AOP ID and Title:

Short Title: DIO2i anterior swim bladder

Authors

Lucia Vergauwen [1], [lucia.vergauwen(at)uantwerpen.be]

Evelyn Stinckens [1], [evelyn.stinckens(at)uantwerpen.be]

Dan Villeneuve [2], [villeneuve.dan(at)epa.gov]

Dries Knapen [1], [dries.knapen (at)uantwerpen.be]

[1] Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium

[2] United States Environmental Protection Agency, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN, USA.

Status

Abstract

Other than the difference in deiodinase (DIO) isoform, the current AOP is identical to the corresponding AOP leading from DIO1 inhibition to increased mortality via anterior swim bladder inflation (https://aopwiki.org/aops/158). The overall importance of DIO1 versus DIO2 in fish is not exactly clear. The current state of the art suggests that DIO2 is more important than DIO1 in regulating swim bladder inflation. Therefore the current AOP may be of higher biological relevance compared to AOP 158. Starting from reduced serum T3 levels, this AOP is identical to the AOP leading from thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation (https://aopwiki.org/aops/159).

This AOP describes the sequence of events leading from deiodinase inhibition to increased mortality via reduced anterior swim bladder inflation. Disruption of the thyroid hormone system is increasingly being recognized as an important toxicity pathway that can cause many adverse outcomes, including disruption of developmental processes. Three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates that activate or inactivate THs and are therefore important mediators of TH action. Type II deiodinase (DIO2) has thyroxine (T4) as a preferred substrate and is mostly important for converting T4 to the more biologically active triiodothyronine (T3). Inhibition of DIO2 therefore reduces T3 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 DIO2 reduces levels of T3, thereby prohibiting proper inflation of the anterior 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 (Cavallin et al., 2017; Godfrey et al., 2017; Stinckens et al., 2020). 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).

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

The development of these AOPs was mainly based on a series of dedicated experiments (using a set of reference chemicals as prototypical stressors) in zebrafish and fathead minnow that form the core of the empirical evidence. Specific literature searches were used to add evidence from other studies, mainly in zebrafish and fathead minnow. No systematic review approach was applied.

Summary of the AOP

Events

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

Key Event Relationships

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 exactly understood.

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

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 (Woolley 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 only applicable to larval development, which is the period where the anterior swim bladder chamber inflates. In all life stages, the conversion of T4 into more biologically active T3 is essential. Inhibition of deiodinase (DIO) therefore impacts swim bladder inflation in both early (https://aopwiki.org/aops/155) and late developmental life stages.

Sex: All key events in this AOP are plausibly applicable to both sexes. Sex differences are not often investigated in tests using early life stages of fish. In Medaka, sex can be morphologically distinguished as soon as 10 days post fertilization. Females appear more susceptible to thyroid‐induced swim bladder dysfunction compared with males (Godfrey et al., 2019). For zebrafish and fathead minnow, it is currently unclear whether sex-related differences are important in determining the magnitude of the changes across the sequence of events in this AOP. Different fish species have different sex determination and differentiation strategies. Zebrafish do not have identifiable heteromorphic sex chromosomes and sex is determined by multiple genes and influenced by the environment (Nagabhushana and Mishra, 2016). 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 in zebrafish, sex differences are expected to play a minor role in the current AOP. Fathead minnow gonad differentiation also occurs during larval development. Fathead minnows utilize a XY sex determination strategy and markers can be used to genotype sex in life stages where the sex is not yet clearly defined morphologically (Olmstead et al., 2011). Ovarian differentiation starts at 10 dph followed by rapid development (Van Aerle et al., 2004). At 25 dph germ cells of all stages up to the primary oocytes stage were present and at 120 dph, vitellogenic oocytes were present. The germ cells (spermatogonia) of the developing testes only entered meiosis around 90–120 dph. Mature testes with spermatozoa are present around 150 dph. Since the anterior chamber inflates around 14 days post fertilization (9 dph) in fathead minnows, 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 evidence from deiodinase knockdowns showing the link with reduced posterior chamber inflation and the essentiality for downstream effects, but anterior chamber inflation was not studied. There is additional indirect evidence that reduced thyroid hormone synthesis causes reduced anterior swim bladder inflation: Chopra et al. (2019) showed that knockdown of dual oxidase, important for thyroid hormone synthesis, reduced anterior swim bladder inflation. It should be noted that dual oxidase also plays a role in oxidative stress. There is also evidence that alleviation of the effect on anterior chamber inflation reduces the effect on swimming performance.

