Upstream eventDecreased, Triiodothyronine (T3) in serum
Reduced, Posterior swim bladder inflation
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Deiodinase 2 inhibition leading to reduced young of year survival via posterior swim bladder inflation||adjacent|
|Deiodinase 1 inhibition leading to reduced young of year survival via posterior swim bladder inflation||adjacent||Moderate||Low|
|fathead minnow||Pimephales promelas||Moderate||NCBI|
Life Stage Applicability
Key Event Relationship Description
Reduced T3 levels in serum prohibit local TH action in the target tissues. Since swim bladder development and/or inflation is regulated by thyroid hormones, this results in impaired posterior chamber inflation.
Evidence Supporting this KER
There is convincing evidence that decreased T3 levels result in impaired posterior chamber inflation, but the underlying mechanisms are not completely understood. The quantitative understanding is currently very limited because T3 levels and posterior inflation are seldom measured in the same study. Therefore the evidence supporting this KER can be considered moderate.
Thyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition (Liu and Chan, 2002) and larval-to-juvenile transition (Brown et al., 1997) in fish. Inflation of the posterior chamber is part of the embryonic-to-larval transition in fish, together with structural and functional maturation of the mouth and gastrointestinal tract, and resorption of the yolk sac (Liu and Chan, 2002). Marelli et al. (2016) showed that thyroid hormone receptor alpha and beta are both expressed in swim bladder tissue of zebrafish at 5 days post fertilization, corresponding to the timing of posterior inflation. this time point has additionally been shown to coincide with increased T3 and T4 levels (Chang et al., 2012), suggesting that posterior inflation is under thyroid hormone regulation.
- Maternal injection of T3, resulting in increased T3 concentrations in the eggs of striped bass (Morone saxatilis) lead to significant increases in both swim bladder inflation and survival (Brown et al., 1988).
- Dong et al. (2013) and Thisse et al. (2003) showed localized expression of DIO1 and DIO2 in the swim bladder tissue of 96 and 120 hpf zebrafish larvae, suggesting that local activation of thryoid hormones (i.e. conversion of T4 to T3) is requried in swim bladder tissue around that time period.
- Marelli et al. (2016) used morpholinos to block translation of thryoid hormone receptor alpha or beta in zebrafish. They found that thyroid hormone receptor alpha and beta knockdowns failed to inflate the posterior chamber of the swim bladder by 120 hpf, indicating that the action of T3 is needed for proper inflation of the posterior chamber. High T3 doses partially rescued the negative impact in partially resistant mutants, further confirming the importance of T3 in this process.
- Stinckens et al. (2018) showed that effects on posterior chamber inflation in zebrafish could be predicted based on in chemico DIO2 inhibition potential with only few false positives and false negatives. While T3 levels were not determined in this study, DIO2 inhibition is expected to result in decreased T3 levels.
- Bagci et al. (2015) and Heijlen et al. (2013, 2014) reported that knockdown of DIO1+2 in zebrafish resulted in impairment of the inflation of the posterior chamber of the swim bladder. DIO1 and 2 knockdown is expected to result in reduced T3 levels. Indeed, Walpita et al. (2009, 2010) showed that T3 supplementation effectively rescued the effects of DIO1 and 2 knockdown, while T4 supplementation did not.
- de Vrieze et al. (2014) found that knockdown of monocarboxylate transporter 8 (mct8) in zebrafish resulted in a dose-dependent impairment of posterior chamber inflation. Since this transporter is known to transport thyroid hormones across cell membranes, this supports the importance of thyroid hormones in regulating posterior chamber inflation.
- Shi et al. (2019) found that exposure of adult zebrafish to 6:2 chlorinated polyfluorinated ether sulfonate (F-53B), an alternative to perfluorooctanesulfonate (PFOS), decreased T3 levels in both male and female zebrafish. Additionally, F-53B was maternally transferred to the offspring. Decreased T3 levels together with impaired posterior chamber inflation was observed in the F1 offspring.
- Wang et al. (2020) observed a decrease of whole-body T3 as well as impaired posterior chamber inflation in zebrafish exposed to perfluorooctanoic acid and perfluoropolyether carboxylic acids from fertilization until the age of 5 days. Exogeneous T3 or T4 supplementation partly rescued PFECA-induced posterior swim bladder malformation, confirming the causal relationship between reduced T3 levels and reduced posterior chamber inflation.
- Molla et al. (2019) showed that T3 supplementation increased posterior chamber diameter in zebrafish larvae. This confirms that T3 plays an important role in posterior swim bladder inflation.
