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Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation
Point of Contact
- Dries Knapen
- Lucia Vergauwen
- Ann-Cathrin Haigis
- Shihori Tanabe
|Handbook Version||OECD status||OECD project|
This AOP was last modified on April 29, 2023 16:02
Revision dates for related pages
|Inhibition, Deiodinase 2||October 07, 2022 07:56|
|Decreased, Triiodothyronine (T3)||October 07, 2022 08:26|
|Reduced, Posterior swim bladder inflation||October 07, 2022 08:42|
|Reduced, Swimming performance||September 08, 2021 06:12|
|Decrease, Population growth rate||January 03, 2023 09:09|
|Increased Mortality||July 08, 2022 07:32|
|Inhibition, Deiodinase 2 leads to Decreased, Triiodothyronine (T3)||October 07, 2022 08:57|
|Decreased, Triiodothyronine (T3) leads to Reduced, Posterior swim bladder inflation||October 07, 2022 09:12|
|Reduced, Posterior swim bladder inflation leads to Reduced, Swimming performance||September 03, 2021 13:02|
|Reduced, Swimming performance leads to Increased Mortality||September 08, 2021 10:05|
|Increased Mortality leads to Decrease, Population growth rate||July 08, 2022 08:29|
|Inhibition, Deiodinase 2 leads to Reduced, Posterior swim bladder inflation||October 07, 2022 09:22|
|Reduced, Posterior swim bladder inflation leads to Increased Mortality||September 08, 2021 10:08|
|iopanoic acid||November 29, 2016 18:42|
This AOP describes the sequence of events leading from deiodinase inhibition to increased mortality via reduced posterior 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 developmental abnormalities. Three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates that activate or inactivate THs and are therefore important mediators of thyroid hormone (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. As in amphibians, the transition between the different developmental phases in fish, including maturation and inflation of the swim bladder, is mediated by THs (Brown et al., 1988; Liu and Chan, 2002). The swim bladder 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. This AOP describes how DIO2 inhibition results in reduced T3 levels, which prohibit normal inflation of the posterior chamber of the swim bladder in the embryonic phase. The posterior chamber is important for regulating buoyancy and thus for swimming performance (Robertson et al., 2007). Reduced swimming performance reduces chances of survival due to a decreased ability to forage and avoid predators. 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 (Jomaa et al., 2014; Cavallin et al., 2017; Stinckens et al., 2018) and on knockdown/knockout and TH supplementation studies in zebrafish embryos where the DIO2 gene is inactivated (Walpita et al., 2009, 2010; Heijlen et al., 2014; Bagci et al., 2015; Houbrechts et al., 2016).
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 posterior swim bladder inflation (https://aopwiki.org/aops/157). The overall importance of DIO1 versus DIO2 in fish is not exactly clear. DIO2 inhibitors are often also inhibitors of DIO1 (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 (Stinckens et al., 2018). Therefore the current AOP may be of higher biological relevance compared to AOP 157.
AOP Development Strategy
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
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
|Type||Event ID||Title||Short name|
|MIE||1002||Inhibition, Deiodinase 2||Inhibition, Deiodinase 2|
|KE||1003||Decreased, Triiodothyronine (T3)||Decreased, Triiodothyronine (T3)|
|KE||1004||Reduced, Posterior swim bladder inflation||Reduced, Posterior swim bladder inflation|
|KE||1005||Reduced, Swimming performance||Reduced, Swimming performance|
|AO||351||Increased Mortality||Increased Mortality|
|AO||360||Decrease, Population growth rate||Decrease, Population growth rate|
Relationships Between Two Key Events (Including MIEs and AOs)
|Inhibition, Deiodinase 2 leads to Decreased, Triiodothyronine (T3)||adjacent||Moderate||Low|
|Decreased, Triiodothyronine (T3) leads to Reduced, Posterior swim bladder inflation||adjacent||Moderate||Low|
|Reduced, Posterior swim bladder inflation leads to Reduced, Swimming performance||adjacent||Moderate||Low|
|Reduced, Swimming performance leads to Increased Mortality||adjacent||Moderate||Low|
|Increased Mortality leads to Decrease, Population growth rate||adjacent||Moderate||Moderate|
|Inhibition, Deiodinase 2 leads to Reduced, Posterior swim bladder inflation||non-adjacent||Moderate||Low|
|Reduced, Posterior swim bladder inflation leads to Increased Mortality||non-adjacent||High||Low|
Life Stage Applicability
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: The current AOP is only applicable to early embryonic development, which is the period where the posterior swim bladder chamber inflates. In all life stages, the conversion of T4 into more biologically active T3 is essential. Inhibition of DIO2 therefore impacts swim bladder inflation in both early and late (https://aopwiki.org/aops/156) developmental life stages.
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. Knowledge could be expanded to physoclistous fish, such as the Japanese rice fish or medaka (Oryzias latipes) that has a single chambered swim bladder that inflates during early development.
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). In zebrafish and fathead minnow, it is currently unclear whether sex-related differences are important in determining the magnitude of the changes of the sequence of events along this AOP. 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. 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 posterior chamber inflates around 5 days post fertilization in zebrafish, when sex differentiation has not started yet, 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 posterior chamber inflates around 6 days post fertilization (1 dph) in fathead minnows, sex differences are expected to play a minor role in the current AOP.
Essentiality of the Key Events
Overall, the support for essentiality of the KEs is high since there is direct evidence from specifically designed experimental studies illustrating essentiality for several of the important KEs in the AOP. This includes ample evidence from knockdown studies in zebrafish that use targeted perturbation of key events and show downstream effects, and evidence from both chemical exposure with TH supplementation and knockdown with TH supplementation showing that blocking a KE prevents downstream KEs from occurring.
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.
Known Modulating Factors
Data to support the quantitative understanding of this AOP is currently lacking.
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. 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 posterior 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).
Bagci, E., Heijlen, M., Vergauwen, L., Hagenaars, A., Houbrechts, A.M., Esguerra, C.V., Blust, R., Darras, V.M., Knapen, D., 2015. Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction. PLOS One 10, e0123285.
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