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 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, deiodinase inhibition can be assessed using an in chemico assay, measurements of T3 levels could be added to the Fish Embryo Acute Toxicity (FET) test (OECD TG 236), the Fish Early Life Stage Toxicity (FELS) Test (OECD TG210) and the Fish Sexual Development Test (FSDT) (OECD TG 234), and assessments of anterior chamber inflation and swimming performance could be added to the FELS Test and FSDT.

Thyroid hormone system disruption causes multiple unspecific effects. Addition of TH measurements could aid in increasing the diagnostic capacity of a battery of endpoints since they are specific to the TH system. A battery of endpoints would ideally include the MIE, the AO and TH levels as the causal link. It is also in this philosophy that TH measurements are currently being considered as one of the endpoints in project 2.64 of the OECD TG work plan, “Inclusion of thyroid endpoints in OECD fish Test Guidelines”. While T3 measurements showed low levels of variation and were highly predictive of downstream effects in dedicated experiments to support this AOP, more variability may be present in other studies. Because of the rapid development in fish, it is important to compare T3 levels within specific developmental stages. For example, clear changes in T3 levels have been observed in zebrafish at 14, 21 and 32 dpf (Stinckens et al., 2020) and in fathead minnows at 4, 6, 10, 14, 18 and 21 dpf (Nelson et al., 2016; Cavallin et al., 2017) using liquid chromatography tandem mass spectrometry (LC−MS/MS).

References

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.

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, Sepulveda MS. 2019. Sex-specific endocrine-disrupting effects of three halogenated chemicals in japanese medaka. Journal of Applied Toxicology. 39(8):1215-1223.

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.

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.

Nagabhushana A, Mishra RK. 2016. Finding clues to the riddle of sex determination in zebrafish. Journal of Biosciences. 41(1):145-155.

Olmstead AW, Villeneuve DL, Ankley GT, Cavallin JE, Lindberg-Livingston A, Wehmas LC, Degitz SJ. 2011. A method for the determination of genetic sex in the fathead minnow, pimephales promelas, to support testing of endocrine-active chemicals. Environmental Science & Technology. 45(7):3090-3095.

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.

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.

van Aerle R, Runnalls TJ, Tyler CR. 2004. Ontogeny of gonadal sex development relative to growth in fathead minnow. Journal of Fish Biology. 64(2):355-369.

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.

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: 1002: Inhibition, Deiodinase 2

Short Name: Inhibition, Deiodinase 2

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

Olker et al. (2019) identified 20 DIO2-specific inhibitors using a human recombinant DIO2 enzyme (e.g., tetramethrin, elzasonan). Another typical inhibitor of DIO2 (and DIO1 and 3) is iopanoic acid (IOP), which acts as a substrate of all three DIO isoforms (Renko et al., 2015). In fact, many compounds inhibit all three DIO isoforms. Olker et al. (2019) identified 93 compounds that inhibit DIOs 1, 2 and 3.

iopanoic acid

Stinckens et al. (2018)

PERFLUOROOCTANOIC ACID

Stinckens et al. (2018)

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic: Deiodination by DIO enzymes is known to exist in a wide range of vertebrates and invertebrates. This KE is plausibly applicable across vertebrates. Reports of inhibition of DIO2 activity are relatively scarce compared to DIO1. Studies reporting DIO2 inhibition have used human recombinant DIO2 enzyme (Olker et al., 2019), primary human astrocytes (Roberts et al., 2015), rat pituitary (Li et al., 2012), pig liver (Stinckens et al., 2018), Nile tilapia (Oreochromis niloticus) liver (Walpita et al., 2007). Evidence for fish (e.g., zebrafish and fathead minnow) is mostly indirect since DIO enzyme activity is usually not measured in chemical exposure experiments. Stinckens et al. (2018) showed that chemicals with DIO inhibitory potential in pig liver impaired swim bladder inflation in zebrafish, a thyroid hormone regulated process. Based on these results, DIO2 seemed to be more important than DIO1. Houbrechts et al. (2016) did however confirm decreased DIO2 activity in a DIO1-DIO2 knockdown zebrafish at the ages of 3 and 7 days post fertilization and Noyes confirmed decreased outer ring deiodination activity in fathead minnows exposed to BDE-209. Walpita et al. (2007) showed decreased DIO2 activity in the liver of Nile tilapia injected with dexamethasone.

