Relationship: 1044



Inhibition, Deiodinase 1 leads to Reduced, Posterior swim bladder inflation

Upstream event


Inhibition, Deiodinase 1

Downstream event


Reduced, Posterior swim bladder inflation

Key Event Relationship Overview


AOPs Referencing Relationship


AOP Name Adjacency Weight of Evidence Quantitative Understanding
Deiodinase 1 inhibition leading to reduced young of year survival via posterior swim bladder inflation non-adjacent Moderate Low

Taxonomic Applicability


Term Scientific Term Evidence Link
zebrafish Danio rerio High NCBI
fathead minnow Pimephales promelas High NCBI

Sex Applicability


Sex Evidence
Unspecific High

Life Stage Applicability


Term Evidence
Embryo High

Key Event Relationship Description


The two major thyroid hormones are thyroxine (T4) and the more biologically active triiodothyronine (T3), both iodinated derivatives of tyrosine. Active and inactive THs are tightly regulated by enzymes called iodothyronine deiodinases (DIO). The activation occurs via outer ring deiodination (ORD), i.e. removing iodine from the outer, phenolic ring of T4 to form T3, while inactivation occurs via inner ring deiodination (IRD), i.e. removing iodine from the inner tyrosol ring of T4 or T3.

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. All deiodinases are integral membrane proteins of the thioredoxin superfamily that contain selenocysteine in their catalytic centre. Type I deiodinase is capable of converting T4 into T3, as well as to convert rT3 to the inactive thyroid hormone 3,3’ T2, through outer ring deiodination. rT3, rather than T4, is the preferred substrate for DIO1. furthermore, DIO1 has a very high Km (µM range, compared to nM range for DIO2) (Darras and Van Herck, 2012). Type II deiodinase (DIO2) is only capable of ORD activity with T4 as a preferred substrate (i.e., activation of T4 tot T3). DIO3 can inner ring deiodinate T4 and T3 to the inactive forms of THs, reverse T3, (rT3) and 3,3’-T2 respectively. (Darras and Van Herck, 2012)

Since swim bladder development and/or inflation is regulated by thyroid hormones, decreased T3 levels are expected to result in impaired posterior chamber inflation. Because of the high Km and preference for rT3 as a substrate, the importance of DIO1 in activating T4 to T3 in a physiological situation is likely limited. The role of inhibition of DIO1 in decreasing T3 levels and impairing posterior chamber inflation may therefore be limited.

Evidence Supporting this KER


There is convincing evidence that inhibition of DIO activity, either through specific knockdown or through chemical exposure, results in impaired posterior chamber inflation, but the underlying mechanisms are not completely understood, including the relative importance of DIO1 and DIO2. Based on current evidence, it seems that DIO2 is more important in regulating posterior chamber inflation. Due to the difficulty of measuring DIO activity in small fish embryos, quantitative linkages and temporal concordance have been difficult to establish. The quantitative understanding is currently based on a relationship between the classification of chemicals according to their in chemico DIO inhibitory potential (using a threshold and uncertainty zone) on the one hand, and occurence of in vivo effects on posterior chamber inflation on the other hand. Predictions based on this relationship have been proven highly successful. Therefore the evidence supporting this KER can be considered moderate.

Biological Plausibility


Inhibition of DIO 1 activity is widely accepted to reduce the conversion of T4 to the more biologically active T3. Thyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition and larval-to-juvenile transition in fish. Inflation of the posterior swim bladder 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. Together with empirical evidence, it is plausible to assume that posterior swim bladder inflation is under thyroid hormone regulation but scientific understanding is incomplete. It follows that disrupted conversion of T4 to T3 is likely to interfere with normal inflation of the posterior swim bladder chamber.

Empirical Evidence


Deiodinases are criticial for normal development. Several defects have already been reported in cases where the TH hormone balance is disturbed. Winata et al. (2009, 2010) reported reduced pigmentation, otic vesicle length and head-trunk angle in DIO1+2 and DIO2 knockdown fish. These effects were rescued after T3 supplementation, indicating the importance of T4 to T3 conversion by deiodinases.

Substantial evidence for the link between deiodinase inhibition and impaired posterior chamber inflation is available:

  • Chang et al., (2012) established a base-line for TH levels during zebrafish development and observed peaks in whole-body T3 content at 5 dpf when the posterior chamber of the swim bladder inflates.
  • 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 DIO2 mRNA has also been shown to be present in zebrafish swim bladder tissue at 96 hpf using whole mount in situ hybridization (Heijlen et al., 2013; Dong et al., 2013), suggesting a tissue-specific role of T3 in the inflation process of the posterior chamber.
  • Exposure to propylthiouracil (PTU), a very potent DIO1 inhibitor, caused thyroid hypertrophy in X. leavis because of the inhibition of the peripheral conversion of T4 to T3 (Degitz et al., 2005), decreased serum T3 levels in the rat (Frumess and Larsen, 1975) and resulted in effects on posterior chamber inflation in zebrafish (Jomaa et al., 2014; Stinckens et al., 2018).
  • After exposure of fathead minnows (Pimephales promelas) to the non-specific deiodinase inhibitor IOP from 1-6 dpf, Incidence and length of inflated posterior swim bladders were significantly reduced (Cavallin et al., 2017).

Uncertainties and Inconsistencies


The mode of action through which reduced DIO1 inhibition results in impaired posterior chamber inflation still needs to be elucidated.

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 pathwat 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). 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).

