Xenopus laevis | High | NCBI |
Life Stage | Evidence |
---|---|
All life stages | High |
Sex | Evidence |
---|---|
Unspecific | High |
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. 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 (TT4). TT4 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 is well established (Manzon and Youson, 1997; Yaoita and Brown, 1990). 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 cautious.
Life stage: Thyroid hormones are essential in all life stages, but elevations of circulating levels are associated with specific developmental events.
Sex: Thyroid hormones are essential in both sexes.
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. One of the most unique characteristics of TH is their ability to regulate their own concentration, not only in the plasma level, but also in the individual cell level, to maintain their homeostasis. This is succeed 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.
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 HLPC 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.
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.
- Brent GA. (2012). Mechanisms of thyroid hormone action. J Clin Invest. 122: 3035-3043.
- Cheng SY, Leonard JL, Davis PJ. (2010).Molecular aspects of thyroid hormone actions. Endocr Rev. 31:139–170.
- 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.
- 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.
- 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.
- 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.
- 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.
- Oetting A, Yen PM. (2007). New insights into thyroid hormone action. Best Pract Res Clin Endocrinol Metab. 21:193–208.
- Schussler, G.C. (2000). The thyroxine-binding proteins. Thyroid 10:141–149.
- 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.
- Visser WE, Friesema EC, Jansen J, Visser TJ. (2008). Thyroid hormone transport in and out of cells. Trends Endocrinol Metab. 19:50-56.
- Williams GR. (2008). Neurodevelopmental and neurophysiological actions of thyroid hormone. J Neuroendocrinol. 20:784–794.
- 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.
- 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: 1007: Reduced, Anterior swim bladder inflation
Short Name: Reduced, Anterior swim bladder inflation
Key Event Component
Process | Object | Action |
---|---|---|
swim bladder inflation | anterior chamber swim bladder | decreased |
AOPs Including This Key Event
Biological Context
Level of Biological Organization |
---|
Organ |
Organ term
Organ term |
---|
swim bladder |
Domain of Applicability
Taxonomic ApplicabilityTerm | Scientific Term | Evidence | Links |
---|---|---|---|
zebrafish | Danio rerio | High | NCBI |
fathead minnow | Pimephales promelas | High | NCBI |
Life Stage | Evidence |
---|---|
larvae | High |
Sex | Evidence |
---|---|
Unspecific | High |
Taxonomic: Teleost fish can be divided in two groups according to swim bladder morphology: physoclistous (e.g., yellow perch) and physostomus (e.g., zebrafish and fathead minnow). Physostomus fish retain a duct between the digestive tract and the swim bladder during adulthood allowing them to gulp air at the surface to fill the swim bladder. In contrast, in physoclistous fish, once initial inflation by gulping atmospheric air at the water surface has occurred, the swim bladder is closed off from the digestive tract and swim bladder volume is regulated by gas secretion into the swim bladder (Woolley and Qin, 2010). The evidence for impaired inflation of the anterior chamber of the swim bladder currently comes from work on zebrafish and fathead minnow (Stinckens et al., 2016; Nelson et al., 2016; Cavallin et al., 2017; Godfrey et al., 2017; Stinckens et al., 2020). While zebrafish and fathead minnows are physostomous fish with a two-chambered swim bladder, the Japanese rice fish (Oryzias latipes) is a physoclistous fish with a single chambered swim bladder that inflates during early development. The key event 'reduced anterior chamber inflation' is not applicable to such fish species.
Life stage: The anterior chamber inflates during a specific developmental time frame. In zebrafish, the anterior chamber inflates around 21 days post fertilization (dpf) which is during the larval stage. In the fathead minnow, the anterior chamber inflates around 14 dpf, also during the larval stage. Therefore this KE is only applicable to the larval life stage.
Sex: 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. Since the anterior chamber inflates around 21 days post fertilization, sex differences are expected to play a minor role.
Key Event Description
The swim bladder of bony fish is evolutionary homologous to the lung (Zheng et al., 2011). The teleost swim bladder is a gas-filled structure that consists of two chambers, the posterior and anterior chamber. In zebrafish, the posterior chamber inflates around 96 h post fertilization (hpf) which is 2 days post hatch, and the anterior chamber inflates around 21 dpf. In fathead minnow, the posterior and anterior chamber inflate around 6 and 14 dpf respectively. Inflation of the anterior swim bladder chamber is part of the larval-to-juvenile transition in fish, together with the development of adult fins and fin rays, ossification of the axial skeleton, formation of an adult pigmentation pattern, scale formation, maturation and remodeling of organs including the lateral line, nervous system, gut and kidneys (McMenamin and Parichy, 2013).
The anterior chamber is formed by evagination from the cranial end of the posterior chamber (Robertson et al., 2007). Dumbarton et al. (2010) showed that the anterior chamber of zebrafish has particularly closely packed and highly organized bundles of muscle fibres, suggesting that contraction of these muscles would reduce swim bladder volume. While it had previously been suggested that the posterior chamber had a more important role as a hydrostatic organ, this implies high importance of the anterior chamber for buoyancy. The anterior chamber has an additional role in hearing (Bang et al., 2002). Weberian ossicles (the Weberian apparatus) connect the anterior chamber to the inner ear resulting in an amplification of sound waves. Reduced inflation of the anterior chamber may manifest itself as either a complete failure to inflate the chamber or reduced size of the chamber. Reduced size is often associated with a deviating morphology.
How it is Measured or Detected
In several fish species, inflation of the anterior chamber can be observed using a stereomicroscope because the larvae are still transparent during the larval stage. This is for example true for zebrafish and fathead minnow. Anterior chamber size can then be measured based on photographs with a calibrator.
References
- Bang, P.I., Yelick, P.C., Malicko, J.J., Sewell, W.F. 2002. High-throughput behavioral screening method for detecting auditory response defects in zebrafish. Journal of Neuroscience Methods. 118, 177-187.
- Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.
- Dumbarton, T.C., Stoyek, M., Croll, R.P., Smith, F.M., 2010. Adrenergic control of swimbladder deflation in the zebrafish (Danio rerio). J. Exp. Biol. 213,2536–2546, http://dx.doi.org/10.1242/jeb.039792.
- Godfrey, A., Hooser, B., Abdelmoneim, A., Horzmann, K.A., Freemanc, J.L., Sepulveda, M.S., 2017. Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish. Aquatic Toxicology 193, 228-235.
- McMenamin, S.K., Parichy, D.M., 2013. Metamorphosis in Teleosts. Animal Metamorphosis 103, 127-165.
- Nelson KR, Schroeder AL, Ankley GT, Blackwell BR, Blanksma C, Degitz SJ, Flynn KM, Jensen KM, Johnson RD, Kahl MD, Knapen D, Kosian PA, Milsk RY, Randolph EC, Saari T, Stinckens E, Vergauwen L, Villeneuve DL. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole – Part I: fathead minnow. Aquatic Toxicology 173: 192-203.
- Roberston, G.N., McGee, C.A.S., Dumbarton, T.C., Croll, R.P., Smith, F.M., 2007. Development of the swim bladder and its innervation in the zebrafish, Danio rerio. J. Morphol. 268, 967–985, http://dx.doi.org/10.1002/jmor.
- 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.
- Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.
- Woolley, L.D., Qin, J.G., 2010. Swimbladder inflation and its implication to the culture of marine finfish larvae. Reviews in Aquaculture 2, 181-190.
- Zheng, W., Wang, Z., Collins, J.E., Andrews, R.M., Stemple, D., Gong, Z. 2011. Comparative transcriptome analyses indicate molecular homology of zebrafish swim bladder and mammalian lung. PLoS One 6, http://dx.doi.org/10.1371/
Event: 1005: Reduced, Swimming performance
Short Name: Reduced, Swimming performance
Key Event Component
Process | Object | Action |
---|---|---|
aquatic locomotion | decreased |
AOPs Including This Key Event
Biological Context
Level of Biological Organization |
---|
Individual |
Domain of Applicability
Taxonomic ApplicabilityTerm | Scientific Term | Evidence | Links |
---|---|---|---|
zebrafish | Danio rerio | High | NCBI |
teleost fish | teleost fish | High | NCBI |
fathead minnow | Pimephales promelas | High | NCBI |
Life Stage | Evidence |
---|---|
All life stages | High |
Sex | Evidence |
---|---|
Unspecific | High |
Taxonomic: Importance of swimming performance for natural behaviour is generally applicable to fish.
Life stage: Importance of swimming performance for natural behaviour is generally applicable across all life stages.
Sex: Importance of swimming performance for natural behaviour is generally applicable across sexes.
Key Event Description
Adequate swimming performance in fish is essential for behaviour such as foraging, predator avoidance and reproduction.
How it is Measured or Detected
For fish larvae, automated observation and tracking systems are commercially available and increasingly used for measuring swimming performance including distance travelled, duration of movements, swimming speed, etc. This kind of measurements is often included in publications describing effects of chemicals in zebrafish larvae (Hagenaars et al., 2014; Stinckens et al., 2016; Vergauwen et al., 2015).
For juvenile and adult fish, measurements of swim performance vary. However, in some circumstances, a swim tunnel has been used to measure various data (Fu et al., 2013).
References
Fu C, Cao ZD, Fu SJ. 2013. The effects of caudal fin loss and regeneration on the swimming performance of three cyprinid fish species with different swimming capactities. The Journal of Experimental Biology 216:3164-3174. doi:10.1242/jeb.084244
Hagenaars, A., Stinckens, E., Vergauwen, L., Bervoets, L., Knapen, D., 2014. PFOSaffects posterior swim bladder chamber inflation and swimming performanceof zebrafish larvae. Aquat. Toxicol. 157, 225–235.
Stinckens, E., Vergauwen, L., Schroeder, A.L., Maho, W., Blackwell, B., Witter, H.,Blust, R., Ankley, G.T., Covaci, A., Villenueve, D.L., Knapen, D., 2016. Disruption of thyroid hormone balance after 2-mercaptobenzothiazole exposure causes swim bladder inflation impairment—part II: zebrafish. Aquat. Toxicol. 173:204-17.
Vergauwen, Lucia; Nørgaard Schmidt, Stine; Maho, Walid; Stickens, Evelyn; Hagenaars, An; Blust, Ronny; Mayer, Philipp; Covaci, Adrian; Knapen, Dries. 2014. A high throughput passive dosing format for the Fish Embryo Acute Toxicity test. Chemosphere. 139: 9-17.
List of Adverse Outcomes in this AOP
Event: 351: Increased Mortality
Short Name: Increased Mortality
Key Event Component
Process | Object | Action |
---|---|---|
mortality | increased |
AOPs Including This Key Event
Biological Context
Level of Biological Organization |
---|
Population |
Domain of Applicability
Taxonomic ApplicabilityTerm | Scientific Term | Evidence | Links |
---|---|---|---|
zebrafish | Danio rerio | High | NCBI |
Gallus gallus | Gallus gallus | High | NCBI |
fathead minnow | Pimephales promelas | High | NCBI |
Life Stage | Evidence |
---|---|
All life stages | High |
Sex | Evidence |
---|---|
Unspecific | High |
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 is typically measured by observation. Lack of any heart beat, gill movement, and body movement are typical signs of death used in the evaluation of mortality of animals.
Mortality can be measured:
- in the lab by recording mortality during prolonged exposure experiments
- in dedicated mesocosms, or in drainable ponds
- in the field, for example by determining age structure after one capture, or by capture-tag-recapture efforts
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
Process | Object | Action |
---|---|---|
population growth rate | population of organisms | decreased |
AOPs Including This Key Event
Biological Context
Level of Biological Organization |
---|
Population |
Domain of Applicability
Taxonomic ApplicabilityTerm | Scientific Term | Evidence | Links |
---|---|---|---|
all species | all species | High | NCBI |
Life Stage | Evidence |
---|---|
All life stages | Not Specified |
Sex | Evidence |
---|---|
Unspecific | Not Specified |
Consideration of population size and changes in population size over time is potentially relevant to all living organisms.