In mammals, DIO2 controls the intracellular concentration of T3. The cells that express DIO2 locally produce T3 that can more rapidly access the thyroid receptors in the nucleus than T3 from plasma (Bianco et al., 2002). For example, DIO2 is highly expressed in the mammalian brain. In teleosts, DIO2 has a markedly higher activity level compared to other vertebrates and it is expressed in liver (Orozco and Valverde, 2005). This could explain why DIO2 inhibition seems to be more important than DIO1 inhibition in determining the adverse outcome in zebrafish (Stinckens et al., 2018).

Life stage: Deiodinase activity is important for all vertebrate life stages. Already during early embryonic development, deiodinase activity is needed to regulate thyroid hormone concentrations and coordinate developmental processes. DIO2 shows more marked changes in expression around the time of the embryo-larval and larval-to-juvenile transition periods during zebrafish development, highlighting its importance for early life stages (Vergauwen et al., 2018).

Sex: This KE is plausibly applicable to both sexes. Deiodinases are important for TH homeostasis and identical in both sexes. Therefore inhibition of deiodinases is not expected to be sex-specific.

Key Event Description

Disruption of the thyroid hormone system is increasingly being recognized as an important toxicity pathway, as it can cause many adverse outcomes. Thyroid hormones do not only play an important role in the adult individual, but they are also critical during embryonic development. Thyroid hormones (THs) play an important role in a wide range of biological processes in vertebrates including growth, development, reproduction, cardiac function, thermoregulation, response to injury, tissue repair and homeostasis. Numerous chemicals are known to disturb thyroid function, for example by inhibiting thyroperoxidase (TPO) or deiodinase (DIO), upregulating excretion pathways or modifying gene expression. The two major thyroid hormones are triiodothyronine (T3) and thyroxine (T4), both iodinated derivatives of tyrosine. Most TH actions depend on the binding of T3 to its nuclear receptors. Active and inactive THs are tightly regulated by enzymes called iodothyronine deiodinases (DIO). The activation occurs via outer ring deiodination (ORD), i.e. removing iodine from the outer, phenolic ring of T4 to form T3, while inactivation occurs via inner ring deiodination (IRD), i.e. removing iodine from the inner tyrosol ring of T4 or T3.

Three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates that activate or inactivate THs and are therefore important mediators of TH action. All deiodinases are integral membrane proteins of the thioredoxin superfamily that contain selenocysteine in their catalytic centre. Type I deiodinase is capable to convert T4 into T3, as well as to convert rT3 to the inactive thyroid hormone 3,3’ T2, through outer ring deiodination. rT3, rather than T4, is the preferred substrate for DIO1. furthermore, DIO1 has a very high Km (µM range, compared to nM range for DIO2) (Darras and Van Herck, 2012). Type II deiodinase (DIO2) is only capable of ORD activity with T4 as a preferred substrate (i.e., activation of T4 to T3). DIO3 can inner ring deiodinate T4 and T3 to the inactive forms of THs, reverse T3, (rT3) and 3,3’-T2 respectively. DIO2 is a transmembrane protein anchored to the endoplasmic reticulum and the active site faces the perinuclear cytosol.

How it is Measured or Detected

At this time, there are no approved OECD or EPA guideline protocols for measurement of DIO inhibition. Deiodination is the major pathway regulating T3 bioavailability in mammalian tissues. In vitro assays can be used to examine inhibition of deiodinase 2 (DIO2) activity upon exposure to thyroid disrupting compounds.