Heijlen et al. (2015) reported histologically abnormal tissue layers in the swim bladder of DIO3 knockdown zebrafish. As reported in Bagci et al. (2015) and Heijlen et al. (2014), posterior chamber inflation was impaired in DIO3 knockdown zebrafish. DIO3 is a thyroid hormone inactivating enzyme, which would result in higher levels of T3 in serum. This indicates that not only too low, but also too high T3 levels, impact posterior chamber inflation. The underlying mechanism is currently unknown.

Quantitative Understanding of the Linkage


In chemico DIO1 inhibition assays were performed using potential thyroid disrupting compounds (Stinckens et al., 2018). Using dose-response curves, the IC50 values were determined and used to caterogize the chemicals according to their potency (strong, weak, uncertainty zone). For DIO1, 7 potent DIO1 inhibitors were selected, of which 6 caused an effect on posterior chamber inflation in zebrafish embryos, resulting in only 1 false positive prediction. One out of two compounds in the uncertainty zone impaired posterior chamber inflation. Finally, 2 out of 5 weak DIO1 inhibitors caused impaired posterior chamber inflation, resulting in 2 false negative predictions. The latter false negative predictions are potentially due to the fact that multiple AOPs can result in impaired swim bladder inflation.

Response-response Relationship




Known modulating factors


Known Feedforward/Feedback loops influencing this KER


Domain of Applicability


The evidence for a relationship between DIO1 inhibition and inflation of the posterior chamber of the swim bladder is currently based on work in zebrafish and fathead minnow.

Mol et al. (1998) concluded concluded that deiodinases in teleosts were more similar to mammalian deiodinases than had been generally accepted, based on the similarities in susceptibility to inhibition and the agreement of the Km values.

This KER is probably not sex-dependent since both females and males rely on activation of THs by deiodinase for regulation of vital processes. Additionally, zebrafish are undifferentiated gonochorists, and gonad differentiation starts only around 23-25 dpf (Uchida et al., 2002), well after the time point of posterior chamber inflation (around 5 dpf).



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, http://dx.doi.org/10.1371/journal.pone.0123285.

Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.

Chang, J., Wang, M., Gui, W., Zhao, Y., Yu, L., Zhu, G., 2012. Changes in thyroidhormone levels during zebrafish development. Zool. Sci. 29, 181–184, http://dx.doi.org/10.2108/zsj.29.181.

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Degitz, S.J., Holcombe, G.W., Flynn, K.M., Kosian, P.A., Korte, J.J., Tietge, J.E., 2005.Progress towards development of an amphibian-based screening assay usinXenopus laevis. Organismal and thyroidal responses to the model compounds6-propylthiouracil, methimazole, and thyroxine. Toxicol. Sci. 87, 353–364.

Dong, W., Macaulay, L., Kwok, K.W.H., Hinton, D.E., Stapleton, H.M., 2013. Using whole mount in situ hydridization to examine thyroid hormone deiodinase expression in embryonic and larval zebrafish: a tool for examining OH-BDE toxicity to early life stages. Aquat. Toxicol. 132–133, 190–199, http://dx.doi.org/10.1016/j.biotechadv.2011.08.021.Secreted.

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Houbrechts, A.M., Delarue, J., Gabriels, I.J., Sourbron, J., Darras, V.M., 2016. Permanent Deiodinase Type 2 Deficiency Strongly Perturbs Zebrafish Development, Growth, and Fertility. Endocrinology 157, 3668-3681.

Jomaa, B., Hermsen, S.A.B., Kessels, M.Y., Van Den Berg, J.H.J., Peijnenburg, A.A.C.M.,Aarts, J.M.M.J.G., Piersma, A.H., Rietjens, I.M.C.M., 2014. Developmental toxicity of thyroid-active compounds in a zebrafish embryotoxicity test. ALTEX 31,303–317, http://dx.doi.org/10.14573/altex.1402011.

Kress, E., Rezza, A., Nadjar, J., Samarut, J., Plateroti, M., 2009. The frizzled-relatedsFRP2 gene is a target of thyroid hormone receptor alfa1 and activates beta-catenin signaling in mouse intestine. J. Biol. Chem. 284, 1234–1241, http://dx.doi.org/10.1074/jbc.M806548200.

Mol, K.A., Van der Geyten, S., Burel, C., Kuhn, E.R., Boujard, T., Darras, V.M., 1998. Comparative study of iodothyronine outer ring and inner ring deiodinase activities in five teleostean fishes. Fish Physiology and Biochemistry 18, 253-266.

Plateroti, M., Kress, E., Mori, J.I., Samarut, J., 2006. Thyroid hormone receptor alpha1 directly controls transcription of the beta-catenin gene in intestinal epithelial cells. Mol. Cell. Biol. 26, 3204–3214, http://dx.doi.org/10.1128/MCB.26.8.3204.

Schneider, M.J., Fiering, S.N., Thai, B., Wu, S.Y., St Germain, E., Parlow, A.F., St Germain, D.L., Galton, V.A., 2006. Targeted disruption of the type 1 selenodeiodinase gene (Dio1) results in marked changes in thyroid hormone economy in mice. Endocrinology 147, 580-589.

Stinckens, E., Vergauwen, L., Ankley, G.T., Blust, R., Darras, V.M., Villeneuve, D.L., Witters, H., Volz, D.C., Knapen, D., 2018. An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish. Aquatic Toxicology 200, 1-12. 10.1016/j.aquatox.2018.04.009.

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