Key Event Description
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
List of Adjacent Key Event Relationships
Relationship: 309: Thyroperoxidase, Inhibition leads to TH synthesis, Decreased
AOPs Referencing Relationship
AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding |
---|---|---|---|
Inhibition of Thyroperoxidase and Subsequent Adverse Neurodevelopmental Outcomes in Mammals | adjacent | High | Low |
Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | Moderate | Low |
Inhibition of thyroid peroxidase leading to impaired fertility in fish | adjacent | High | High |
Thyroperoxidase inhibition leading to altered amphibian metamorphosis | adjacent | High | Moderate |
Thyroperoxidase inhibition leading to increased mortality via altered eye structure | adjacent |
Evidence Supporting Applicability of this Relationship
Life Stage | Evidence |
---|---|
All life stages | High |
Sex | Evidence |
---|---|
Male | High |
Female | High |
Taxonomic: Inhibition of TPO activity is widely accepted to directly impact TH synthesis. This is true for both rats and humans, as well as some fishes, frogs and birds. Most of the data supporting a causative relationship between TPO inhibition and altered TH synthesis is derived from animal studies, in vitro thyroid microsomes from rats or pigs, and a limited number of human ex vivo (Nagasaka and Hidaka, 1976; Vickers et al., 2012) and clinical studies. There are data to support that gene mutations in TPO result in congenital hypothyroidism, underscoring the essential role of TPO in human thyroid hormone synthesis.
Life stage: Applicability to certain life stages may depend on the species and their dependence on maternally transferred thyroid hormones during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. Therefore, it is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH disruptors.
Key Event Relationship 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 (Taurog, 2005). Two commonly used reference chemicals, propylthiouracil (PTU) and methimazole (MMI), are drugs that inhibit the ability of TPO to: a) activate iodine and transfer it to thyroglobulin (Tg) (Davidson et al., 1978); and, b) couple thyroglobulin (Tg)-bound iodotyrosyls to produce Tg-bound thyroxine (T4) and triiodothyronine (T3) (Taurog, 2005).
Evidence Supporting this KER
The weight of evidence supporting a direct linkage between the MIE, TPO inhibition, and the KE of decreased TH synthesis, is strong and supported by more than three decades of research in animals, including humans (Cooper et al., 1982; Cooper et al.,1983; Divi and Doerge, 1994).
Biological PlausibilityThe biological plausibility for this KER is rated Strong. TPO is the only enzyme capable of de novo systhesis of TH. TPO catalyzes several reactions, including the oxidation of iodide, nonspecific iodination of tyrosyl residues of thyroglobulin (Tg) to form monoiodotyrosyl (MIT) or diiodotyrosyl (DIT) residues, and the coupling of these Tg-bound iodotyrosyls to produce Tg-bound T3 and T4 (Divi and Doerge, 1994; Kessler et al., 2008; Ruf et al., 2006; Taurog et al., 1996, 2005). Therefore, inhibition of TPO activity is widely accepted to directly impact TH synthesis.
Empirical EvidenceEmpirical support for this KER is strong. There are several papers that have measured alterations in TPO and subsequent effects on TH synthesis. Taurog et al. (1996) showed decreased guicaol activity, decreased bound I125, and subsequent decreases in newly formed T3 and T4 per molecule of Tg, following exposure to PTU, MMI and some antibiotics. Following in vivo exposure to PTU in rats (Cooper et al., 1982; 1983), there are concentration and time-dependent decreases in thyroid protein bound iodine and serum T4 and T3 that recovered one month after cessation of PTU exposure. In addition, measures of thyroidal iodine content were highly correlated with intra-thyroidal PTU concentration. Vickers et al. (2012) demonstrated dose- and time- dependent inhibition of TPO activity in both human and rat thyroid homogenates exposed to MMI. Tietge et al (2010) recently showed decreases in thyroidal T4 following MMI exposure in Xenopus. Also in Xenopus, Haselman et al (2020) showed decreases in thyroidal iodotyrosines (MIT/DIT) and iodothyronines (T4/T3) following exposure to MMI. Doerge et al (1998) showed that a tryphenylmethane dye, malachite green, inhibited TPO and lowered thyroxine production. A recent paper used a series of benzothiazoles and showed TPO inhibition (guicaol assay) and inhibition of TSH stimulated thyroxine release from Xenopus thyroid gland explant cultures (Hornung et al., 2015). Chemically induced Inhibition of TPO has also been shown to result in reduced TH synthesis in zebrafish (Raldua and Babin, 2009; Thienpont et al., 2011; Rehberger et al., 2018).
Temporal Evidence: The temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005). The impact of decreased TPO activity on thyroidal hormone synthesis is similar across all ages. Good evidence for the temporal relationship of the KER comes from thyroid system modeling (e.g., Degon et al., 2008; Fisher et al., 2013) using data using data from studies of iodine deficiency and chemicals that inhibit NIS. In addition, there is ample evidence of the temporal impacts of TPO inhibition on TH synthesis, using ex vivo and in vitro measures that demonstrate the time course of inhibition following chemical exposures, including some data from human thyroid microsomes and ex vivo thyroid slices (Vickers et al., 2012). Future work is needed that measures both TPO inhibition and TH production during development.
Dose-Response Evidence: Dose-response data is available from a number of studies that correlate TPO inhibition with decreased TH production measured using a variety of endpoints including iodine organification (e.g., Taurog et al., 1996), inhibition of guicaol oxidation in thyroid microsomes (e.g., Doerge and Chang, 2002), and direct measure of thyroid gland T4 concentrations (e.g., Hornung et al., 2015). However, there is a lack of dose-response data from developmental studies showing direct linkages from TPO inhibition to thyroidal TH synthesis.
Uncertainties and InconsistenciesWhile it is clear that TPO inhibition will lead to altered hormone synthesis, there is a need for data that will inform quantitative modeling of the relationship between TPO inhibition and the magnitude of effects on thyroid hormone synthesis.
It is important to note that data from studies on genistein highlight this uncertainty. Doerge and colleagues have demonstrated that for this compound up to 80% TPO inhibition did not result in decreased serum T4 in rats (Doerge and Chang, 2002). This is not consistent with other prototypical TPO inhibitors (e.g., PTU, MMI). It remains to be determined, if for some presently unknown reason, that genistein is an outlier or not. This again points to the need for quantitative modeling of the relationship between TPO inhibtion and downstream KEs.
Quantitative Understanding of the Linkage
In Xenopus laevis, Haselman et al.(2020) demonstrated temporal profiles of thyroidal iodotyrosines (MIT/DIT) and iodothyronines (T4/T3), the products of TPO activity, following exposure to three different model TPO inhibitors (MMI, PTU, MBT) at multiple concentrations. This study established that, in Xenopus, measurable decreases in the products of TPO activity can occur as early as 2 days of exposure during pro-metamorphosis. However, despite consistent profiles of some iodo-species across chemicals, other iodo-species showed inconsistent profiles across chemicals. This highlights the multiple mechanisms of TPO (iodination and coupling) and differential susceptiblity to inhibition of those mechanisms depending on the chemical's type of interaction with TPO. The most consistent concentration-response relationship across chemicals and over time was demonstrated by thyroidal T4, which is the most relevant product to subsequent key events. At the highest concentrations tested for each chemcial, thyroidal T4 was below detection by 7 days of exposure across all three TPO inhibitors. Keeping in mind that the thyroid gland has follicular lumen space where thyroglobulin/T4 is stored until proteolysis and release to the blood, full inhibition of TPO would result in a delayed measureable response due to the time it takes to deplete stored hormone. Regardless of the delay, the results from this study imply full inhibition of TPO by each of these three chemicals at the highest test concentrations, but would require chemical residue analysis and/or toxicokinetic modeling to relate cellular/tissue concentrations at the site of TPO catalysis to levels of inhibition via Michaelis-Menten kinetic descriptions.
Profiles of thyroidal iodinated species demonstrated by Haselman et al. (2020) across three different TPO inhibitors suggests that a high level of TPO inhibition must occur in order to elicit responses in subsequent key events. Although the level of TPO inhibition is not directly quantifiable from this study, these data suggest that at least 90-100% inhibition was occurring since circulating T4 was not detectable at 10 days of exposure to the highest concentrations of MMI and MBT. However, additional efforts would be necessary to determine the minimum level of TPO inhibition that leads to a measurable decrease in thyroidal T4 and subsequently circulating T4.
Response-response relationshipThere are only a limited number of studies where both TPO inhibition and iodine organification have been measured in vivo, and there are not enough data available to make any definitive quantitative correlations. One in vivo study in rats exposed to the TPO inhibitor genistein found no in vivo impact on serum thyroid hormone concentrations, even when TPO was inhibited up to 80% (Chang and Doerge, 2000).
Given that this is an MIE to KE relationship, there is only one response to evaluate in the relationship. Decreased TH synthesis, as measured by responses of iodinated species in the thyroid gland, is the result of TPO inhibition, which cannot be measured directly in vivo.
Time-scaleIn vivo, evaluations of TPO inhibition are limited to evaluation of the iodinated species, or products of TPO activity, present in the thyroid gland at a particular time. However, as stated previously, any measurable response in these iodinated species is not a discreet assessment of TPO activity given that the gland maintains storage of hormone in the follicular lumen space and any alteration of TPO activity would be detected once the stores begin to be depleted. In Xenopus laevis, Haselman et al. (2020) showed a decrease in thyroidal iodinated species after only 2 days of exposure to potent TPO inhibitor MMI during thyroid-mediated metamorphosis and within 4 days for PTU and MBT, both model TPO inhibitors.
Known modulating factorsIodine availability will impact the ability of TPO to iodinate tyrosine residues on thyroglobulin. Iodine availability to TPO can be impacted a number of ways. First, environmental availability of iodine can vary greatly depending on whether and how much iodine exists in surface waters for aquatic organisms (gill respirators) and in the diets of both terrestrial and aquatic organisms. Second, somewhat regardless of iodine availability through environmental uptake (i.e., barring extremely high iodine exposure), iodine is actively transported into the thyroid follicular cell from the blood via sodium-iodide symporter (NIS), which has been shown to be susceptible to inhibition by, for example, perchlorate. As such, iodine availability to TPO is mediated by functional NIS. Finally, iodine is not fully available to TPO on the apical surface of the thyroid follicular cell until it is transported through the apical membrane by pendrin, an anion exchange protein - mutations or inhibition of pendrin could affect iodine availability to TPO.
Hydrogen peroxide is also needed by TPO to mediate the oxidation of iodide, which is produced locally by dual oxidase (DUOX). A mutation or inhibition of DUOX will impact local production of H2O2 leading to lower oxidizing potential of TPO and less organification of iodide.
Known Feedforward/Feedback loops influencing this KERThyroid stimulating hormone (TSH) released from the pituitary positively regulates the synthesis and release of thyroid hormones from the thyroid gland. As such, when TPO is inhibited and thyroid hormone synthesis is decreased, lower systemic levels of hormone cause feedback from the pituitary via TSH to upregulate a number of processes in the thyroid gland as a means of compensation, including (but not limited to) enhanced gene expression of NIS and thyrocyte cell proliferation (Tietge et al., 2010; Haselman et al., 2020).
References
Chang HC, Doerge DR. Dietary genistein inactivates rat thyroid peroxidase in vivo without an apparent hypothyroid effect. Toxicol Appl Pharmacol 168:244–252 (2000).
Chang, J., Wang, M., Gui, W., Zhao, Y., Yu, L., Zhu, G., 2012. Changes in Thyroid Hormone Levels during Zebrafish Development. Zoological Science 29, 181-184.
Cooper DS, Kieffer JD, Halpern R, Saxe V, Mover H, Maloof F, Ridgway EC (1983) Propylthiouracil (PTU) pharmacology in the rat. II. Effects of PTU on thyroid function. Endocrinology 113:921-928.
Cooper DS, Saxe VC, Meskell M, Maloof F, Ridgway EC. Acute effects of propylthiouracil (PTU) on thyroidal iodide organification and peripheral iodothyronine deiodination: correlation with serum PTU levels measured by radioimmunoassay. J Clin Endocrinol Metab. 1982 54(1):101-7.
Davidson, B., Soodak, M., Neary, J.T., Strout, H.V., and Kieffer, J.D. (1978). The irreversible inactivation of thyroid peroxidase by methylmercaptoimidazole, thiouracil, and propylthiouracil in vitro and its relationship to in vivo findings. Endocrinology 103:871–882.