Evidence of dose-concordance:
- Rehberger et al. (2018) observed decreased T3 levels in the thyroid follicles (which is expected to result in decreased T3 levels in serum) of 120 hpf zebrafish embryos after exposure to PTU starting from 10 mg/L PTU. Stinckens et al.(2018) showed that the downstream KE, impaired posterior chamber inflation, occured at much higher concentrations (EC10: 184 mg/L)
Uncertainties and Inconsistencies
The mechanism through which altered TH levels result in impaired posterior chamber inflation still needs to be elucidated. It is currently unclear which aspect of swim bladder development and inflation is affected by TH disruption. Based on the developmental stages of the posterior chamber, several hypotheses could explain effects on posterior chamber inflation due to disrupted TH levels. A first hypothesis includes effects on the budding of the posterior chamber inflation. Secondly, the effect on posterior chamber inflation could also be caused by disturbing the formation and growth of the three tissue layers of this organ. It has been reported that the Hedgehog signalling pathway plays an essential role in swim bladder development and is required for growth and differentiation of cells of the swim bladder. The Wnt/β-catenin signalling pathway is required for the organization and growth of all three tissue layers (Yin et al., 2011, 2012, Winata 2009, Kress et al., 2009). Both signalling pathways have been related to THs in amphibian and rodent species (Kress et al., 2009; Plateroti et al., 2006; Stolow and Shi, 1995). Molla et al. (2019) showed that insulin-like growth factor (IGF‐1) plays a role in swim bladder inflation/maturation in zebrafish. Several other hypotheses include effects on the successful initial inflation of the posterior chamber, effects on lactic acid production that is required for the maintenance of the swim bladder volume, or effects on the production of surfactant that is crucial to maintain the surface tension necessary for swim bladder inflation.
Another uncertainty lies in the relative importance of the different T4 actvating iodothyronine deiodinases (DIO1, DIO2) in regulating swim bladder inflation. Stinckens et al. (2018) showed that exposure of zebrafish embryos to seven strong DIO1 inhibitors (measured using in chemico enzyme inhibition assays), six out of seven compounds impaired posterior chamber inflation. Exposure to strong DIO2 inhibitors on the other hand affected posterior chamber inflation and/or surface area in all cases. These results suggest that DIO2 enzymes may play a more important role in swim bladder inflation compared to DIO1 enzymes. it has been previously suggested that DIO2 is the major contributor to TH activation in developing zebrafish embryos (Darras et al., 2015; Walpita et al., 2010). It has been shown that a morpholino knockdown targeting DIO1 mRNA alone did not affect embryonic development in zebrafish, while knockdown of DIO2 delayed progression of otic vesicle length, head-trunk angle and pigmentation index (Houbrechts et al., 2016; Walpita et al., 2010, 2009). DIO1 inhibition may only become essential in hypothyroidal circumstances, for example when DIO2 is inhibited or in case of iodine deficiency, in zebrafish (Walpita et al., 2010) and mice (Galton et al., 2009; Schneider et al., 2006).
As reported by Bagci et al. (2015) and Heijlen et al. (2014), posterior chamber inflation was impaired in DIO3 knockdown zebrafish. Heijlen et al. (2014) additionally reported histologically abnormal tissue layers in the swim bladder of DIO3 knockdown zebrafish. DIO3 is a thyroid hormone inactivating enzyme, which would result in higher levels of T3 in serum. Wei et al. (2018) showed that exposure to bisphenol S in adult zebrafish decreased T4 levels and increased T3 levels, and these changes in thyroid hormone levels were transferred to the offspring, in which impaired swim bladder inflation was observed. This indicates that not only too low, but also too high T3 levels, impact posterior chamber inflation. The underlying mechanism is currently unknown.
In the study of Cavallin et al. (2017) fathead minnow embryos were exposed to IOP, a model iodothyronine deiodinase inhibitor that is assumed to inhibit all three deiodinase enzymes (DIO1,2,3). The authors observed increased whole body T3 concentrations in 4 and 6 day old embryos, together with impaired posterior chamber inflation. Transcript levels of DIO1, 2 and 3 remained unaltered and thus offered no proof of a compensatory mechanism that could explain these results.
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, posterior swim bladder chamber inflation, which occurs early during development, appears to be less sensitive to inhibition of TH synthesis than to inhibition of the conversion of T4 to T3 (Stinckens et al., 2016, 2018; Nelson et al., 2016). There have however been a few reports of reduced posterior inflation upon inhibition of TH synthesis (Liu and Chan, 2002). It must however be noted that these observations could reflect delayed inflation due to a general delay in development rather than a direct effect on the swim bladder. Longer observations would have to clarify this.
Quantitative Understanding of the Linkage
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
The indirect relationship between deiodinase inhibition, expected to lead to reduced T3 levels, and reduced posterior chamber inflation has been confirmed in early zebrafish larvae around the time of posterior chamber inflation, i.e. around 5 days post fertilization in zebrafish (Stinckens et al., 2018) and around 6 days post fertilization in fathead minnows (Cavallin et al., 2017).
The relationship between reduced T3 levels and reduced posterior chamber inflation is not applicable to older larvae that successfully inflated the posterior chamber but show impaired anterior chamber inflation after chronic exposure to low concentrations of thyroid hormone system disruptors. In 32 day old zebrafish exposed to methimazole, propylthiouracil, 2-mercaptobenzothiazole or iopaonic acid (Stinckens et al., 2016, 2020) as well as in 14-21 day old fathead minnows exposed to iopaonic acid (Cavallin et al., 2017), a clear inverse relationship was found. With decreasing whole body T3 concentrations, posterior chamber volume increased, suggesting a possible compensatory mechanism for the observed decrease in anterior chamber volume. As a result, the sum of both chamber surfaces, reflecting the total amount of gas, was equal to controls for most treatments (Stinckens et al., 2016; Stinckens et al., 2020)
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