Deiodinase 2 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 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 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 (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). 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. DIO2 inhibitors are often also inhibitors of DIO1 (Olker et al., 2019; Stinckens et al. 2018). In the ToxCast DIO2 inhibition single concentration assay, 304 out of 1820 chemicals were positive and 177 of these were also positive for DIO1 inhibition (viewed on 5/7/2022). This complicates the distinction between the relative contribution of DIO1 and DIO2 inhibition to reduced swim bladder inflation. The current state of the art suggests that DIO2 is more important than DIO1 in regulating swim bladder inflation. Six out of seven DIO1 inhibitors impaired posterior chamber inflation, but almost all of these compounds also inhibit DIO2 (Stinckens et al., 2018)). Tetrachlorobisphenol A (TCBPA), the only compound that inhibits DIO1 and not DIO2, had no effect on the posterior swim bladder. Exposure to strong DIO2 inhibitors on the other hand affected posterior chamber inflation and/or surface area in all cases. Therefore the current AOP may be of higher biological relevance compared to AOP 158. Starting from reduced 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).

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 exactly 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, 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).

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