Divi, R. L., and Doerge, D. R. (1994). Mechanism-based inactivation of lactoperoxidase and thyroid peroxidase by resorcinol derivatives. Biochemistry 33(32), 9668-74.
Doerge DR, Chang HC, Divi RL, Churchwell Mechanism for inhibition of thyroid peroxidase by leucomalachite green. Chem Res Toxicol. 1998 11(9):1098-104.
Doerge DR, Chang HC. Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo. J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Sep 25;777(1-2):269-79
Hornung MW, Kosian PA, Haselman JT, Korte JJ, Challis K, Macherla C, Nevalainen E, Degitz SJ. In Vitro, Ex Vivo, and In Vivo Determination of Thyroid Hormone Modulating Activity of Benzothiazoles.Toxicol Sci. 2015 146(2):254-64.
Haselman, J.T., Olker, J.H., Kosian, P.A., Korte, J.J., Swintek, J.A., Denny, J.S., Nichols, J.W., Tietge, J.E., Hornung, M.W. and Degitz, S.J., 2020. Targeted pathway-based in vivo testing using thyroperoxidase inhibition to evaluate plasma thyroxine as a surrogate metric of metamorphic success in model amphibian Xenopus laevis. Toxicological Sciences, 175(2), pp.236-250.
Kessler, J., Obinger, C., and Eales, G. (2008). Factors influencing the study of peroxidase-generated iodine species and implications for thyroglobulin synthesis. Thyroid 18(7), 769-74, 10.1089/thy.2007.0310.
Nagasaka, A., and Hidaka, H. (1976). Effect of antithyroid agents 6-propyl-2-thiouracil and 1-mehtyl-2-mercaptoimidazole on human thyroid iodine peroxidase. J. Clin. Endocrinol. Metab. 43:152–158.
Opitz, R., Maquet, E., Zoenen, M., Dadhich, R., Costagliola, S., 2011. TSH Receptor Function Is Required for Normal Thyroid Differentiation in Zebrafish. Molecular Endocrinology 25, 1579-1599.
Power, D.M., Llewellyn, L., Faustino, M., Nowell, M.A., Bjornsson, B.T., Einarsdottir, I.E., Canario, A.V., Sweeney, G.E., 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol 130, 447-459.
Raldua, D., Babin, P.J., 2009. Simple, Rapid Zebrafish Larva Bioassay for Assessing the Potential of Chemical Pollutants and Drugs to Disrupt Thyroid Gland Function. Environmental Science & Technology 43, 6844-6850.
Rehberger, K., Baumann, L., Hecker, M., Braunbeck, T., 2018. Intrafollicular thyroid hormone staining in whole-mount zebrafish (Danio rerio) embryos for the detection of thyroid hormone synthesis disruption. Fish Physiology and Biochemistry 44, 997-1010.
Ruf, J., and Carayon, P. (2006). Structural and functional aspects of thyroid peroxidase. Archives of biochemistry and biophysics 445(2), 269-77, 10.1016/j.abb.2005.06.023.
Taurog, A., Dorris, M. L., and Doerge, D. R. (1996). Mechanism of simultaneous iodination and coupling catalyzed by thyroid peroxidase. Archives of biochemistry and biophysics 330(1), 24-32,
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.
Thienpont, B., Tingaud-Sequeira, A., Prats, E., Barata, C., Babin, P.J., Raldua, D., 2011. Zebrafish Eleutheroembryos Provide a Suitable Vertebrate Model for Screening Chemicals that Impair Thyroid Hormone Synthesis. Environmental Science & Technology 45, 7525-7532.
Tietge JE, Butterworth BC, Haselman JT, Holcombe GW, Hornung MW, Korte JJ, Kosian PA, Wolfe M, Degitz SJ. Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis. Aquat Toxicol. 2010 98(1):44-50
Vickers AE, Heale J, Sinclair JR, Morris S, Rowe JM, Fisher RL. Thyroid organotypic rat and human cultures used to investigate drug effects on thyroid function, hormone synthesis and release pathways. Toxicol Appl Pharmacol. 2012 260(1):81-8.
Walter, K.M., Miller, G.W., Chen, X.P., Yaghoobi, B., Puschner, B., Lein, P.J., 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (Danio rerio). General and Comparative Endocrinology 272, 20-32.
Relationship: 305: TH synthesis, Decreased leads to T4 in serum, Decreased
AOPs Referencing Relationship
AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding |
---|---|---|---|
Inhibition of Thyroperoxidase and Subsequent Adverse Neurodevelopmental Outcomes in Mammals | adjacent | High | Moderate |
XX Inhibition of Sodium Iodide Symporter and Subsequent Adverse Neurodevelopmental Outcomes in Mammals | adjacent | High | Moderate |
Sodium Iodide Symporter (NIS) Inhibition and Subsequent Adverse Neurodevelopmental Outcomes in Mammals | adjacent | High | High |
Inhibition of Na+/I- symporter (NIS) leads to learning and memory impairment | adjacent | High | Moderate |
Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | Moderate | Low |
Thyroperoxidase inhibition leading to altered amphibian metamorphosis | adjacent | High | Moderate |
Sodium Iodide Symporter (NIS) Inhibition leading to altered amphibian metamorphosis | adjacent | High | High |
Thyroperoxidase inhibition leading to increased mortality via altered eye structure | adjacent |
Evidence Supporting Applicability of this Relationship
Life Stage | Evidence |
---|---|
All life stages | High |
Sex | Evidence |
---|---|
Male | High |
Female | High |
While a majority of the empirical evidence comes from work with laboratory rodents, there is a large amount of supporting data from humans (with anti-hyperthyroidism drugs including propylthiouracil and methimazole), some amphibian species (e.g., frog), and some avian species (e.g, chicken). The following are samples from a large literature that supports this concept: Cooper et al. (1982; 1983); Hornung et al. (2010); Van Herck et al. (2013); Paul et al. (2013); Alexander et al. (2017).
Key Event Relationship Description
Thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) are synthesized by NIS and TPO in the thyroid gland as iodinated thyroglobulin (Tg) and stored in the colloid of thyroid follicles. Secretion from the follicle into serum is a multi-step process. The first involves thyroid stimulating hormone (TSH) stimulation of the separation of the peptide linkage between Tg and TH. The next steps involve endocytosis of colloid, fusion of the endosome with the basolateral membrane of the thyrocyte, and finally release of TH into blood. More detailed descriptions of this process can be found in reviews by Braverman and Utiger (2012) and Zoeller et al. (2007).
Evidence Supporting this KER
The weight of evidence linking these two KEs of decreased TH synthesis and decreased T4 in serum is strong. It is commonly accepted dogma that decreased synthesis in the thyroid gland will result in decreased circulating TH (serum T4).
Biological PlausibilityThe biological relationship between two KEs in this KER is well understood and documented fact within the scientific community.
Empirical EvidenceIt is widely accepted that TPO inhibition leads to declines in serum T4 levels in adult mammals. This is due to the fact that the sole source for circulating T4 derives from hormone synthesis in the thyroid gland. Indeed, it has been known for decades that insufficient dietary iodine will lead to decreased serum TH concentrations due to inadequate synthesis. Strong qualitative and quantitative relationships exist between reduced TH synthesis and reduced serum T4 (Ekerot et al., 2013; Degon et al., 2008; Cooper et al., 1982; 1983; Leonard et al., 2016; Zoeller and Tan, 2007). There is more limited evidence supporting the relationship between decreased TH synthesis and lowered circulating hormone levels during development. Lu and Anderson (1994) followed the time course of TH synthesis, measured as thyroxine secretion rate, in non-treated pregnant rats and correlated it with serum T4 levels More recently, modeling of TH in the rat fetus demonstrates the quantitative relationship between TH synthesis and serum T4 concentrations (Hassan et al., 2017). Furthermore, a wide variety of drugs and chemicals that inhibit TPO are known to result in decreased release of TH from the thyroid gland, as well as decreased circulating TH concentrations. This is evidenced by a very large number of studies that employed a wide variety of techniques, including thyroid gland explant cultures, tracing organification of 131-I and in vivo treatment of a variety of animal species with known TPO inhibitors (King and May,1984; Atterwill et al., 1990; Brown et al., 1986; Brucker-Davis, 1998; Haselman et al., 2020; Hornung et al., 2010; Hurley et al., 1998; Kohrle, 2008; Tietge et al., 2010).
Temporal Evidence: The temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005). There are currently no studies that measured both TPO synthesis and TH production during development. However, the impact of decreased TH synthesis on serum hormones is similar across all ages. Good evidence for the temporal relationship comes from thyroid system modeling of the impacts of iodine deficiency and NIS inhibition (e.g., Degon et al., 2008; Fisher et al., 2013). In addition, recovery experiments have demonstrated that serum thyroid hormones recovered in athyroid mice following grafting of in-vitro derived follicles (Antonica et al., 2012). In Xenopus, it has been shown that depression of TH synthesis in the thyroid gland precedes depression of circulating TH within 7 days of exposure during pro-metamorphosis (Haselman et al., 2020).
Dose-response Evidence: Dose-response data is lacking from studies that include concurrent measures of both TH synthesis and serum TH concentrations. However, data is available demonstrating correlations between thyroidal TH and serum TH concentrations during gestation and lactation during development (Gilbert et al., 2013). This data was used to develop a rat quantitative biologically-based dose-response model for iodine deficiency (Fisher et al., 2013). In Xenopus, dose-responses were demonstrated in both thyroidal T4 and circulating T4 following exposure to three TPO inhibitors (Haselman et al., 2020).
Uncertainties and InconsistenciesThere are no inconsistencies in this KER, but there are some uncertainties. The first uncertainty stems from the paucity of data for quantitative modeling of the relationship between the degree of synthesis decrease and resulting changes in circulating T4 concentrations. In addition, most of the data supporting this KER comes from inhibition of TPO, and there are a number of other processes (e.g., endocytosis, lysosomal fusion, basolateral fusion and release) that are not as well studied.
Quantitative Understanding of the Linkage
In rats, Hassan et al. (2020) demonstrated in vitro:ex vivo correlations of TPO inhibition using PTU and MMI and constructed a quantitative model relating level of TPO inhibition with changes in circulating T4 levels. They determined that 30% inhibition of TPO was sufficient to decrease circulating T4 levels by 20%.
In Xenopus, Haselman et al. (2020) collected temporal and dose-response data for both thyroidal and circulating T4 which showed strong qualitative concordance of the response-response relationship. A quantitative relationship exists therein, but is yet to be demonstrated mathematically in this species.
Response-response relationshipFisher et al. (2013) published a quantitative biologically-based dose-response model for iodine deficiency in the rat. This model provides quantitative relationships for thyroidal T4 synthesis (iodine organification) and predictions of serum T4 concentrations in developing rats. There are other computational models that include thyroid hormone synthesis. Ekerot et al. (2012) modeled TPO, T3, T4 and TSH in dogs and humans based on exposure to myeloperoxidase inhibitors that also inhibit TPO. This model was recently adapted for rat (Leonard et al., 2016) and Hassan et al (2017) have extended it to include the pregnant rat dam in response to TPO inhibition induced by PTU. While the original model predicted serum TH and TSH levels as a function of oral dose, it was not used to explicitly predict the relationship between serum hormones and TPO inhibition, or thyroidal hormone synthesis. Leonard et al. (2016) recently incorporated TPO inhibition into the model. Degon et al (2008) developed a human thyroid model that includes TPO, but does not make quantitative prediction of organification changes due to inhibition of the TPO enzyme. Further empirical support for the response-response relationship has been demonstrated in the amphibian model, Xenopus laevis, exposed to TPO inhibitors during pro-metamorphosis (Haselman et al., 2020) wherein temporal profiles were measured for both thyroidal and circulating T4.
Time-scaleGiven that the thyroid gland contains follicular lumen space filled with stored thyroglobulin/T4, complete inhibition of thyroid hormone synthesis at a given point in time will not result in an instantaneous decrease in circulating T4. The system will be capable of maintaining sufficient circulating T4 levels until the gland stores are depleted. The time it takes to deplete stored hormone will greatly depend on species, developmental status and numerous other factors.
