Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation

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.

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., 2007).

This AOP describes how inhibition of TPO results in decreased synthesis of THs 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 results in a decreased ability to forage and avoid predators, this reduces chances of survival. The final adverse outcome is a decrease of the population growth rate. 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). Convincing evidence comes from a TPO knockout study in zebrafish confirming that anterior inflation was reduced (Fang et al. 2022). 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, another enzyme that is important for TH synthesis since it provides hydrogen peroxide for iodide oxidation (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). Apart from the upstream part, the current AOP is identical to the corresponding AOPs leading from DIO1 and DIO2 inhibition to increased mortality via anterior swim bladder inflation (https://aopwiki.org/aops/156, https://aopwiki.org/aops/158).

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.

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.

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. While the AOP is only applicable to fish, some of the upstream KEs are relevant across vertebrates. The taxonomic domain of applicability call of the KEs can be found on the respective pages. 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 in fish, 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), the Fish Early Life Stage Toxicity (FELS) Test (OECD TG210) and the Fish Sexual Development Test (FSDT), 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 thyroid hormone 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 thyroid hormone levels within specific developmental stages. For example, clear changes in thyroid hormone levels have been observed in zebrafish at 5, 14, 21 and 32 dpf (Stinckens et al., 2016; 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).

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