To the extent possible under law, AOP-Wiki has waived all copyright and related or neighboring rights to KER:1039
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||Evgeniia Kazymova (send email)||Under Development: Contributions and Comments Welcome||EAGMST Approved|
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 Collection Strategy
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.
- Chang et al. (2012) observed an increase of whole body T4 concentrations in zebrafish larvae at 21 days post fertilization, corresponding to the timing of anterior swim bladder inflation.
- Nelson et al. (2016) showed reduced whole body T4 levels at 6 days post fertilization and delayed anterior inflation (lower proportion of inflation at 14 dpf compared to controls) after exposure of fathead minnow embryos to 2-mercaptobenzothiazole. All anterior chambers eventually inflated but their size was reduced and morphology deviated.
- Stinckens et al. (2016) showed reduced whole body T4 levels both at 5 (before anterior inflation) and 32 days post fertilization (after anterior inflation) when zebrafish were exposed to 2-mercaptobenzothiazole (a thyroperoxidase inhibitor) from 0 to 32 days post fertilization. A large percentage of MBT-exposed fish had an uninflated anterior swim bladder, although some recovery was observed over time.
- Stinckens et al. (2016) further showed a significant correlation between whole body T4 levels and anterior chamber volume, with reduced T4 levels leading to smaller anterior chambers.
- Stinckens et al. (2020) established a significant correlation between reduced whole body T4 levels and reduced anterior chamber volume in 32 day old zebrafish across two compound exposures. This includes methimazole and propylthiouracil, two inhibitors of TH synthesis. Godfrey et al. (2017) also observed impaored anterior chamber inflation after exposure of zebrafish to Methimazole. Stinckens et al. (2020) continued the follow-up until 32 dpf and observed no recovery.
- Chopra et al. (2019) found that a nonsense mutation of the duox gene, coding for the enzyme dual oxidase involved in thyroid hormone synthesis, resulted in decreased intrafollicular T4 levels and impaired anterior chamber inflation until at least 54 dpf in zebrafish. It should be noted that dual oxidase is not only involved in thyroid hormone synthesis, but also in the production of reactive oxygen species (ROS) (Flores et al. 2010; Niethammer et al. 2009). In zebrafish, ROS can also be induced e.g. by copper (Zhou et al. 2016), which has also been shown to impair swim bladder development (Xu et al. 2017). Impaired production of ROS after dual oxidase knockdown may contribute to an impairment of swim bladder development.
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
Quantitative Understanding of the Linkage
Stinckens et al. (2016) showed a significant linear quantitative relationship between whole body T4 levels and anterior chamber volume (measured as surface in 2D images) in 32 day old juvenile zebrafish, with reduced T4 levels leading to smaller anterior chambers after continuous exposure to 2-mercaptobenzothiazole, a thyroid hormone synthesis inhibitor.
Stinckens et al. (2020, supplementary information) established a significant linear quantitative relationship between reduced T4 levels and reduced anterior chamber volume (measured as surface in 2D images) in 32 day old juvenile zebrafish across two compound exposures. This includes methimazole and propylthiouracil, two inhibitors of TH synthesis.
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.
Alt, B., Reibe, S., Feitosa, N.M., Elsalini, O.A., Wendl, T., Rohr, K.B., 2006. Analysis of origin and growth of the thyroid gland in zebrafish. Dev. Dyn. 235, 1872–1883, http://dx.doi.org/10.1002/dvdy.20831.
Brown, C.L., Doroshov, S.I., Nunez, J.M., Hadley, C., Vaneenennaam, J., Nishioka, R.S. and Bern, H.A. 1988. Maternal triiodothyronine injections cause increases in swimbladder inflation and survival rates in larval striped bass, Morone saxatilis. J. Exp. Zool. 248: 168–176.
Brown, C.L., Sullivan, C.V., Bern, H.A. and Dickhoff, W.W. 1987. Occurrence of thyroid hormones in early developmental stages of teleost fish. Trans. Am. Fish. Soc. Symp. 2: 144–150.
Brown, D.D., 1997. The role of thyroid hormone in zebrafish and axolotl development. Proc. Natl. Acad. Sci. U. S. A. 94, 13011–13016, http://dx.doi.org/ 10.1073/pnas.94.24.13011.
Campinho, M.A., Saraiva, J., Florindo, C., Power, D.M., 2014. Maternal Thyroid Hormones Are Essential for Neural Development in Zebrafish. Molecular Endocrinology 28, 1136-1149.
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 thyroid hormone levels during zebrafish development. Zool. Sci. 29, 181–184, http:// dx.doi.org/10.2108/zsj.29.181.
Chopra, K., Ishibashi, S., Amaya, E., 2019. Zebrafish duox mutations provide a model for human congenital hypothyroidism. Biology Open 8(2):bio.037655, DOI:10.1242/bio.037655.
Elsalini, O.A., Rohr, K.B., 2003. Phenylthiourea disrupts thyroid function in developing zebrafish. Dev. Genes Evol. 212, 593–598, http://dx.doi.org/10. 1007/s00427-002-0279-3.
Flores MV, Crawford KC, Pullin LM, Hall CJ, Crosier KE, Crosier PS. 2010. Dual oxidase in the intestinal epithelium of zebrafish larvae has anti-bacterial properties. Biochemical and Biophysical Research Communications. 400(1):164-168.