In Xenopus, Haselman et al. (2020) demonstrated an approximately 5 day difference between a significant decrease in thyroidal T4 preceding a significant decrease in circulating T4 while exposed to a potent TPO inhibitor (MMI) continuously during pro-metamorphosis.
Known modulating factorsDuring Xenopus metamorphosis, circulating T4 steadily increases to peak levels at metamorphic climax. Therefore, during Xenopus metamorphosis, this KER is operable at an increased rate as compared to a system that is maintaining steady circulating T4 levels through homeostatic control. In this case, developmental status is a modulating factor for the rates and trajectories of these KEs.
Known Feedforward/Feedback loops influencing this KERThis KER is entirely influenced by the feedback loop between circulating T4 originating from the thyroid gland and circulating TSH originating from the pituitary. Intermediate biochemical processes exist within the hypothalamus to affirm feedback and coordinately release TSH from the pituitary. However, quantitative representations of these feedback processes are limited to models discussed previously.
In Xenopus, circulating levels of T4 increase through pro-metamorphosis indicating a "release" of feedback to allow circulating levels of T4 to increase and drive metamorphic changes (Sternberg et al., 2011). This provides evidence that homeostatic control of feedback can be developmentally dependent, and likely species dependent.
References
Alexander EK, Pearce EN, Brent GA, Brown RS, Chen H, Dosiou C, Grobman WA, Laurberg P, Lazarus JH, Mandel SJ, Peeters RP, Sullivan S. 2017 Guidelines of the American Thyroid Association for the Diagnosis and Management of Thyroid Disease During Pregnancy and the Postpartum. Thyroid. 2017 Mar;27(3):315-389.
Antonica F, Kasprzyk DF, Opitz R, Iacovino M, Liao XH, Dumitrescu AM, Refetoff S, Peremans K, Manto M, Kyba M, Costagliola S. Generation of functional thyroid from embryonic stem cells. Nature. 2012 491(7422):66-71.
Atterwill CK, Fowler KF. A comparison of cultured rat FRTL-5 and porcine thyroid cells for predicting the thyroid toxicity of xenobiotics. Toxicol In Vitro. 1990. 4(4-5):369-74.
Braverman, L.E. and Utiger, R.D. (2012). Werner and Ingbar's The Thyroid: A Fundamental and Clinical Text (10 ed.). Philadelphia, PA: Lippincott Williams & Wilkins. pp. 775-786. ISBN 978-1451120639.
Brown CG, Fowler KL, Nicholls PJ, Atterwill C. Assessment of thyrotoxicity using in vitro cell culture systems. Food Chem Toxicol. 1986 24(6-7):557-62.
Brucker-Davis F. Effects of environmental synthetic chemicals on thyroid function. Thyroid. 1998 8(9):827-56.
Cooper DS, Kieffer JD, Halpern R, Saxe V, Mover H, Maloof F, Ridgway EC (1983) Propylthiouracil (PTU) pharmacology in the rat. II. Effects of PTU on thyroid function. Endocrinology 113:921-928.
Cooper DS, Saxe VC, Meskell M, Maloof F, Ridgway EC.Acute effects of propylthiouracil (PTU) on thyroidal iodide organification and peripheral iodothyronine deiodination: correlation with serum PTU levels measured by radioimmunoassay. J Clin Endocrinol Metab. 1982 54(1):101-7.
Degon, M., Chipkin, S.R., Hollot, C.V., Zoeller, R.T., and Chait, Y. (2008). A computational model of the human thyroid. Mathematical Biosciences 212, 22–53
Ekerot P, Ferguson D, Glämsta EL, Nilsson LB, Andersson H, Rosqvist S, Visser SA. Systems pharmacology modeling of drug-induced modulation of thyroid hormones in dogs and translation to human. Pharm Res. 2013 30(6):1513-24.
Fisher JW, Li S, Crofton K, Zoeller RT, McLanahan ED, Lumen A, Gilbert ME. Evaluation of iodide deficiency in the lactating rat and pup using a biologically based dose-response model. Toxicol Sci. 2013 132(1):75-86.
Gilbert ME, Hedge JM, Valentín-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett JM, Fisher JW. An animal model of marginal iodine deficiency during development: the thyroid axis and neurodevelopmental outcome. Toxicol Sci. 2013 132(1):177-95.
Haselman, J.T., Olker, J.H., Kosian, P.A., Korte, J.J., Swintek, J.A., Denny, J.S., Nichols, J.W., Tietge, J.E., Hornung, M.W. and Degitz, S.J., 2020. Targeted pathway-based in vivo testing using thyroperoxidase inhibition to evaluate plasma thyroxine as a surrogate metric of metamorphic success in model amphibian Xenopus laevis. Toxicological Sciences, 175(2), pp.236-250.
Hassan, I., El-Masri, H., Ford, J., Brennan, A., Handa, S., Paul Friedman, K. and Gilbert, M.E., 2020. Extrapolating in vitro screening assay data for thyroperoxidase inhibition to predict serum thyroid hormones in the rat. Toxicological Sciences, 173(2), pp.280-292.
Hassan, I, El-Masri, H., Kosian, PA, Ford, J, Degitz, SJ and Gilbert, ME. Quantitative Adverse Outcome Pathway for Neurodevelopmental Effects of Thyroid Peroxidase-Induced Thyroid Hormone Synthesis Inhibition. Toxicol Sci. 2017 Nov 1;160(1):57-73
Hornung MW, Degitz SJ, Korte LM, Olson JM, Kosian PA, Linnum AL, Tietge JE. Inhibition of thyroid hormone release from cultured amphibian thyroid glands by methimazole, 6-propylthiouracil, and perchlorate. Toxicol Sci. 2010 118(1):42-51.
Hurley PM. Mode of carcinogenic action of pesticides inducing thyroid follicular cell tumors in rodents. Environ Health Perspect. 1998 106(8):437-45.
King DB, May JD. Thyroidal influence on body growth. J Exp Zool. 1984 Dec;232(3):453-60.
Köhrle J. Environment and endocrinology: the case of thyroidology. Ann Endocrinol (Paris). 2008 69(2):116-22.
Leonard JA, Tan YM, Gilbert M, Isaacs K, El-Masri H. Estimating margin of exposure to thyroid peroxidase inhibitors using high-throughput in vitro data, high-throughput exposure modeling, and physiologically based pharmacokinetic/pharmacodynamic modeling. Toxicol Sci. 2016 151(1):57-70.
Lu, M-H, and Anderson, RR. Thyroxine secretion rats during pregnancy in the rat. Endo Res. 1994. 20(4):343-364.
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.
Sternberg, R.M., Thoemke, K.R., Korte, J.J., Moen, S.M., Olson, J.M., Korte, L., Tietge, J.E. and Degitz Jr, S.J., 2011. Control of pituitary thyroid-stimulating hormone synthesis and secretion by thyroid hormones during Xenopus metamorphosis. General and comparative endocrinology, 173(3), pp.428-437.
Tietge, J.E., Butterworth, B.C., Haselman, J.T., Holcombe, G.W., Hornung, M.W., Korte, J.J., Kosian, P.A., Wolfe, M. and Degitz, S.J., 2010. Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis. Aquatic Toxicology, 98(1), pp.44-50.
Van Herck SL, Geysens S, Delbaere J, Darras VM. Regulators of thyroid hormone availability and action in embryonic chicken brain development. Gen Comp Endocrinol. 2013. 190:96-104.
Zoeller, R. T., Tan, S. W., and Tyl, R. W. (2007). General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical reviews in toxicology 37(1-2), 11-53.
Relationship: 2038: T4 in serum, Decreased leads to Decreased, Triiodothyronine (T3) in serum
AOPs Referencing Relationship
AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding |
---|---|---|---|
Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | Moderate | Moderate |
Thyroperoxidase inhibition leading to increased mortality via altered eye structure | adjacent |
Evidence Supporting Applicability of this Relationship
Life Stage | Evidence |
---|---|
Juvenile | Moderate |
larvae | Moderate |
Sex | Evidence |
---|---|
Unspecific | High |
Taxonomic: The evidence for a relationship between circulating T4 and T3 levels currently comes from work on zebrafish and fathead minnow.
Life stage: This key event relationship is applicable to late larvae and juveniles rather than to embryos, because of the presence of maternal TH in embryos.
Uncertainties during embryonic lifestage:
- A decrease in T4 was observed in fathead minnows exposed to 1 mg/L 2-mercaptobenzothiazole (MBT), a thyroperoxidase inhibitor, through 6 dpf (Nelson et al., 2016). In contrast, there was no observed effect on T3 in fathead minnows exposed to MBT through 6 dpf. Comparably, zebrafish exposed to 0.4 or 0.7 mg/L MBT through 120 hpf showed decreased T4 but not T3 (Stinckens et al., 2016). During this early larval life stage, T3 may have been derived from maternal T4. In addition, it could be produced from further depletion of any T4 still produced by the thyroid gland (as thyroperoxidase may not have been fully inhibited at the tested exposure concentrations).
- Since exposure to PFAS did result in decreased whole-body T4 and T3 in 5 day old zebrafish, the life-stage specificity possibly depends on the mechanism that lies at the basis of the TH changes (Wang et al., 2020). The exact mechanisms by which PFAS disrupt the thyroid hormone system remain uncertain. Compounds that directly reduce T3 levels (e.g., deiodinase inhibitors) in addition to reducing T4 levels via another mechanism can be expected to result in decreased T4 and T3 levels.
Sex: This KER is probably not sex-dependent since both females and males have the same mechanisms of TH synthesis and conversion. Additionally, zebrafish are undifferentiated gonochorists, and gonad differentiation starts only around 23-25 dpf (Uchida et al., 2002), and the current data is based on larvae younger than or around that age.
Key Event Relationship Description
When serum thyroxine (T4) levels are decreased, less T4 is available for conversion to the more biologically active triiodothyronine (T3). While some thyroid hormone (TH) disrupting mechanisms can immediately affect T3 levels, including deiodinase inhibition, other mechanisms reduce T4 levels, for example through inhibition of TH synthesis, leading to decreased T3 levels.
Since in fish early life stages TH are typically measured on a whole body level, it is currently uncertain whether TH levels changes occur at the serum and/or tissue level. Pending more dedicated studies, whole body TH levels are considered a proxy for serum TH levels.
This key event relationship is not always evident. This could be due to feedback/compensatory mechanisms that in some cases seem to be able to maintain T3 levels even though T4 levels are reduced, for example through increased conversion of T4 to T3 by deiodinases.
Evidence Supporting this KER
Biological PlausibilityWhen serum thyroxine (T4) levels are decreased, less T4 is available for conversion to the more biologically active triiodothyronine (T3). It is plausible to assume that while some thyroid hormone (TH) disrupting mechanisms can immediately affect T3 levels, including deiodinase inhibition, other mechanisms reduce T4 levels, for example through inhibition of TH synthesis, leading to decreased T3 levels.
Empirical Evidence- A decrease in whole-body T4 and T3 was observed in zebrafish exposed to methimazole from fertilization until the age of 21 and 32 days and to propylthiouracil until the age of 14, 21 and 32 days (Stinckens et al., 2020). Additionally, a strong correlation was observed between T4 and T3 levels. Both compounds are thyroperoxidase inhibitors expected to inhibit thyroid hormone synthesis.
- A dose-dependent decrease in whole-body T4 and T3 was observed in zebrafish exposed to perfluorooctanoic acid and perfluoropolyether carboxylic acids from fertilization until the age of 5 days (Wang et al., 2020). The exact mechanisms by which PFAS disrupt the thyroid hormone system remain uncertain.
- While T4 measurements could not be acquired in fathead minnows exposed to 1 mg/L 2-mercaptobenzothiazole, a thyroperoxidase inhibitor, for 14 days, a significant decrease in T3 was observed (Nelson et al., 2016). The decreased T3 levels were likely the result of reduced T4 synthesis.
- Since in fish early life stages THs are typically measured on a whole body level, it is currently uncertain whether TH levels changes occur at the serum and/or tissue level. Pending more dedicated studies, whole body TH levels are considered a proxy for serum TH levels.