Several methods for deiodinase activity measurements are available. A first in vitro assay measures deiodinase activities by quantifying the radioactive iodine release from iodine-labelled substrates, depending on the preferred substrates of the isoforms of deiodinases (Forhead et al., 2006; Pavelka, 2010; Houbrechts et al., 2016; Stinckens et al., 2018). Each of these assays requires a source of deiodinase which can be obtained for example using unexposed pig liver tissue (available from slaughterhouses) or rat liver tissue. Olker et al. (2019) on the other hand used an adenovirus expression system to produce the DIO2 enzyme and developed an assay for nonradioactive measurement of iodide released using the Sandell-Kolthoff method in a 96-well plate format. This assay was then used to screen the ToxCast Phase 1 chemical library. The specific synthesis of DIO2 through the adenovirus expression system provides an important advantage over other methods where activity of the different deiodinase isoforms needs to be distinguished in other ways, such as based on differences in enzyme kinetics.

Measurements of in vivo deiodinase activity in tissues collected from animal experiments are scarce. Noyes et al. (2011) showed decreased rate of outer ring deiodination (mediated by DIO1 and DIO2) in whole fish microsomes after exposure to BDE-209. After incubation with the substrate, thyroid hormone levels were measured using LC-MS/MS. Houbrechts et al. (2016) confirmed DIO2 deiodination activity in a DIO1-DIO2 knockdown zebrafish at the ages of 3 and 7 days post fertilization. Decreased T3 levels are often used as evidence of DIO inhibition, for example after exposure to iopanoic acid, in fish species such as zebrafish (Stinckens et al., 2020) and fathead minnow (Cavallin et al., 2017). It should be noted that it is difficult to make the distinction between decreased T3 levels caused by outer ring deiodination mediated by DIO2 inhibition or DIO1 inhibition.

References

Bianco, A.C., Salvatore, D., Gereben, B., Berry, M.J., Larsen, P.R., 2002. Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocrine Reviews 23, 38-89.

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

Darras, V.M., Van Herck, S.L.J., 2012. Iodothyronine deiodinase structure and function: from ascidians to humans. Journal of Endocrinology 215, 189-206.

Forhead, A.J., Curtis, K., Kaptein, E., Visser, T.J., Fowden, A.L., 2006. Developmental control of iodothyronine deiodinases by cortisol in the ovine fetus and placenta near term. Endocrinology 147, 5988-5994.

Houbrechts, A.M., Delarue, J., Gabriels, I.J., Sourbron, J., Darras, V.M., 2016. Permanent Deiodinase Type 2 Deficiency Strongly Perturbs Zebrafish Development, Growth, and Fertility. Endocrinology 157, 3668-3681.

Li, N.N., Jiang, Y.Q., Shan, Z.Y., Teng, W.P., 2012. Prolonged high iodine intake is associated with inhibition of type 2 deiodinase activity in pituitary and elevation of serum thyrotropin levels. British Journal of Nutrition 107, 674-682.

Noyes PD, Hinton DE, Stapleton HM. 2011. Accumulation and debromination of decabromodiphenyl ether (bde-209) in juvenile fathead minnows (pimephales promelas) induces thyroid disruption and liver alterations. Toxicological Sciences. 122(2):265-274.

Olker, J.H., Korte, J.J., Denny, J.S., Hartig, P.C., Cardon, M.C., Knutsen, C.N., Kent, P.M., Christensen, J.P., Degitz, S.J., Hornung, M.W., 2019. Screening the ToxCast Phase 1, Phase 2, and e1k Chemical Libraries for Inhibitors of Iodothyronine Deiodinases. Toxicological Sciences 168, 430-442.

Orozco, A., Valverde, R.C., 2005. Thyroid hormone deiodination in fish. Thyroid 15, 799-813.

Pavelka, S., 2010. Radiometric enzyme assays: development of methods for extremely sensitive determination of types 1, 2 and 3 iodothyronine deiodinase enzyme activities. Journal of Radioanalytical and Nuclear Chemistry 286, 861-865.

Renko, K., Schache, S., Hoefig, C.S., Welsink, T., Schwiebert, C., Braun, D., Becker, N.P., Kohrle, J., Schomburg, L., 2015. An Improved Nonradioactive Screening Method Identifies Genistein and Xanthohumol as Potent Inhibitors of Iodothyronine Deiodinases. Thyroid 25, 962-968.

Roberts, S.C., Bianco, A.C., Stapleton, H.M., 2015. Disruption of Type 2 Iodothyronine Deiodinase Activity in Cultured Human Glial Cells by Polybrominated Diphenyl Ethers. Chemical Research in Toxicology 28, 1265-1274.

