Upstream eventInhibition, Deiodinase 2
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||non-adjacent||Moderate||Low|
|fathead minnow||Pimephales promelas||High||NCBI|
Life Stage Applicability
Key Event Relationship Description
Iodothyronine deiodinase or DIO is a peroxidase enzyme that is involved in the activation or deactivition of thyroid hormones. Currently, three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates that locally activate or inactivate THs and are therefore important mediators of TH action. All deiodinases are integral membrane proteins of the thioredoxin superfamily that contain the amino acid selenocysteine in their catalytic centre. DIO1 and DIO2 are capable of converting T4 into the more biologically active T3. DIO3 on the other hand converts T4 and T3 to the inactive forms of THs. The inhibition of DIO 1 and 2 enzymes results in decreased serum T3 levels and decreased T3 levels at the site of action. 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 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.
Inhibition of DIO activity is widely accepted to reduce the T3 levels in serum and is expected to reduce local T3 levels in target tissues, since the conversion of T4 to T3 is inhibited. In fish, many different adverse effects during early development resulting from disruption of the TH endocrine system have been reported, including effects on swim bladder inflation. As in amphibians, the transition in fish between the different developmental phases, including maturation and inflation of the swim bladder, have been shown to be mediated by THs.
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 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). PTU also 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). It should be noted that there are some uncertainties related to the species-specific susceptibility of DIO1 to inhibition by PTU (Kuiper et al., 2006; Orozco et al., 2012).
- 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.
- 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 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). 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 activating iodothyronine deiodinases (DIO1, DIO2) in regulating swim bladder inflation. Stinckens et al. (2018) showed that when exposing 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 DIO2 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 DIO2, 6 potent DIO2 inhibitors were selected, all of which caused an effect on posterior chamber inflation in zebrafish embryos. Two out of five compounds in the uncertainty zone impaired posterior chamber inflation. Finally, 1 out of 3 weak DIO2 inhibitors caused impaired posterior chamber inflation, resulting in 1 false negative prediction. The latter false negative prediction is potentially due to the fact that multiple AOPs can result in impaired swim bladder inflation.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
The evidence for a relationship between DIO2 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 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.
Darras, V.M., Houbrechts, A.M., Van Herck, S.L.J., 2015. Intracellular thyroid hormone metabolism as a local regulator of nuclear thyroid hormone receptor-mediated impact on vertebrate development. Biochimica Et Biophysica Acta-Gene Regulatory Mechanisms 1849, 130-141.
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.
Frumess, R.D., Larsen, P.R. 1975. Correlation of serum triiodothyronine (T3) and thyroxine (T4) with biological effects of thyroid hormone replacement in propylthiouracil-treated rats. Metabolism 24:4.
Galton, V.A., Schneider, M.J., Clark, A.S., St Germain, D.L., 2009. Life without thyroxine to 3,5,3'-triiodothyronine conversion: studies in mice devoid of the 5'-deiodinases. Endocrinology 150, 2957-2963.
Heijlen, M., Houbrechts, A.M., Bagci, E., Van Herck, S.L.J., Kersseboom, S., Esguerra,C.V., Blust, R., Visser, T.J., Knapen, D., Darras, V.M., 2014. Knockdown of type 3iodothyronine deiodinase severely perturbs both embryonic and early larval development in zebrafish. Endocrinology 155, 1547–1559, http://dx.doi.org/10.1210/en.2013-1660.
Heijlen, M., Houbrechts, A.M., Darras, V.M., 2013. Zebrafish as a model to study peripheral thyroid hormone metabolism in vertebrate development. Gen.Comp. Endocrinol. 188, 289–296, http://dx.doi.org/10.1016/j.ygcen.2013.04.004.
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.
Kuiper, G., Klootwijk, W., Dubois, G.M., Destree, O., Darras, V.M., Van der Geyten, S., Demeneix, B., Visser, T.J., 2006. Characterization of recombinant Xenopus laevis type I iodothyronine deiodinase: substitution of a proline residue in the catalytic center by serine (Pro132Ser) restores sensitivity to 6-propyl-2-thiouracil. Endocrinology 147, 3519-3529.
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.
Orozco, A., Valverde, C., Olvera, A., Garcia, C., 2012. Iodothyronine deiodinases: a functional and evolutionary perspective. Journal of Endocrinology 215, 207-219.
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.
Stolow, M.A., Shi, Y.B., 1995. Xenopus sonic hedgehog as a potential morphogen during embryogenesis and thyroid hormone-dependent metamorphosis.Nucleic Acids Res. 23, 2555–2562, http://dx.doi.org/10.1093/nar/23.13.2555.
Uchida, D., Yamashita, M., Kitano, T., Iguchi, T., 2002. Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish. Journal of Experimental Biology 205, 711-718.
Walpita, C.N., Crawford, A.D., Darras, V.M., 2010. Combined antisense knockdown of type 1 and type 2 iodothyronine deiodinases disrupts embryonic development in zebrafish (Danio rerio). General and Comparative Endocrinology 166, 134-141.
Walpita, C.N., Crawford, A.D., Janssens, E.D., Van der Geyten, S., Darras, V.M., 2009. Type 2 iodothyronine deiodinase is essential for thyroid hormone-dependent embryonic development and pigmentation in zebrafish. Endocrinology 150, 530-539.
Winata, C.L., Korzh, S., Kondrychyn, I., Korzh, V., Gong, Z. 2010. The role of vasulature and blood circulation in zebrafish swim bladder development. Dev. Biol. 10:3.
Winata, C.L., Korzh, S., Kondrychyn, I., Zheng, W., Korzh, V., Gong, Z. 2009. Development of zebrafish swimbladder: the requirement of Hedgehog signaling in specification and organization of the three tissue layers. Dev. Biol.331, 222–236, http://dx.doi.org/10.1016/j.ydbio.2009.04.035.
Yin, A., Korzh, S., Winata, C.L., Korzh, V., Gong, Z., 2011. Wnt signaling is required for early development of zebrafish swimbladder. PLoS One 6, http://dx.doi.org/10.1371/journal.pone.0018431.
Yin, A., Korzh, V., Gong, Z., 2012. Perturbation of zebrafish swim bladder development by enhancing Wnt signaling in Wif1 morphants. Biochim.Biophys. Acta—Mol. Cell Res. 1823, 236–244, http://dx.doi.org/10.1016/j.bbamcr.2011.09.018