- This relationship depends on the MIE that is causing the decrease in T3. For example, deiodinase inhibition results in reduced activation of T4 to T3 and thus in reduced T3 levels; increased T4 levels have been observed, probably as a compensatory mechanism in response to the lower T3 levels. For example, Cavallin et al. (2017) exposed fathead minnows to iopanoic acid, a deiodinase inhibitor, and observed T4 increases together with T3 decreases.
- This key event relationship is not always evident. This could be due to feedback/compensatory mechanisms that in some cases seem to be able to maintain T3 levels even though T4 levels are reduced, for example through increased conversion of T4 to T3 by deiodinases. Examples of studies showing reduced T4 levels in the absence of reduced T3 levels:
- Zebrafish exposed to 0.35 mg/L 2-mercaptobenzothiazole, a thyroperoxidase inhibitor, through 32 dpf showed decreased whole-body T4, but T3 levels showed particularly large variation and overall were not significantly decreased (Stinckens et al., 2016).
- Although T4 content of 28 dpf larval fathead minnows exposed to 32 or 100 µg/l methimazole, a thyroperoxidase inhibitor, was reduced, these fish showed no change in whole body T3 content (Crane et al., 2006). Significantly higher T3/T4 ratios in fish held in 100 µg/l methimazole suggest an increased conversion of T4 to T3 or reduced degradation and conjugation during continued exposure to methimazole
Quantitative Understanding of the Linkage
Stinckens et al. (2020, supplementary information) showed a significant linear relationship between whole body T3 and T4 concentrations at 21 and 32 days post fertilization after continuous exposure of zebrafish to methimazole and propylthiouracil, two inhibitors of TH synthesis.
Known Feedforward/Feedback loops influencing this KERThis key event relationship is not always evident. This could be due to feedback/compensatory mechanisms that in some cases seem to be able to maintain T3 levels even though T4 levels are reduced, for example through increased conversion of T4 to T3 by deiodinases. Examples of studies showing reduced T4 levels in the absence of reduced T3 levels:
- Zebrafish exposed to 0.35 mg/L 2-mercaptobenzothiazole, a thyroperoxidase inhibitor, through 32 dpf showed decreased whole-body T4, but T3 levels showed particularly large variation and overall were not significantly decreased (Stinckens et al., 2016).
- Although T4 content of 28 dpf larval fathead minnows exposed to 32 or 100 µg/l methimazole, a thyroperoxidase inhibitor, was reduced, these fish showed no change in whole body T3 content (Crane et al., 2006). Significantly higher T3/T4 ratios in fish held in 100 µg/l methimazole suggest an increased conversion of T4 to T3 or reduced degradation and conjugation during continued exposure to methimazole
This relationship depends on the MIE that is causing the decrease in T3. For example, deiodinase inhibition results in reduced activation of T4 to T3 and thus in reduced T3 levels; increased T4 levels have been observed, probably as a compensatory mechanism in response to the lower T3 levels. For example, Cavallin et al. (2017) exposed fathead minnows to iopanoic acid, a deiodinase inhibitor, and observed T4 increases together with T3 decreases.
References
Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.
Crane, H.M., Pickford, D.B., Hutchinson, T.H., Brown, J.A., 2006. The effects of methimazole on development of the fathead minnow, pimephales promelas, from embryo to adult. Toxicological Sciences 93, 278-285.
Nelson, K., Schroeder, A., Ankley, G., Blackwell, B., Blanksma, C., Degitz, S., Flynn, K., Jensen, K., Johnson, R., Kahl, M., Knapen, D., Kosian, P., Milsk, R., Randolph, E., Saari, T., Stinckens, E., Vergauwen, L., Villeneuve, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquatic Toxicology 173, 192-203.
Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.
Stinckens, E., Vergauwen, L., Schroeder, A., Maho, W., Blackwell, B., Witters, H., Blust, R., Ankley, G., Covaci, A., Villeneuve, D., Knapen, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish. Aquatic Toxicology 173, 204-217.
Wang, J.X., Shi, G.H., Yao, J.Z., Sheng, N., Cui, R.N., Su, Z.B., Guo, Y., Dai, J.Y., 2020. Perfluoropolyether carboxylic acids (novel alternatives to PFOA) impair zebrafish posterior swim bladder development via thyroid hormone disruption. Environment International 134.
Relationship: 1035: Decreased, Triiodothyronine (T3) in serum leads to Reduced, Anterior swim bladder inflation
AOPs Referencing Relationship
AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding |
---|---|---|---|
Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | Moderate | Moderate |
Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | Moderate | Moderate |
Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | Moderate | Moderate |
Evidence Supporting Applicability of this Relationship
Life Stage | Evidence |
---|---|
larvae | High |
Sex | Evidence |
---|---|
Unspecific | High |
Taxonomic: Teleost fish can be divided in two groups according to swim bladder morphology: physoclistous (e.g., yellow perch) and physostomus (e.g., zebrafish and fathead minnow). Physostomus fish retain a duct between the digestive tract and the swim bladder during adulthood allowing them to gulp air at the surface to fill the swim bladder. In contrast, in physoclistous fish, once initial inflation by gulping atmospheric air at the water surface has occurred, the swim bladder is closed off from the digestive tract and swim bladder volume is regulated by gas secretion into the swim bladder (Woolley and Qin, 2010). The evidence for impaired inflation of the anterior chamber of the swim bladder currently comes from work on zebrafish and fathead minnow (Stinckens et al., 2016; Nelson et al., 2016; Cavallin et al., 2017; Godfrey et al., 2017; Stinckens et al., 2020).
Life stage: The anterior chamber inflates during a specific developmental time frame. In zebrafish, the anterior chamber inflates around 21 days post fertilization (dpf) which is during the larval stage. In the fathead minnow, the anterior chamber inflates around 14 dpf, also during the larval stage. Therefore this KER is only applicable to the larval life stage.
Sex: This KER is probably not sex-dependent since both females and males rely on THs for regulation of vital processes. Additionally, zebrafish are undifferentiated gonochorists, and gonad differentiation starts only around 23-25 dpf (Uchida et al., 2002), after the time point of anterior chamber inflation (around 21 dpf in zebrafish).
Key Event Relationship Description
Thyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition and larval-to-juvenile transition in fish. Inflation of the anterior swim bladder chamber is part of the larval-to-juvenile transition in fish, together with the development of adult fins and fin rays, ossification of the axial skeleton, formation of an adult pigmentation pattern, scale formation, maturation and remodeling of organs including the lateral line, nervous system, gut and kidneys.
Evidence Supporting this KER
Biological PlausibilityThyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition (Liu and Chan, 2002) and larval-to-juvenile transition (Brown et al., 1997) in fish. Inflation of the anterior swim bladder chamber is part of the larval-to-juvenile transition in fish, together with the development of adult fins and fin rays, ossification of the axial skeleton, formation of an adult pigmentation pattern, scale formation, maturation and remodeling of organs including the lateral line, nervous system, gut and kidneys (Brown, 1997; Liu and Chan, 2002; McMenamin and Parichy, 2013).
Empirical Evidence- In a study in which embryo-larval fathead minnows (Pimephales promelas) were exposed to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole (MBT), T3 concentrations measured at 14dpf were reduced at the same concentration (1 mg/L) that significantly reduced anterior swim bladder inflation at the same time-point (Nelson et al. 2016).
- Maternal injection of T3, resulting in increased T3 concentrations in the eggs of striped bass (Morone saxatilis) lead to significant increases in both swim bladder inflation and survival (Brown et al., 1988).
- In the study of Cavallin et al. (2017) fathead minnow larvae were exposed to IOP, a model iodothyronine deiodinase inhibitor that is assumed to inhibit all three deiodinase enzymes (DIO1,2,3). The authors observed pronounced decreases of whole body T3 concentrations and increases of whole body T4 concentrations, together with impaired inflation of the anterior swim bladder chamber. More specifically, inflation was delayed and the size of the swim bladder chamber was reduced until the end of the exposure experiment. Since exposure was started after inflation of the posterior chamber, this study shows that DIO inhibition can directly affect anterior chamber inflation.
- In the study of Stinckens et al. (2020) a strong correlation between reduced T3 levels and reduced anterior chamber inflation was observed in zebrafish exposed to iopanoic acid, a deiodinase inhibitor, as well as methimazole and propylthiouracil, both thyroperoxidase inhibitors, from fertilization until the age of 32 days. Anterior chamber inflation was delayed and a number of larvae did not manage to inflate the anterior chamber by the end of the 32 day exposure period. Additionally, exposed fish that had inflated the swim bladder had reduced anterior chamber sizes.
- Since in fish early life stages THs are typically measured on a whole body level, it is currently uncertain whether TH levels changes occur at the serum and/or tissue level. Pending more dedicated studies, whole body TH levels are considered a proxy for serum TH levels.
- Reduced anterior chamber inflation upon disruption of the thyroid hormone system is in most cases, but not always, accompanied by reduced whole body T3 levels. Stinckens et al. (2016) found a consistent relationship between reduced whole body T4 levels, but not T3 levels, and reduced anterior chamber inflation. Possibly, local T4 levels in the swim bladder tissue were too low to allow for enough local activation to T3. This relates to the general uncertainty on serum versus tissue TH levels.
- The mechanism underlying the link between reduced T3 and reduced anterior chamber inflation remains unclear, but several hypotheses exist (Stinckens et al., 2020). For example, altered gas distribution between chambers could be the result of impaired development of smooth muscle fibers, delayed and/or impaired evagination of the anterior chamber, impaired anterior budding through altered Wnt and hedgehog signalling, etc.
- Increased T3 levels also seem to result in reduced swim bladder inflation. For example, Li et al. (2011) reported impairment of swim bladder inflation in Chinese rare minnows (Gobiocypris rarus) exposed to exogenous T3.
References
- Brown, C. L., Doroshov, S. I., Nunez, J. M., Hadley, C., Vaneenennaam, J., Nishioka, R. S. and Bern, H. A. (1988), Maternal triiodothyronine injections cause increases in swimbladder inflation and survival rates in larval striped bass, Morone saxatilis. J. Exp. Zool., 248: 168–176. doi: 10.1002/jez.1402480207
- Brown, D.D., 1997. The role of thyroid hormone in zebrafish and axolotl development. Proceedings of the National Academy of Sciences of the United States of America 94, 13011-13016.
- Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.
- Godfrey, A., Hooser, B., Abdelmoneim, A., Horzmann, K.A., Freemanc, J.L., Sepulveda, M.S., 2017. Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish. Aquatic Toxicology 193, 228-235.
- Li W, Zha J, Yang L, Li Z, Wang Z. Regulation of thyroid hormone related genes mRNA expression by exogenous T₃ in larvae and adult Chinese rare minnow (Gobiocypris rarus). Environ Toxicol Pharmacol. 2011 Jan;31(1):189-97. doi: 10.1016/j.etap.2010.10.007.
- Liu, Y.W., Chan, W.K., 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 70, 36-45.
- McMenamin, S.K., Parichy, D.M., 2013. Metamorphosis in Teleosts. Animal Metamorphosis 103, 127-165.
- Nelson KR, Schroeder AL, Ankley GT, Blackwell BR, Blanksma C, Degitz SJ, Flynn KM, Jensen KM, Johnson RD, Kahl MD, Knapen D, Kosian PA, Milsk RY, Randolph EC,Saari T, Stinckens E, Vergauwen L, Villeneuve DL. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquat Toxicol. 2016 Apr;173:192-203. doi: 10.1016/j.aquatox.2015.12.024.
- 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.
- Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.
- Uchida, D., Yamashita, M., Kitano, T., Iguchi, T., 2002. Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish. Journal of Experimental Biology 205, 711-718.
Relationship: 1034: Reduced, Anterior swim bladder inflation leads to Reduced, Swimming performance
AOPs Referencing Relationship
AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding |
---|---|---|---|
Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | Moderate | Low |
Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | Moderate | Low |
Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | Moderate | Low |
Evidence Supporting Applicability of this Relationship
Life Stage | Evidence |
---|---|
larvae | High |
Sex | Evidence |
---|---|
Unspecific | High |
Taxonomic: Importance of proper functioning of the swim bladder for supporting natural swimming behaviour can be plausibly assumed to be generally applicable to fish possessing an anterior chamber. Evidence exists for the role of the posterior chamber in swimming performance comes from a wide variety of freshwater and marine fish species. Evidence for the specific role of the anterior chamber is however less abundant.