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

Vergauwen, L., Cavallin, J.E., Ankley, G.T., Bars, C., Gabriels, I.J., Michiels, E.D.G., Fitzpatrick, K.R., Periz-Stanacev, J., Randolph, E.C., Robinson, S.L., Saari, T.W., Schroeder, A.L., Stinckens, E., Swintek, J., Van Cruchten, S.J., Verbueken, E., Villeneuve, D.L., Knapen, D., 2018. Gene transcription ontogeny of hypothalamic-pituitary-thyroid axis development in early-life stage fathead minnow and zebrafish. General and Comparative Endocrinology 266, 87-100.

Walpita, C.N., Grommen, S.V., Darras, V.M., Van der Geyten, S., 2007. The influence of stress on thyroid hormone production and peripheral deiodination in the Nile tilapia (Oreochromis niloticus). Gen Comp Endocrinol 150, 18-25.

List of Key Events in the AOP

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

Short Name: Decreased, Triiodothyronine (T3) in serum

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic: The overall evidence supporting taxonomic applicability is strong. With few exceptions vertebrate species have circulating T3 and T4 that are bound to transport proteins in blood. Therefore, the current key event is plausibly applicable to vertebrates in general. Clear species differences exist in transport proteins (Yamauchi and Isihara, 2009). Specifically, the majority of supporting data for TH decreases in serum come from rat studies, and the predominant iodothyronine binding protein in rat serum is transthyretin (TTR). TTR demonstrates a reduced binding affinity for T4 when compared with thyroxine binding globulin (TBG), the predominant serum binding protein for T4 in humans. This difference in serum binding protein affinity for THs is thought to modulate serum half-life for T4; the half-life of T4 in rats is 12-24 hr, wherease the half-life in humans is 5-9 days (Capen, 1997). While these species differences impact hormone half-life, possibly regulatory feedback mechanisms, and quantitative dose-response relationships, measurement of serum THs is still regarded as a measurable key event causatively linked to downstream adverse outcomes.

THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in amphibian and larbean metamorphoses (Manzon and Youson, 1997; Yaoita and Brown, 1990) as well as fish development, embryo-to-larval transition and larval-to-juvenile transition (Thienpont et al., 2011; Liu and Chan, 2002) is well established. Their existence and importance has been also described in many differrent animal and plant kingdoms (Eales, 1997; Heyland and Moroz, 2005), while their role as environmental messenger via exogenous routes in echinoderms confirms the hypothesis that these molecules are widely distributed among the living organisms (Heyland and Hodin, 2004). However, the role of TH in the different species may differ depending on the expression or function of specific proteins (e.g receptors or enzymes) that are related to TH function, and therefore extrapolation between species should be done with caution.

Life stage: Thyroid hormones are essential in all life stages, but decreases of circulating levels are associated with specific developmental events. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, T4 levels are not expected to decrease in response to exposure to inhibitors of TH synthesis during these earliest stages of development. However, T3 levels are expected to decrease upon exposure to deiodinase inhibitors in any life stage, since maternal T4 needs to be activated to T3 by deiodinases similar to embryonically synthesized T4.

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

 

Key Event Description

There are two biologically active thyroid hormones (THs), triiodothyronine (T3) and thyroxine (T4), and a few less active iodothyronines (rT3, 3,5-T2), which are all derived from the modification of tyrosine molecules (Hulbert, 2000). However, the plasma concentrations of the other iodothyronines are significantly lower than those of T3 and T4. The different iodothyronines are formed by the sequential outer or inner ring monodeiodination of T4 by the deiodinating enzymes, Dio1, Dio2, and Dio3 (Gereben et al., 2008). Deiodinase structure is considered to be unique, as THs are the only molecules in the body that incorporate iodide.

The circulatory system serves as the major transport and delivery system for THs from synthesis in the gland to delivery to tissues. The majority of THs in the blood are bound to transport proteins (Bartalena and Robbins, 1993). In humans, the major transport proteins are TBG (thyroxine binding globulin), TTR (transthyretin) and albumin. The percent bound to these proteins in adult humans is about 75, 15 and 10 percent, respectively (Schussler 2000). Unbound (free) hormones are approximately 0.03 and 0.3 percent for T4 and T3, respectively. In serum, it is the free form of the hormone that is active.

