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T4 in serum, Decreased leads to Reduced, Anterior swim bladder inflation
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation||non-adjacent||Moderate||Moderate||Dries Knapen (send email)||Open for adoption||EAGMST Under Review|
Life Stage Applicability
Key Event Relationship Description
Reduced T4 levels in serum prohibit local production of active T3 hormone by deiodinases expressed in the target tissues. There is evidence suggesting that anterior swim bladder inflation relies on increased thyroid hormone levels at this specific developmental time point.
Evidence Supporting this KER
There is convincing evidence that decreased T4 levels result in impaired anterior chamber inflation, but the underlying mechanisms are not completely understood. A convincing linear quantitative relationship between reduced T4 levels and reduced anterior chamber volume was shown in zebrafish across exposure to a limited set of three compounds. 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. The formation of the anterior chamber coincides with the second transition phase (Winata et al., 2009) and with a peak in T4 synthesis (Chang et al., 2012) suggesting that anterior inflation is under thyroid hormone regulation. Since most of the more biologically active T3 originates from the conversion of T4, decreased circulatory T4 levels are plausibly linked to reduced anterior chamber inflation.
Uncertainties and Inconsistencies
Reduced anterior chamber inflation upon disruption of the thyroid hormone system is in most cases, but not always, accompanied by reduced whole body T3 levels. Stinckens et al. (2016) found a consistent relationship between reduced whole body T4 levels, but not T3 levels, and reduced anterior chamber inflation after exposure to 2-mercaptobenzothiazole (MBT). Possibly, local T4 levels in the swim bladder tissue were too low to allow for enough local activation to T3. This relates to the general uncertainty on serum versus tissue TH levels. Alternatively, differences in timing between T3/T4 measurements (at 120hpf and 32dpf), the moment when there is a need for T3 to inflate the swim bladder (unknown but probably in between 120hpf and 32dpf) and the observation of the phenotype (32dpf), could lead to the hypothesis that T3 concentration was reduced in between the two measurements. There is also a possibility that the effect of MBT on anterior chamber inflation is not directly caused by decreased thyroid hormone levels, but rather by another mechanism such as oxidative stress. MBT is known to elevate the production of reactive oxygen species (ROS) levels in fish cells (Zeng et al., 2016). In general, chemicals may have multiple modes of action and effects on autophagy, ROS, cardiac function may impact swim bladder inflation.
The mechanism through which reduced T4 hormone concentrations in serum result in anterior chamber inflation impairment is not yet understood. The anterior chamber is formed by evagination from the cranial end of the posterior chamber (Robertson et al., 2007, Winata et al., 2009). Several hypotheses could explain effects on anterior chamber inflation due to reduced T4 levels:
- Evagination from the posterior chamber could be impaired. Villeneuve et al. (unpublished results) showed that although the anterior bud was present after exposure to a deiodinase 2 inhibitor, the anterior chamber did not inflate.
- The formation of the tissue layers of the anterior swim bladder could be affected, although Villeneuve et al. (unpublished results) observed intact tissue layers of the anterior swim bladder after exposure to a deiodinase 2 inhibitor.
- The anterior chamber is inflated with gas from the posterior chamber through the communicating duct. Impaired gas exchange between the two chambers could be at the basis of impaired anterior inflation. Both Nelson et al. (2016) and Stinckens et al. (2016) found that posterior chambers were larger when anterior chambers were smaller or not inflated at all. The sum of the areas of the posterior and anterior chambers remained constant independent of inflation of the anterior chamber (Stinkens et al., 2016). These results suggest retention of the gas in the posterior chamber.
- Since gas exchange relies on a functional communicating duct between the posterior and anterior chamber, and the communicating duct is known to progressively narrow and eventually close during development, a dysfunctional communicating duct or a closure prior to anterior inflation could inhibit inflation. However, Villeneuve et al. (unpublished results) showed that the communicating duct was anatomically intact and open after exposure to iopanoic acid (a deiodinase 2 inhibitor), still leading to impaired anterior inflation.
- Lactic acid production which is essential for producing gas to fill the swim bladder could be affected, although the observation that the total amount of gas in both chambers is not affected when anterior inflation is impaired seems to contradict this (Stinckens et al., 2016).
- Possibly there is an effect on the production of surfactant, which is crucial to maintain the surface tension necessary for swim bladder inflation.
- Reinwald et al. (2021) showed that T3 and propylthiouracil treatment of zebrafish embryos altered expression of genes involved in muscle contraction and functioning in an opposing fashion. The authors suggested impaired muscle function as an additional key event between decreased T3 levels and reduced swim bladder inflation.
In some cases indirect effects may play a role in the impact of chemical exposure or genetic knockdown/knockout on swim bladder inflation. For example, dual oxidase also plays a role in oxidative stress.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Reduced anterior chamber inflation upon disruption of the thyroid hormone system is consistently accompanied by reduced whole body T4 levels when fish are exposed to thyroid hormone synthesis inhibitors. however, when fish are exposed to deiodinase inhibitors, in the absence of feedback processes, stable T4 levels would be expected. Stable T4 levels were indeed observed in 14, 21 and 32 day old zebrafish exposed to iopanoic acid, a deiodinase inhibitor. While Cavallin et al. (2017) found a consistent relationship between reduced whole body T3 levels, and reduced anterior chamber inflation after exposure of fathead minnows to iopanoic acid, they observed increased T4 levels. Possibly, the inhibition of the conversion of T4 to T3 resulted in a compensatory mechanism that increased T4 levels. This was accompanied by increases of deiodinase 2 and 3 mRNA levels, which also indicate a compensatory response to deiodinase inhibition.
Domain of 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 physostomus (e.g., zebrafish and fathead minnow). Physostomus 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. This KER 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 KER 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.
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