Life stage: In zebrafish, the anterior chamber inflates around 21 days post fertilization (dpf) which is during the larval stage. In the fathead minnow, the anterior chamber inflates around 14 dpf, also during the larval stage. Therefore this KER is only applicable to the larval life stage. To what extent fish can survive and swim with partly inflated swim bladders during later life stages is unknown.
Sex: 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. Since the anterior chamber inflates around 21 days post fertilization, sex differences are expected to play a minor role.
Key Event Relationship Description
Effects on swim bladder inflation can alter swimming performance and buoyancy of fish, which is essential for predator avoidance, energy sparing, migration, reproduction and feeding behaviour, resulting in lower young-of-year survival.
Evidence Supporting this KER
The weight of evidence supporting a direct linkage between these two KEs, i.e. reduced anterior swim bladder inflation and reduced swimming performance, is weak.
Biological PlausibilityThe anterior chamber of the swim bladder has a function in regulating the buoyancy of fish, by altering the volume of the swim bladder (Roberston et al., 2007). Fish rely on the lipid and gas content in their body to regulate their position within the water column, with the latter being more efficient at increasing body buoyancy. Therefore, fish with functional swim bladders have no problem supporting their body (Brix 2002), while it is highly likely that impaired inflation severely impacts swimming performance. Fish with no functional swim bladder can survive, but are severely disadvantaged., making the likelihood of surviving smaller.
Several studies in zebrafish and fathead minnow showed that a smaller AC was associated with a larger posterior chamber (Nelson et al., 2016; Stinckens et al., 2016; Cavallin et al., 2017, Stinckens et al., submitted) suggesting a possible compensatory mechanism. As shown by Stoyek et al. (2011) however, the AC volume is highly dynamic under normal conditions due to a series of regular corrugations running along the chamber wall, and is in fact the main driver for adjusting buoyancy while the basic PC volume remains largely invariable. Therefore, it is plausible to assume that functionality of the swim bladder is affected when AC inflation is incomplete, even when the PC appears to fully compensate the gas volume of the swim bladder.
Empirical Evidence- Lindsey et al. (2010) showed that zebrafish started swimming deeper down in the water column upon inflation of the anterior chamber, confirming a role of the anterior chamber in supporting swimming performance.
- After exposure to 2-mercaptobenzothiazole, a TPO inhibitor, from 0 to 32 days post fertilization (dpf) in zebrafish, the swimming activity of fish was impacted starting at 26 dpf if the inflation of the anterior chamber of the swim bladder was impaired or had no normal structure/size (Stinckens et al., 2016).
- Methimazole (MMI) and propylthiouracil (PTU), two thyroperoxidase inhibitors, and iopanoic acid (IOP), a deiodinase inhibitor, each reduced both anterior chamber inflation and swimming distance in zebrafish exposed from fertlization until the age of 32 days (Stinckens et al., 2020). Stinckens et al. (2020) showed a specific, direct link between reduced anterior chamber inflation and reduced swimming performance. First, after 21 d of exposure to 111 mg/L propylthiouracil around 30% of anterior chambers were not inflated and swimming distance was reduced, while by 32 days post fertilization all larvae had inflated their anterior chamber (although chamber surface was still smaller) and the effect on swimming distance had disappeared. The most direct way to assess the role of anterior chamber inflation in swimming performance, however, is to compare larvae with and without inflated anterior chamber at the same time point and within the same experimental treatment. Both in the propylthiouracil exposure at 21 days post fertilization and in the iopanoic acid exposure at 21 and 32 days post fertilization, swimming distance was clearly reduced in larvae lacking an inflated anterior chamber, while the swimming distance of larvae with inflated anterior chamber was equal to that of controls.
- It has also been reported that larvae that fail to inflate their swim bladder use additional energy to maintain buoyancy (Lindsey et al., 2010, Goodsell et al. 1996), possibly contributing to reduced swimming activity. Furthermore, Chatain (1994) associated larvae with non-inflated swim bladders with numerous complications, such as spinal deformities and lordosis and reduced growth rates, adding to the impact on swimming behaviour.
- An increasing incidence of swim bladder non-inflation has also been reported in Atlantic salmon (Poppe et al. 1997). Affected fish had severely altered balance and buoyancy, observed through a specific swimming behaviour, as the affected fish were swimming upside down in an almost vertical position (Poppe et al. 1997).
After exposure to 100 mg/L methimazole, 95% of the zebrafish larvae failed to inflate their anterior chamber at 32 dpf and swimming distance was reduced (Stinckens et al., 2020). On the other hand, there was no effect of impaired anterior chamber inflation on swimming distance in the methimazole exposure of 50 mg/L. Also, inflated but smaller anterior chambers did not result in a decreased swimming performance in this study. A similar result, where non-inflated anterior chambers did not consistently lead to reduced swimming performance, was previously found after exposure to 2-mercaptobenzothiazole (Stinckens et al., 2016). In summary, the precise relationship between these two KEs is not easy to determine and may be different for different chemicals. Swimming capacity can be affected via other processes which may or may not depend on the HPT axis, such as decreased cardiorespiratory function, energy metabolism and growth.
As Robertson et al., (2007) reported, the swim bladder only starts regulating buoyancy actively from 32 dpf onward in zebrafish, possibly explaining the lack of effect on swimming capacity in some cases.
The anterior chamber is also important for producing and transducing sound through the Weberian Apparatus (Popper, 1974; Lechner and Ladich, 2008). It is highly plausible that impaired inflation or size of the anterior swim bladder could lead to a reduction in young-of-year survival as hearing loss would affect their ability to respond to their surrounding environment, thus impacting ecological relevant endpoints such as predator avoidance or prey seeking (Wisenden et al., 2008; Fay, 2009).
References
Brix O (2002) The physiology of living in water. In: Hart PJ, Reynolds J (eds) Handbook of Fish Biology and Fisheries, Vol. 1, pp. 70–96. Blackwell Publishing, Malden, USA.
Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.
Chatain, B., 1994. Abnormal swimbladder development and lordosis in sea bass (Dicentrarchus labrax) and sea bream (Sparus auratus). Aquaculture 119:371–379.
Czesny, S.J., Graeb, B.D.S., Dettmersn, J.M., 2005. Ecological consequences of swimbladder noninflation for larval yellow perch. Trans. Am. Fish. Soc. 134,1011–1020, http://dx.doi.org/10.1577/T04-016.1.
Fay, R., 2009. Soundscapes and the sense of hearing of fishes. Integrative Zool. 4,26–32.
Goodsell, D.S., Morris, G.M., Olsen, A.J. 1996. Automated docking of fleixble ligands. Applications of Autodock. J. Mol. Recogonition, 9:1-5.
Kurata, M., Ishibashi, Y., Takii, K., Kumai, H., Miyashita, S., Sawada, Y., 2014.Influence of initial swimbladder inflation failure on survival of Pacific bluefintuna, Thuunus orientalis (Temminck and Schlegl) larvae. Aquacult. Res. 45,882–892.
Lechner, W., Ladich, F., 2008. Size matters: diversity in swimbladders andWeberian ossicles affects hearing in catfishes. J. Exp. Biol. 211, 1681–1689.
Lindsey, B.W., Smith, F.M., Croll, R.P., 2010. From inflation to flotation: contribution of the swimbladder to whole-body density and swimming depth during development of the zebrafish (Danio rerio). Zebrafish 7, 85–96, http://dx.doi.org/10.1089/zeb.2009.0616.
Maack, G., Segner, H., 2003. Morphological development of the gonads in zebrafish. Journal of Fish Biology 62, 895-906.
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.
Poppe, T.T., Hellberg, H., Griffiths, D., Mendal, H. 1977. Swim bladder abnormality in farmed Atlantic salmon, Salmo salar. Diseases of aquatic organisms 30:73-76.
Roberston, G.N., McGee, C.A.S., Dumbarton, T.C., Croll, R.P., Smith, F.M., 2007.Development of the swim bladder and its innervation in the zebrafish, Danio rerio. J. Morphol. 268, 967–985, http://dx.doi.org/10.1002/jmor.
Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.
Stinckens, E., Vergauwen, L., Schroeder, A.L., Maho, W., Blackwell, B., Witter, H.,Blust, R., Ankley, G.T., Covaci, A., Villenueve, D.L., Knapen, D., 2016. Disruption of thyroid hormone balance after 2-mercaptobenzothiazole exposure causes swim bladder inflation impairment—part II: zebrafish. Aquat. Toxicol. 173:204-17.
Stoyek, M.R., Smith, F.M., Croll, R.P., 2011. Effects of altered ambient pressure on the volume and distribution of gas within the swimbladder of the adult zebrafish, Danio rerio. Journal of Experimental Biology 214, 2962-2972.
Wisenden, B.D., Pogatschnik, J., Gibson, D., Bonacci, L., Schumacher, A., Willet, A.,2008. Sound the alarm: learned association of predation risk with novelauditory stimuli by fathead minnows (Pimephales promelas) and glowlighttetras (Hemigrammus erythrozonus) after single simultaneous pairings withconspecific chemical alarm cues. Environ. Biol. Fish 81, 141–147.
Woolley, L.D., Qin, J.G., 2010. Swimbladder inflation and its implication to theculture of marine finfish larvae. Rev. Aquac. 2, 181–190, http://dx.doi.org/10.1111/j.1753-5131.2010.01035.x.
Relationship: 2212: Reduced, Swimming performance leads to Increased Mortality
AOPs Referencing Relationship
AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding |
---|---|---|---|
Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation | adjacent | Moderate | Low |
Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | Moderate | Low |
Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation | adjacent | Moderate | Low |
Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | Moderate | Low |
Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | Moderate | Low |
Evidence Supporting Applicability of this Relationship
Life Stage | Evidence |
---|---|
Adult | Moderate |
Juvenile | Moderate |
larvae | Moderate |
Sex | Evidence |
---|---|
Unspecific | Moderate |
Importance of swimming performance on survival is generally applicable to all hatched fish across life stages and sexes.
Key Event Relationship Description
Reduced swimming performance is likely to affect essential endpoints such as predator avoidance, feeding behaviour and reproduction. These parameters are biologicaly plausible to affect survival, especially in a non-laboratory environment where food is scarce and predators are abundant.
Evidence Supporting this KER
A direct relationship between reduced swimming performance and reduced survival is difficult to establish. There is however a lot of indirect evidence linking reduced swim bladder inflation to reduced survival (https://aopwiki.org/relationships/2213), which can be plausibly assumed to be related to reduced swimming performance.
For example, all zebrafish larvae that failed to inflate the posterior chamber after exposure to 2 mg/L iopanoic acid (IOP), died by the age of 9 dpf (Stinckens et al., 2020). Since larvae from the same group that were able to inflate the posterior chamber survived and the test was performed in the laboratory in optimal conditions, it is plausible to assume that the cause of death was the inability to swim and find food due to the failure to inflate the posterior swim bladder chamber.
Biological PlausibilityReduced swimming performance is likely to affect essential endpoints such as predator avoidance, feeding behaviour and reproduction. These parameters are biologicaly plausible to affect survival, especially in a non-laboratory environment where food is scarce and predators are abundant.
Empirical EvidenceA direct relationship between reduced swimming performance and reduced survival is difficult to establish. There is however a lot of indirect evidence linking reduced swim bladder inflation to reduced survival (see non-adjacent KER 1041), which can be plausibly assumed to be related to reduced swimming performance.
For example, all zebrafish larvae that failed to inflate the posterior chamber after exposure to 2 mg/L iopanoic acid (IOP), died by the age of 9 dpf (Stinckens et al., 2020). Since larvae from the same group that were able to inflate the posterior chamber survived and the test was performed in the laboratory in optimal conditions, it is plausible to assume that the cause of death was the inability to swim and find food due to the failure to inflate the posterior swim bladder chamber.
Uncertainties and InconsistenciesA direct relationship between reduced swimming performance and reduced survival is difficult to establish in a laboratory environment where food is abundant and there are no predators.