There are major species differences in the predominant binding proteins and their affinities for THs (see section below on Taxonomic applicability). However, there is broad agreement that changes in serum concentrations of THs is diagnostic of thyroid disease or chemical-induced disruption of thyroid homeostasis (Zoeller et al., 2007).

It is notable that the changes measured in the TH concentration reflect mainly the changes in the serum transport proteins rather than changes in the thyroid status. These thyroid-binding proteins serve as hormonal store which ensure their even and constant distribution in the different tissues, while they protect the most sensitive ones in the case of severe changes in thyroid availability, like in thyroidectomies (Obregon et al., 1981). Until recently, it was believed that all of the effects of TH were mediated by the binding of T3 to the thyroid nuclear receptors (TRa and TRb), a notion which is now questionable due to the increasing evidence that support the non-genomic action of TH (Davis et al., 2010, Moeller et al., 2006). Many non-nuclear TH binding sites have been identified to date and they usually lead to rapid cellular response in TH-effects (Bassett et al., 2003), but the specific pathways that are activated in this regard need to be elucidated.

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

In regards to the brain, the TH concentration involves also an additional level of regulation, namely the hormonal transport through the Blood Brain Barrier (BBB) (Williams, 2008). The TRH and the TSH are actually regulating the production of pro-hormone T4 and in a lesser extent of T3, which is the biologically active TH. The rest of the required amount of T3 is produced by outer ring deiodination of T4 by the deiodinating enzymes D1 and D2 (Bianco et al., 2006), a process which takes place mainly in liver and kidneys but also in other target organs such as in the brain, the anterior pituitary, brown adipose tissue, thyroid and skeletal muscle (Gereben et al., 2008; Larsen, 2009). Both hormones exert their action in almost all tissues of mammals and they are acting intracellularly, and thus the uptake of T3 and T4 by the target cells is a crucial step of the overall pathway. The trans-membrane transport of TH is performed mainly through transporters that differ depending on the cell type (Hennemann et al., 2001; Friesema et al., 2005; Visser et al., 2008). Many transporter proteins have been identified up to date but the monocarboxylate transporters (Mct8, Mct10) and the anion-transporting polypeptide (OATP1c1) show the highest degree of affinity towards TH (Jansen et al., 2005).

T3 and T4 have significant effects on normal development, neural differentiation, growth rate and metabolism (Yen, 2001; Brent, 2012; Williams, 2008), with the most prominent ones to occur during the fetal development and early childhood. The clinical features of hypothyroidism and hyperthyroidism emphasize the pleiotropic effects of these hormones on many different pathways and target organs. The thyroidal actions though are not only restricted to mammals, as their high significance has been identified also for other vertebrates, with the most well-studied to be the amphibian metamorphosis (Furlow and Neff, 2006). The importance of the thyroid-regulated pathways becomes more apparent in iodine deficient areas of the world, where a higher rate of cretinism and growth retardation has been observed and linked to decreased TH levels (Gilbert et al., 2012). Another very common cause of severe hypothyroidism in human is the congenital hypothyroidism, but the manifestation of these effects is only detectable in the lack of adequate treatment and is mainly related to neurological impairment and growth retardation (Glinoer, 2001), emphasizing the role of TH in neurodevelopment in all above cases. In adults, the thyroid-related effects are mainly linked to metabolic activities, such as deficiencies in oxygen consumption, and in the metabolism of the vitamin, proteins, lipids and carbohydrates, but these defects are subtle and reversible (Oetting and Yen, 2007). Blood tests to detect the amount of thyroid hormone (T4) and thyroid stimulating hormone (TSH) are routinely done for newborn babies for the diagnosis of congenital hypothyroidism at the earliest stage possible.

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

Although the HPT axis is highly conserved, there are some differences between fish and mammals (Blanton and Specker, 2007; Deal and Volkoff, 2020). For example, in fish, corticotropin releasing hormone (CRH) often plays a more important role in regulating thyrotropin (TSH) secretion by the pituitary and thus thyroid hormone synthesis compared to TSH-releasing hormone (TRH). TTRs from fish have low sequence identity with human TTR, for example seabream TTR has 54% sequence identity with human TTR but the only amino acid difference within the thyroxine-binding site is the conservative substitution of Ser117 in human TTR to Thr117 in seabream TTR (Santos and Power, 1999; Yamauchi et al., 1999; Eneqvist et al., 2004). In vitro binding experiments showed that TH disrupting chemicals bind with equal or weaker affinity to seabream TTR than to the human TTR with polar TH disrupting chemicals, in particular, showing a more than 500-fold lower affinity for seabream TTR compared to human TTR (Zhang et al., 2018).