Godfrey A, Hooser B, Abdelmoneim A, Sepulveda MS. 2019. Sex-specific endocrine-disrupting effects of three halogenated chemicals in japanese medaka. Journal of Applied Toxicology. 39(8):1215-1223.
Godfrey, A., Hooser, B., Abdelmoneim, A., Horzmann, K.A., Freemanc, J.L., Sepulveda, M.S., 2017. Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish. Aquatic Toxicology 193, 228-235.
Hsu, C.W., Tsai, S.C., Shen, S.C., Wu, S.M., 2014. Profiles of thyrotropin, thyroid hormones, follicular cells and type I deiodinase gene expression during ontogenetic development of tilapia larvae and juveniles. Fish Physiology and Biochemistry 40, 1587-1599.
Liu, Y.W., Chan, W.K., 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 70, 36–45, http://dx.doi. org/10.1046/j.1432-0436.2002.700104.x.
Nagabhushana A, Mishra RK. 2016. Finding clues to the riddle of sex determination in zebrafish. Journal of Biosciences. 41(1):145-155.
Nelson KR, Schroeder AL, Ankley GT, Blackwell BR, Blanksma C, Degitz SJ, Flynn KM, Jensen KM, Johnson RD, Kahl MD, Knapen D, Kosian PA, Milsk RY, Randolph EC, Saari T, Stinckens E, Vergauwen L, Villeneuve DL. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole – Part I: fathead minnow. Aquatic Toxicology 173: 192-203.
Niethammer P, Grabher C, Look AT, Mitchison TJ. 2009. A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature. 459(7249):996-U123.
Olmstead AW, Villeneuve DL, Ankley GT, Cavallin JE, Lindberg-Livingston A, Wehmas LC, Degitz SJ. 2011. A method for the determination of genetic sex in the fathead minnow, pimephales promelas, to support testing of endocrine-active chemicals. Environmental Science & Technology. 45(7):3090-3095.
Power DM, Llewellyn L, Faustino M, Nowell MA, Björnsson BT, Einarsdottir IE, Canario AV, Sweeney GE. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. 2001 Dec; 130(4):447-59.
Reider, M., Connaughton, V.P., 2014. Effects of Low-Dose Embryonic Thyroid Disruption and Rearing Temperature on the Development of the Eye and Retina in Zebrafish. Birth Defects Res. Part B Dev. Reprod. Toxicol. 101, 347–354, http://dx.doi.org/10.1002/bdrb.21118.
Reinwald H, Konig A, Ayobahan SU, Alvincz J, Sipos L, Gockener B, Bohle G, Shomroni O, Hollert H, Salinas G et al. 2021. Toxicogenomic fin(ger)prints for thyroid disruption aop refinement and biomarker identification in zebrafish embryos. Science of the Total Environment. 760.
Roberston, G.N., McGee, C.A.S., Dumbarton, T.C., Croll, R.P., Smith, F.M., 2007. Development of the swim bladder and its innervation in the zebrafish, Danio rerio. J. Morphol. 268, 967–985, http://dx.doi.org/10.1002/jmor.
Ruuskanen, S., Hsu, B.Y., 2018. Maternal Thyroid Hormones: An Unexplored Mechanism Underlying Maternal Effects in an Ecological Framework. Physiological and Biochemical Zoology 91, 904-916.
Stinckens E, Vergauwen L, Schroeder AL, Maho W, Blackwell BR, Witters H, Blust R, Ankley GT, Covaci A, Villeneuve DL, Knapen D. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole – Part II: zebrafish. Aquatic Toxicology 173:204-217.
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.
Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.
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.
van Aerle R, Runnalls TJ, Tyler CR. 2004. Ontogeny of gonadal sex development relative to growth in fathead minnow. Journal of Fish Biology. 64(2):355-369.
Walpita, C.N., Van der Geyten, S., Rurangwa, E., Darras, V.M., 2007. The effect of 3,5,3′-triiodothyronine supplementation on zebrafish (Danio rerio) embryonic development and expression of iodothyronine deiodinases and thyroid hormone receptors. Gen. Comp. Endocrinol. 152, 206–214, http://dx.doi.org/ 10.1016/j.ygcen.2007.02.020.
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.
Woolley LD, Qin JG. 2010. Swimbladder inflation and its implication to the culture of marine finfish larvae. Reviews in Aquaculture. 2(4):181-190.
Xu JP, Zhang RT, Zhang T, Zhao G, Huang Y, Wang HL, Liu JX. 2017. Copper impairs zebrafish swimbladder development by down-regulating wnt signaling. Aquatic Toxicology. 192:155-164.
Zeng FX, Sherry JP, Bols NC. 2016. Evaluating the toxic potential of benzothiazoles with the rainbow trout cell lines, rtgill-w1 and rtl-w1. Chemosphere. 155:308-318.
Zhou XY, Zhang T, Ren L, Wu JJ, Wang WM, Liu JX. 2016. Copper elevated embryonic hemoglobin through reactive oxygen species during zebrafish erythrogenesis. Aquatic Toxicology. 175:1-11.