Quantitative Understanding of the Linkage
Quantitative understanding of this linkage is currently limited.
Time-scaleReduced swimming performance is not expected to immediately lead to mortality. Depending on the extent of the reduction in swimming performance and depending on the cause of death (e.g., starvation due to the inability to find food, being caught by a predator) the lag time may vary.
As an example, Stinckens et al. (2020) found that zebrafish larvae that failed to inflate the swim bladder at 5 dpf and did not manage to inflate it during the days afterwards died by the age of 9 dpf. Since zebrafish initiate exogenous feeding around day 5 when the yolk is almost completely depleted, there was a lag period of around 4 days after which reduced feeding resulted in mortality. Obviously, in a laboratory setup there is no increased risk of being caught by a predator.
References
Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.
Relationship: 2013: Increased Mortality leads to Decrease, Population trajectory
AOPs Referencing Relationship
AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding |
---|---|---|---|
Acetylcholinesterase Inhibition leading to Acute Mortality via Impaired Coordination & Movement | adjacent | ||
Acetylcholinesterase inhibition leading to acute mortality | adjacent | Moderate | Moderate |
Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation | adjacent | High | Moderate |
Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | High | Moderate |
Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation | adjacent | High | Moderate |
Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | High | Moderate |
Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation | adjacent | High | Moderate |
Thyroperoxidase inhibition leading to increased mortality via altered eye structure | adjacent |
Evidence Supporting Applicability of this Relationship
Life Stage | Evidence |
---|---|
All life stages | High |
Sex | Evidence |
---|---|
Unspecific | High |
Taxonomic: All organisms must survive to reproductive age in order to reproduce and sustain populations. The additional considerations related to survival made above are applicable to other fish species in addition to zebrafish and fathead minnows with the same reproductive strategy (r-strategist as described in the theory of MaxArthur and Wilson (1967)). The impact of reduced survival on population size is even greater for k-strategists that invest more energy in a lower number of offspring.
Life stage: Density dependent effects start to play a role in the larval stage of fish when free-feeding starts (Hazlerigg et al., 2014).
Sex: This linkage is independent of sex.
Key Event Relationship Description
- Increased mortality in the reproductive population may lead to a declining population.
- Increased mortality may lead to a declining population. This depends on the excess mortality due to the applied stressor and the environmental parameters such as food availability and predation rate. Most fish species are r-strategist, meaning they produce a lot of offspring instead of investing in parental care. This results in natural high larval mortality causing only a small percentage of the larvae to survive to maturity. If the excess larval mortality due to a stressor is small, the population dynamics might result in constant population size. Should the larval excess be more significant, or last on the long-term, this will affect the population. To calculate the long-term persistence of the population, population dynamic models should be used.
Evidence Supporting this KER
Survival rate is an obvious determinant of population size and is therefore included in population modeling (e.g., Miller et al., 2020).
Biological Plausibility- Survival to reproductive maturity is a parameter of demographic significance. Assuming resource availability (i.e., food, habitat, etc.) is not limiting to the extant population, sufficient mortality in the reproductive population may ultimately lead to declining population trajectories.
- Under some conditions, reduced larval survival may be compensated by reduced predation and increased food availability, and therefore not result in population decline (Stige et al., 2019).
- According to empirical data, combined with population dynamic models, feeding larvae are the crucial life stage in zebrafish (and other r-strategists) for the regulation of the population. (Schäfers et al., 1993)
- In fathead minnow, natural survival of early life stages has been found to be highly variable and influential on population growth (Miller and Ankley, 2004)
-
The extent to which larval mortality affects population size could depend on the fraction of surplus mortality compared to a natural situation.
-
There are scenarios in which individual mortality may not lead to declining population size. These include instances where populations are limited by the availability of habitat and food resources, which can be replenished through immigration. Effects of mortality in the larvae can be compensated by reduced competition for resources (Stige et al., 2019).
-
The direct impact of pesticides on migration behavior can be difficult to track in the field, and documentation of mortality during migration is likely underestimated (Eng 2017).
-
In general, there is not enough empirical data on the relationships between survival and population level effects in fish (Rearick et al., 2018) to optimize population models.
References
Beaudouin, R., Goussen, B., Piccini, B., Augustine, S., Devillers, J., Brion, F., Pery, A.R., 2015. An individual-based model of zebrafish population dynamics accounting for energy dynamics. PloS one 10, e0125841.
Caswell, H., 2000. Matrix population models. Sinauer Sunderland, MA, USA.
Eng, M.L., Stutchbury, B.J.M. & Morrissey, C.A. Imidacloprid and chlorpyrifos insecticides impair migratory ability in a seed-eating songbird. Sci Rep 7, 15176 (2017)
Hazlerigg, C.R., Lorenzen, K., Thorbek, P., Wheeler, J.R., Tyler, C.R., 2012. Density-dependent processes in the life history of fishes: evidence from laboratory populations of zebrafish Danio rerio. PLoS One 7, e37550.
MacArthur, R., Wilson, E., 1967. The Theory of Island Biogeography. Princeton: Princeton Univ. Press. 203 p.
Miller, D.H., Ankley, G.T., 2004. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17β-trenbolone as a case study. Ecotoxicology and Environmental Safety 59, 1-9.
Miller, D.H., Clark, B.W., Nacci, D.E. 2020. A multidimensional density dependent matrix population model for assessing risk of stressors to fish populations. Ecotoxicology and environmental safety 201, 110786
Pinceel, T., Vanschoenwinkel, B., Brendonck, L., Buschke, F., 2016. Modelling the sensitivity of life history traits to climate change in a temporary pool crustacean. Scientific reports 6, 29451.
Rearick, D.C., Ward, J., Venturelli, P., Schoenfuss, H., 2018. Environmental oestrogens cause predation-induced population decline in a freshwater fish. Royal Society open science 5, 181065.
Schäfers, C., Oertel, D., Nagel, R., 1993. Effects of 3, 4-dichloroaniline on fish populations with differing strategies of reproduction. Ecotoxicology and Ecophysiology, 133-146.
Stige, L.C., Rogers, L.A., Neuheimer, A.B., Hunsicker, M.E., Yaragina, N.A., Ottersen, G., Ciannelli, L., Langangen, Ø., Durant, J.M., 2019. Density‐and size‐dependent mortality in fish early life stages. Fish and Fisheries 20, 962-976.Hazlerigg, C.R.E., Tyler, C.R., Lorenzen, K., Wheeler, J.R., Thorbek, P., 2014. Population relevance of toxicant mediated changes in sex ratio in fish: An assessment using an individual-based zebrafish (Danio rerio) model. Ecological Modelling 280, 76-88.
Stige, L.C., Rogers, L.A., Neuheimer, A.B., Hunsicker, M.E., Yaragina, N.A., Ottersen, G., Ciannelli, L., Langangen, O., Durant, J.M., 2019. Density- and size-dependent mortality in fish early life stages. Fish and Fisheries 20, 962-976.
List of Non Adjacent Key Event Relationships
Relationship: 366: Thyroperoxidase, Inhibition leads to T4 in serum, Decreased
AOPs Referencing Relationship
AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding |
---|---|---|---|
Inhibition of Thyroperoxidase and Subsequent Adverse Neurodevelopmental Outcomes in Mammals | non-adjacent | High | Moderate |
Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation | non-adjacent | High | Low |
Thyroperoxidase inhibition leading to altered amphibian metamorphosis | non-adjacent | High | High |
Evidence Supporting Applicability of this Relationship
Life Stage | Evidence |
---|---|
All life stages | High |
Sex | Evidence |
---|---|
Male | High |
Female | High |
Use of TPO inhibitors as anti-hyperthyroidism drugs in humans and pets (Emiliano et al., 2010; Trepanier, 2006) and effects of these drugs on serum TH concentrations in rats (US EPA, 2005), amphibian, fish and avian species (Coady et al., 2010; Grommen et al., 2011; Nelson et al., 2016; Rosebrough et al., 2006; Stinckens et al.; 2020; Tietge et al., 2012), strongly supports a causative linkage between inhibition of TPO and decreased serum T4 across species.
Key Event Relationship Description
Thyroperoxidase (TPO) is the enzyme that catalyzes iodine organification of thyroglobulin to produce thyroglobulin (Tg)-bound T3 and T4 in the lumen of thyroid follicles. Tg-bound THs are endocytosed across the apical lumen-follicular cell membrane, undergo thyroglobulin proteolysis, followed by hormone section into the blood stream (see Taurog, 2005 for review). This indirect KER describes the relationship of TPO inhibition to reduced circulating levels of thyroid hormone (TH) in the serum.
Evidence Supporting this KER
The weight of evidence linking thyroperoxidase inhibition to reductions in circulating serum TH is strong. Many studies support this basic linkage. There is no inconsistent data.
Biological PlausibilityIt is a well-accepted fact that inhibition of the only enzyme capable of synthesizing THs, TPO, results in subsequent decrease in serum TH concentrations. A large amount of evidence from clinical and animal studies clearly support the commonly accepted dogma that inhibition of TPO leads to decreased serum THs.
Empirical EvidenceThe majority of research in support of this KER involve exposure to known TPO inhibitors and measurement of serum hormones. There are many in vivo studies that link decreases in serum TH concentrations with exposure to xenobiotics that inhibit thyroperoxidase (TPO) (Brucker-Davis, 1998; Hurley, 1998; Boas et al., 2006; Crofton, 2008; Kohrle, 2008; Pearce and Braverman, 2009; Murk et al., 2013).
While these studies support the connection between exposure to a known TPO inhibitor and decreased TH, many of these studies do not empirically measure TPO inhibition or decreased TH synthesis. Thus, many studies support the indirect linkage between TPO inhibition (for chemicals identified as TPO inhibitors in in vivo or ex vivo studies) and decreased TH, with the well accepted theory that these proceed via decreased TH synthesis. That exposure to TPO inhibitors leads to decreased serum TH concentrations, via decreased TH synthesis is strongly supported by decades of mechanistic research in a variety of species.
This indirect relationship is also evidenced by the use of clinically-relevant anti-hyperthyroidism drugs, MMI and PTU (Laurberg & Anderson, 2014; Sundaresh et al., 2013). These drugs are both recognized TPO inhibitors and are part of a standard drug-based regimen of care for clinically hyperthyroid patients including those with Grave's disease. Serum THs are measured as the bioindicator of successful treatment with anti-hyperthyroidism drugs; the actual decrease in TH synthesis in the thyroid gland is implied in the efficacious use of these drugs (Trepanier, 2006).
In rats, MMI and PTU are often used as control chemicals to decrease serum THs to study biological phenomena related to disruption of TH homeostasis (many examples, including Zoeller and Crofton, 2005; Morreale de Escobar et al, 2004; Schwartz et al., 1997; Herwig et al., 2014; Wu et al., 2013; Pathak et al., 2011). Further, MMI is recommended as a positive control for use in the Amphibian Metamorphosis (Frog) Assay within Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program (US EPA, 2009; Coady et al., 2010), an assay used to evaluate the potential for chemicals to disrupt TH homeostasis. PTU has been suggested a positive control chemical in the guidance for the Comparative Developmental Thyroid Assay (US EPA, 2005), a non-guideline assay used to evaluate the potential for chemicals to disrupt TH homeostasis during gestation and early neonatal development.
Additionally, evidence is available from studies investigating responses to TPO inhibitors in fish. For example, Stinckens et al. (2020) reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L of methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.
Thus, an indirect key event relationship between TPO inhibition and decreased serum THs is strongly supported by a large database of clinical medicine and investigative research with whole animals (with a great deal of supporting evidence in rats and frogs).