Zebrafish and fathead minnows are oviparous fish species in which maternal thyroid hormones are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic thyroid hormone synthesis is initiated. Maternal transfer of thyroid hormones, both T4 and T3, to the eggs has been demonstrated in zebrafish (Walpita et al., 2007; Chang et al., 2012) and fathead minnows (Crane et al., 2004; Nelson et al., 2016).

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

How it is Measured or Detected

T3 and T4 can be measured as free (unbound) or total (bound + unbound). Free hormone are considered more direct indicators of T4 and T3 activities in the body. The majority of T3 and T4 measurements are made using either RIA or ELISA kits. In animal studies, total T3 and T4 are typically measured as the concentrations of free hormone are very low and difficult to detect. Historically, the most widely used method in toxicology is RIA. The method is routinely used in rodent endocrine and toxicity studies. The ELISA method has become more routine in rodent studies. The ELISA method is a commonly used as a human clinical test method.

Recently, analytical determination of iodothyronines (T3, T4, rT3, T2) and their conjugates through methods employing HPLC and mass spectrometry have become more common (DeVito et al., 1999; Miller et al., 2009; Hornung et al., 2015; Nelson et al., 2016; Stinckens et al., 2016).

Any of these measurements should be evaluated for fit-for-purpose, relationship to the actual endpoint of interest, repeatability, and reproducibility. All three of the methods summarized above would be fit-for-purpose, depending on the number of samples to be evaluated and the associated costs of each method. Both RIA and ELISA measure THs by a an indirect methodology, whereas analytical determination is the most direct measurement available. All of these methods, particularly RIA, are repeatable and reproducible.

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

References

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

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

Bianco AC, Kim BW. (2006). Deiodinases: implications of the local control of thyroid hormone action. J Clin Invest. 116: 2571–2579.

Blanton ML, Specker JL. 2007. The hypothalamic-pituitary-thyroid (hpt) axis in fish and its role in fish development and reproduction. Crit Rev Toxicol. 37(1-2):97-115.

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

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

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

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

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

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

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

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

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

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

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

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

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

Gereben B, Zavacki AM, Ribich S, Kim BW, Huang SA, Simonides WS, Zeöld A, Bianco AC. (2008). Cellular and molecular basis of deiodinase-regulated thyroid hormone signalling. Endocr Rev. 29:898–938.

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

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

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

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

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

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

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

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

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

Larsen PR. (2009).Type 2 iodothyronine deiodinase in human skeletal muscle: new insights into its physiological role and regulation. J Clin Endocrinol Metab. 94:1893-1895.

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

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

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

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

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

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

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

Power DM, Llewellyn L, Faustino M, Nowell MA, Bjornsson BT, Einarsdottir IE, Canario AV, Sweeney GE. 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. 130(4):447-459.

Ruuskanen S, Hsu BY. 2018. Maternal thyroid hormones: An unexplored mechanism underlying maternal effects in an ecological framework. Physiological and Biochemical Zoology. 91(3):904-916.

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

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

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

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

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

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

Walpita CN, Van der Geyten S, Rurangwa E, Darras VM. 2007. The effect of 3,5,3'-triiodothyronine supplementation on zebrafish (danio rerio) embryonic development and expression of iodothyronine deiodinases and thyroid hormone receptors. Gen Comp Endocrinol. 152(2-3):206-214.

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

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

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

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

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

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

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

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

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

Event: 1007: Reduced, Anterior swim bladder inflation

Short Name: Reduced, Anterior swim bladder inflation

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic: Teleost fish can be divided in two groups according to swim bladder morphology: physoclistous (e.g., yellow perch, sea bass, striped bass) and physostomous (e.g., zebrafish and fathead minnow). Physostomous 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. Therefore, the current key event is plausibly applicable to physostomous fish in general.

