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Relationship: 1035

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Decreased, Triiodothyronine (T3) leads to Reduced, Anterior swim bladder inflation

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Decreased, Triiodothyronine (T3)

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Reduced, Anterior swim bladder inflation

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation	adjacent	Moderate	Moderate	Dries Knapen (send email)	Under Development: Contributions and Comments Welcome	WPHA/WNT Endorsed
Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation	adjacent	Moderate	Moderate	Dries Knapen (send email)	Under Development: Contributions and Comments Welcome	WPHA/WNT Endorsed
Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation	adjacent	Moderate	Moderate	Dries Knapen (send email)	Under Development: Contributions and Comments Welcome	WPHA/WNT Endorsed

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

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

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Unspecific	Moderate

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Larvae	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

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, including anterior chamber inflation in fish. Reduced T3 levels prohibit local TH action in the target tissues. Since swim bladder development and/or inflation is regulated by thyroid hormones, this results in impaired anterior chamber inflation.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

There is convincing evidence that decreased T3 levels result in impaired anterior chamber inflation, but the underlying mechanisms are not completely understood. A very convincing linear quantitative relationship between reduced T3 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.

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

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 anterior swim bladder chamber is part of the larval-to-juvenile transition in fish, together with the development of adult fins and fin rays, ossification of the axial skeleton, formation of an adult pigmentation pattern, scale formation, maturation and remodelling of organs including the lateral line, nervous system, gut and kidneys (Brown, 1997; Liu and Chan, 2002; McMenamin and Parichy, 2013).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Dedicated studies with two different experimental setups have been conducted to investigate the link between reduced T3 levels and reduced anterior chamber inflation:

1. Studies applying larval exposures initiated after posterior chamber inflation

In a study in which larval fathead minnows (Pimephales promelas) were exposed to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole (MBT), T3 concentrations measured at 14dpf were reduced at the same concentration (1 mg/L) that significantly reduced anterior swim bladder inflation at the same time-point (Nelson et al. 2016).
In the study of Cavallin et al. (2017) fathead minnow larvae were exposed to IOP, a model iodothyronine deiodinase inhibitor that is assumed to inhibit all three deiodinase enzymes (DIO1,2,3). The authors observed pronounced decreases of whole-body T3 concentrations and increases of whole-body T4 concentrations, together with impaired inflation of the anterior swim bladder chamber. More specifically, inflation was delayed and the size of the swim bladder chamber was reduced until the end of the exposure experiment.

Since exposures were started after inflation of the posterior chamber, these studies show that DIO inhibition can directly affect anterior chamber inflation.

2. Studies applying continuous exposure initiated immediately after fertilization and thus including both posterior and anterior chamber inflation

In the study of Stinckens et al. (2020) exposure concentrations were chosen where the posterior chamber inflates. A strong correlation between reduced T3 levels and reduced anterior chamber inflation was observed in zebrafish exposed to iopanoic acid, a deiodinase inhibitor, as well as methimazole and propylthiouracil, both thyroperoxidase inhibitors, from fertilization until the age of 32 days. Anterior chamber inflation was delayed and a number of larvae did not manage to inflate the anterior chamber by the end of the 32 day exposure period. Additionally, exposed fish that had inflated the swim bladder had reduced anterior chamber sizes.

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Since in fish early life stages THs are typically measured on a whole-body level, it is currently uncertain whether TH levels changes occur at the serum and/or tissue level.
The mechanism underlying the link between reduced T3 and reduced anterior chamber inflation remains unclear, but several hypotheses exist (Stinckens et al., 2020). For example, altered gas distribution between chambers could be the result of impaired development of smooth muscle fibers, delayed and/or impaired evagination of the anterior chamber, impaired anterior budding through altered Wnt and hedgehog signalling, etc. 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.
Increased T3 levels also seem to result in reduced swim bladder inflation. For example, Li et al. (2011) reported impairment of swim bladder inflation in Chinese rare minnows (Gobiocypris rarus) exposed to exogenous T3.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Stinckens et al. (2020, supplementary information) established a very convincing linear quantitative relationship between reduced T3 levels and reduced anterior chamber volume (measured as surface in 2D images) in 32 day old zebrafish across three compound exposures. This includes methimazole and propylthiouracil, two inhibitors of TH synthesis on the one hand, and iopanoic acid, a deiodinase inhibitor, on the other hand.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Taxonomic: Teleost fish can be divided in two groups according to swim bladder morphology: physoclistous (e.g., yellow perch, sea bass, striped bass, medaka) and physostomous (e.g., zebrafish and fathead minnow). Physostomous 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 or medaka (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 KER plausibly applicable to both sexes. Sex differences are not often investigated in tests using early life stages of fish. For zebrafish and fathead minnow, it is currently unclear whether sex-related differences are important in determining the magnitude of the changes in this 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 KER.

References

List of the literature that was cited for this KER description. More help

Brown, D.D., 1997. The role of thyroid hormone in zebrafish and axolotl development. Proceedings of the National Academy of Sciences of the United States of America 94, 13011-13016.

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.

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.

Li W, Zha J, Yang L, Li Z, Wang Z. Regulation of thyroid hormone related genes mRNA expression by exogenous T₃ in larvae and adult Chinese rare minnow (Gobiocypris rarus). Environ Toxicol Pharmacol. 2011 Jan;31(1):189-97. doi: 10.1016/j.etap.2010.10.007.

Liu, Y.W., Chan, W.K., 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 70, 36-45.

McMenamin, S.K., Parichy, D.M., 2013. Metamorphosis in Teleosts. Animal Metamorphosis 103, 127-165.

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. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquat Toxicol. 2016 Apr;173:192-203. doi: 10.1016/j.aquatox.2015.12.024.

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.

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.

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.

Stinckens, E., Vergauwen, L., Schroeder, A., Maho, W., Blackwell, B., Witters, H., Blust, R., Ankley, G., Covaci, A., Villeneuve, D., 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.

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.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.