Temporal Evidence: The temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005). The qualitative impact of thyroperoxidase inhibition on serum hormones is similar across all ages. The temporal nature of the impact on serum THs by TPO inhibitors in developmental exposure studies is evidenced by the duration of exposure and developmental age (Goldey et al., 1995; Ahmed et al., 2010; Tietge et al., 2010), as well as recovery after cessation of exposure (Cooke et al., 1993; Goldey et al., 1995; Sawin et al., 1998; Axelstad et al., 2008; Shibutani et al., 2009; Lasley and Gilbert, 2011). The temporal relationship between TPO inhibitor exposure duration and serum hormone decreases in adult organisms has been widely demonstrated (e.g., Hood et al., 1999; Mannisto et al., 1979). In addition, MMI and PTU induced decreases in serum T4 are alleviated by thyroid hormone replacement in both fetal and postnatal age rats (Calvo et al., 1990; Sack et al., 1995; Goldey and Crofton, 1998). Computational modeling of the thyroid also provides evidence for the indirect temporal relationship between these two KEs (e.g., Degon et al., 2008; Fisher et a., 2013).
Dose-Response Evidence: Empirical data is available from enough studies in animals treated with TPO inhibitors during development to make it readily accepted dogma that a dose-response relationship exists between TPO inhibition and serum TH concentrations. Again, these studies do not empirically measure TPO inhibition or decreased TH synthesis, but rely on the strong support of decades of mechanistic research in a variety of species of the causative relationship between these KEs. Examples of dose-responsive changes in TH concentrations following developmental exposure to TPO inhibitors include studies a variety of species, including: rodents (Blake and Henning, 1985; Goldey et al., 1995; Sawin et al., 1998); frogs (Tietge et al., 2013); fish tissue levels (Elsalini and Rohr, 2003.); and, chickens (Wishe et al., 1979). Computational modeling of the thyroid also provides evidence for the indirect dose-response relationship between these two KEs (e.g., Leonard et al., 2016; Fisher et a., 2013).
Uncertainties and InconsistenciesThere are no inconsistencies in this KER, but there are some uncertainties. The predominant uncertainty regarding the indirect key event relationship between inhibition of TPO activity and decreased serum T4 is the quantitative nature of this relationship, i.e., to what degree must TPO be inhibited in order to decrease serum T4 by a certain magnitude. Many animal (rat) studies typically employ relatively high exposures of TPO-inhibiting chemicals that result in hypothyroidism (severe decrements in T4 and T3). Thus, a dose-response relationship between TPO inhibition and decreased serum T4 is not typically defined. However, there are numerous publications demonstrating clear dose- and duration- dependent relationships between TPO inhibitors dose and reduced serum T3 and T4 in rodent models (see for example: Cooper et al., 1983; Hood et al., 1999; Goldey et al., 2005; Gilbert, 2011). The relationship between maternal and fetal levels of hormone following chemically-induced TPO inhibiton has not been well characterized and may differ based on kinetics. Reductions in serum TH in the fetus, in rat and human is derived a chemical’s effect on the maternal thyroid gland as well as the fetal thyroid gland.
Quantitative Understanding of the Linkage
Response-response relationshipThe indirect linkage between exposure to known TPO inhibitors and decreased serum TH has not been defined quantitatively. The two key event relationships that mediate this relationship (TPO inhibition leading to decreased TH synthesis, and decreased TH synthesis leading to decreased serum TH) have been incorporated into some quantitative models. A quantitative biologically-based dose-response model for iodine deficiency in the rat includes relationships between thyroidal T4 synthesis and serum T4 concentrations in developing rats Fisher et al. (2013). Ekerot et al. (2012) modeled TPO, T3, T4 and TSH in dogs and humans based on exposure to myeloperoxidase inhibitors that also inhibit TPO and was has recently adapted for rat (Leonard et al., 2016). While the original model predicted serum TH and TSH levels as a function of oral dose, it was not used to explicitly predict the relationship between serum hormones and TPO inhibition, or thyroidal hormone synthesis. Leonard et al. (2016) recently incorporated TPO inhibition into the model. Degon et al (2008) developed a human thyroid model that includes TPO but does not make quantitative prediction of organification changes due to inhibition of the TPO enzyme.
References
Ahmed OM, Abd El-Tawab SM, Ahmed RG. Effects of experimentally induced maternal hypothyroidism and hyperthyroidism on the development of rat offspring: I. The development of the thyroid hormones-neurotransmitters and adenosinergic system interactions. Int J Dev Neurosci. 2010 28(6):437-54
Axelstad M, Hansen PR, Boberg J, Bonnichsen M, Nellemann C, Lund SP, Hougaard KS, Hass U. Developmental neurotoxicity of propylthiouracil (PTU) in rats: relationship between transient hypothyroxinemia during development and long-lasting behavioural and functional changes. Toxicol Appl Pharmacol. 2008 232(1):1-13.
Blake HH, Henning SJ. Effect of propylthiouracil dose on serum thyroxine, growth, and weaning in young rats. Am J Physiol. 1985 248(5 Pt 2):R524-30.
Boas M, Feldt-Rasmussen U, Skakkebaek NE, Main KM. Environmental chemicals and thyroid function. Eur J Endocrinol. 2006 154:599-611.
Brucker-Davis F. Effects of environmental synthetic chemicals on thyroid function. Thyroid. 1998 8:827-56.
Calvo R, Obregón MJ, Ruiz de Oña C, Escobar del Rey F, Morreale de Escobar G. Congenital hypothyroidism, as studied in rats. Crucial role of maternal thyroxine but not of 3,5,3'-triiodothyronine in the protection of the fetal brain. J Clin Invest. 1990 Sep;86(3):889-99.
Coady K, Marino T, Thomas J, Currie R, Hancock G, Crofoot J, McNalley L, McFadden L, Geter D, Klecka G. 2010. Evaluation of the amphibian metamorphosis assay: exposure to the goitrogen methimazole and the endogenous thyroid hormone L-thyroxine. Environmental toxicology and chemistry / SETAC. Apr;29:869-880.
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Relationship: 1039: T4 in serum, Decreased leads to Reduced, Anterior swim bladder inflation
AOPs Referencing Relationship
AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding |
---|---|---|---|
Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation | non-adjacent | Moderate | Moderate |
Evidence Supporting Applicability of this Relationship
Sex | Evidence |
---|---|
Unspecific | High |
The evidence for a relationship between circulating T4 levels and inflation of the anterior chamber of the swim bladder currently comes from work on zebrafish and fathead minnow.
This KER is probably not sex-dependent since both females and males rely on synthesis of THs for regulation of vital processes. Additionally, zebrafish are undifferentiated gonochorists, and gonad differentiation starts only around 23-25 dpf (Uchida et al., 2002), after the time point of anterior chamber inflation (around 21 dpf).
Key Event Relationship Description
Reduced T4 levels in serum prohibit local production of active T3 hormone by deiodinases expressed in the target tissues. There is evidence suggesting that anterior swim bladder inflation relies on increased thyroid hormone levels at this specific developmental time point.
Evidence Supporting this KER
Biological PlausibilityThyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition (Liu and Chan, 2002) and larval-to-juvenile transition (Brown et al., 1997) in fish. The formation of the anterior chamber coincides with the second transition phase (Winata et al., 2009) and with a peak in T4 synthesis (Chang et al., 2012) suggesting that anterior inflation is under thyroid hormone regulation. Since most of the more biologically active T3 originates from the conversion of T4, decreased circulatory T4 levels are plausibly linked to reduced anterior chamber inflation.
Empirical Evidence- Chang et al. (2012) observed an increase of whole body T4 concentrations in zebrafish larvae at 21 days post fertilization, corresponding to the timing of anterior swim bladder inflation.
- Nelson et al. (2016) showed reduced whole body T4 levels at 6 days post fertilization and delayed anterior inflation (lower proportion of inflation at 14 dpf compared to controls) after exposure of fathead minnow embryos to 2-mercaptobenzothiazole. All anterior chambers eventually inflated but their size was reduced and morphology deviated.
- Stinckens et al. (2016) showed reduced whole body T4 levels both at 5 (before anterior inflation) and 32 days post fertilization (after anterior inflation) when zebrafish were exposed to 2-mercaptobenzothiazole (a thyroperoxidase inhibitor) from 0 to 32 days post fertilization. A large percentage of MBT-exposed fish had an uninflated anterior swim bladder, although some recovery was observed over time.
- Stinckens et al. (2016) further showed a significant correlation between whole body T4 levels and anterior chamber volume, with reduced T4 levels leading to smaller anterior chambers.
- Stinckens et al. (2020) established a significant correlation between reduced whole body T4 levels and reduced anterior chamber volume in 32 day old zebrafish across two compound exposures. This includes methimazole and propylthiouracil, two inhibitors of TH synthesis. Godfrey et al. (2017) also observed impaored anterior chamber inflation after exposure of zebrafish to Methimazole. Stinckens et al. (2020) continued the follow-up until 32 dpf and observed no recovery.
- Chopra et al. (2019) found that a nonsense mutation of the duox gene, coding for the enzyme dual oxidase involved in thyroid hormone synthesis, resulted in decreased intrafollicular T4 levels and impaired anterior chamber inflation until at least 54 dpf in zebrafish.
The mechanism through which reduced T4 hormone concentrations in serum result in anterior chamber inflation impairment is not yet understood. The anterior chamber is formed by evagination from the cranial end of the posterior chamber (Robertson et al., 2007, Winata et al., 2009). Several hypotheses could explain effects on anterior chamber inflation due to reduced T4 levels:
- Evagination from the posterior chamber could be impaired. Villeneuve et al. (unpublished results) showed that although the anterior bud was present after exposure to a deiodinase 2 inhibitor, the anterior chamber did not inflate.
- The formation of the tissue layers of the anterior swim bladder could be affected, although Villeneuve et al. (unpublished results) observed intact tissue layers of the anterior swim bladder after exposure to a deiodinase 2 inhibitor.
- The anterior chamber is inflated with gas from the posterior chamber through the communicating duct. Impaired gas exchange between the two chambers could be at the basis of impaired anterior inflation. Both Nelson et al. (2016) and Stinckens et al. (2016) found that posterior chambers were larger when anterior chambers were smaller or not inflated at all. The sum of the areas of the posterior and anterior chambers remained constant independent of inflation of the anterior chamber (Stinkens et al., 2016). These results suggest retention of the gas in the posterior chamber.
- Since gas exchange relies on a functional communicating duct between the posterior and anterior chamber, and the communicating duct is known to progressively narrow and eventually close during development, a dysfunctional communicating duct or a closure prior to anterior inflation could inhibit inflation. However, Villeneuve et al. (unpublished results) showed that the communicating duct was anatomically intact and open after exposure to iopanoic acid (a deiodinase 2 inhibitor), still leading to impaired anterior inflation.
- Lactic acid production which is essential for producing gas to fill the swim bladder could be affected, although the observation that the total amount of gas in both chambers is not affected when anterior inflation is impaired seems to contradict this (Stinckens et al., 2016).
- Possibly there is an effect on the production of surfactant, which is crucial to maintain the surface tension necessary for swim bladder inflation.
Quantitative Understanding of the Linkage
Stinckens et al. (2016) showed a significant linear quantitative relationship between whole body T4 levels and anterior chamber volume (measured as surface in 2D images) in 32 day old juvenile zebrafish, with reduced T4 levels leading to smaller anterior chambers after continuous exposure to 2-mercaptobenzothiazole, a thyroid hormone synthesis inhibitor.
Stinckens et al. (2020, supplementary information) established a significant linear quantitative relationship between reduced T4 levels and reduced anterior chamber volume (measured as surface in 2D images) in 32 day old juvenile zebrafish across two compound exposures. This includes methimazole and propylthiouracil, two inhibitors of TH synthesis.
Known Feedforward/Feedback loops influencing this KERReduced anterior chamber inflation upon disruption of the thyroid hormone system is consistently accompanied by reduced whole body T4 levels when fish are exposed to thyroid hormone synthesis inhibitors. however, when fish are exposed to deiodinase inhibitors, in the absence of feedback processes, stable T4 levels would be expected. Stable T4 levels were indeed observed in 14, 21 and 32 day old zebrafish exposed to iopanoic acid, a deiodinase inhibitor. While Cavallin et al. (2017) found a consistent relationship between reduced whole body T3 levels, and reduced anterior chamber inflation after exposure of fathead minnows to iopanoic acid, they observed increased T4 levels. Possibly, the inhibition of the conversion of T4 to T3 resulted in a compensatory mechanism that increased T4 levels. This was accompanied by increases of deiodinase 2 and 3 mRNA levels, which also indicate a compensatory response to deiodinase inhibition.
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