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: This KE/KER plausibly applicable to both sexes. Sex differences are not often investigated in tests using early life stages of fish. In Medaka, sex can be morphologically distinguished as soon as 10 days post fertilization. Females appear more susceptible to thyroid‐induced swim bladder dysfunction compared with males (Godfrey et al., 2019). For zebrafish and fathead minnow, it is currently unclear whether sex-related differences are important in determining the magnitude of the changes in this KE/KER. Different fish species have different sex determination and differentiation strategies. Zebrafish do not have identifiable heteromorphic sex chromosomes and sex is determined by multiple genes and influenced by the environment (Nagabhushana and Mishra, 2016). 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 in zebrafish, sex differences are expected to play a minor role. Fathead minnow gonad differentiation also occurs during larval development. Fathead minnows utilize a XY sex determination strategy and markers can be used to genotype sex in life stages where the sex is not yet clearly defined morphologically (Olmstead et al., 2011). Ovarian differentiation starts at 10 dph followed by rapid development (Van Aerle et al., 2004). At 25 dph germ cells of all stages up to the primary oocytes stage were present and at 120 dph, vitellogenic oocytes were present. The germ cells (spermatogonia) of the developing testes only entered meiosis around 90–120 dph. Mature testes with spermatozoa are present around 150 dph. Since the anterior chamber inflates around 14 days post fertilization (9 dph) in fathead minnows, sex differences are expected to play a minor role in the current AOP.

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, Sepulveda MS. 2019. Sex-specific endocrine-disrupting effects of three halogenated chemicals in japanese medaka. Journal of Applied Toxicology. 39(8):1215-1223.

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.

Nagabhushana A, Mishra RK. 2016. Finding clues to the riddle of sex determination in zebrafish. Journal of Biosciences. 41(1):145-155.

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.

Olmstead AW, Villeneuve DL, Ankley GT, Cavallin JE, Lindberg-Livingston A, Wehmas LC, Degitz SJ. 2011. A method for the determination of genetic sex in the fathead minnow, pimephales promelas, to support testing of endocrine-active chemicals. Environmental Science & Technology. 45(7):3090-3095.

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., 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.

van Aerle R, Runnalls TJ, Tyler CR. 2004. Ontogeny of gonadal sex development relative to growth in fathead minnow. Journal of Fish Biology. 64(2):355-369.

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

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic: Importance of swimming performance for natural behaviour is generally applicable to fish and tho other taxa that rely on swimming to support vital behaviours.

Life stage: Importance of swimming performance for natural behaviour is generally applicable across all free-swimming life stages, i.e., post-embryonic 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, swim tunnels have been used to measure various data (Fu et al., 2013).

Little and Finger (1990) discussed swimming behavior as an indicator of sublethal toxicity in fish.

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. PFOS affects posterior swim bladder chamber inflation and swimming performanceof zebrafish larvae. Aquat. Toxicol. 157, 225–235.

Little EE, Finger SE. 1990. Swimming behavior as an indicator of sublethal toxicity in fish. Environmental Toxicology and Chemistry. 9(1):13-19.

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

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

All living things are susceptible to mortality.

Key Event Description

Increased mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time.

How it is Measured or Detected

Mortality of animals is generally observed as cessation of the heart beat, breathing (gill or lung movement) and locomotory movements. Mortality is typically measured by observation. Depending on the size of the organism, instruments such as microscopes may be used. The reported metric is mostly the mortality rate: the number of deaths in a given area or period, or from a particular cause.

Depending on the species and the study setup, mortality can be measured:

• in the lab by recording mortality during exposure experiments
• in dedicated setups simulating a realistic situation such as mesocosms or drainable ponds for aquatic species
• in the field, for example by determining age structure after one capture, or by capture-mark-recapture efforts. The latter is a method commonly used in ecology to estimate an animal population's size where it is impractical to count every individual.

Regulatory Significance of the AO

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

Event: 360: Decrease, Population trajectory

Short Name: Decrease, Population trajectory

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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

Key Event Description

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

How it is Measured or Detected

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

Regulatory Significance of the AO

Maintenance of sustainable fish and wildlife populations (i.e., adequate to ensure long-term delivery of valued ecosystem services) is a widely accepted regulatory goal upon which risk assessments and risk management decisions are based.

References

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

Appendix 2

List of Key Event Relationships in the AOP