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AOP ID and Title:

AOP 155: Deiodinase 2 inhibition leading to reduced young of year survival via posterior swim bladder inflation
Short Title: DIO2i posterior swim bladder

Graphical Representation

Authors

Dries Knapen [1], [dries.knapen (at)uantwerpen.be]

Lucia Vergauwen [1], [lucia.vergauwen(at)uantwerpen.be]

Evelyn Stinckens [1], [evelyn.stinckens(at)uantwerpen.be]

Dan Villeneuve [2], [villeneuve.dan*(at)epa.gov]

[1] Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium

[2]United States Environmental Protection Agency, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN, USA.


Status

Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite Under Development 1.35 Included in OECD Work Plan

Abstract

The AOP describes the effects of inhibition of deiodinase 2 (DIO2) on posterior swim bladder inflation leading to reduced young of year survival and population trajectory decline. The inhibition of DIO2 is the molecular-initiating event (MIE), which results in decreased circulating concentrations of triiodothyronine (T3) in serum. Disruption of the thyroid hormone (TH) system is increasingly being recognized as an important MoA that can lead to adverse outcomes, especially during embryonic development. In fish, many different adverse effects during early development resulting from disruption of the TH endocrine system have been reported (e.g.,effects on body and eye size, head-to-trunk angle, heartbeat, otolith formation, pigmentation index, swim bladder inflation, hatching time, somite formation, escape response and photoreceptor development). 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. Chemicals interfering with the conversion of T4 to T3 have the potential to inhibit posterior chamber inflation which may result in reduced swimming capacity of the fish, a relevant adverse outcome that can affect feeding behaviour and predator avoidance, resulting in lower survival probability and ultimately population trajectory decline (Czesny et al., 2005; Woolley and Qin, 2010).



Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Sequence Type Event ID Title Short name
1 MIE 1002 Inhibition, Deiodinase 2 Inhibition, Deiodinase 2
2 KE 1003 Decreased, Triiodothyronine (T3) in serum Decreased, Triiodothyronine (T3) in serum
3 KE 1004 Reduced, Posterior swim bladder inflation Reduced, Posterior swim bladder inflation
4 KE 1005 Reduced, Swimming performance Reduced, Swimming performance
5 KE 1006 Reduced, Young of year survival Reduced, Young of year survival
6 KE 1007 Reduced, Anterior swim bladder inflation Reduced, Anterior swim bladder inflation
7 KE 1008 Reduced, Hearing Reduced, Hearing
8 AO 360 Decrease, Population trajectory Decrease, Population trajectory

Key Event Relationships

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
Inhibition, Deiodinase 2 adjacent Decreased, Triiodothyronine (T3) in serum
Decreased, Triiodothyronine (T3) in serum adjacent Reduced, Posterior swim bladder inflation
Reduced, Posterior swim bladder inflation adjacent Reduced, Swimming performance
Reduced, Swimming performance adjacent Reduced, Young of year survival
Reduced, Young of year survival adjacent Decrease, Population trajectory
Reduced, Posterior swim bladder inflation adjacent Reduced, Anterior swim bladder inflation
Reduced, Anterior swim bladder inflation adjacent Reduced, Hearing
Reduced, Hearing adjacent Reduced, Young of year survival
Reduced, Anterior swim bladder inflation adjacent Reduced, Swimming performance
Reduced, Posterior swim bladder inflation non-adjacent Reduced, Young of year survival High Low
Inhibition, Deiodinase 2 non-adjacent Reduced, Posterior swim bladder inflation

Overall Assessment of the AOP

Domain of Applicability

Life Stage Applicability
Life Stage Evidence
Development
Taxonomic Applicability
Term Scientific Term Evidence Links
fathead minnow Pimephales promelas NCBI
zebrafish Danio rerio NCBI
Sex Applicability
Sex Evidence
Unspecific

References


Appendix 1

List of MIEs in this AOP

Event: 1002: Inhibition, Deiodinase 2

Short Name: Inhibition, Deiodinase 2

Key Event Component

Process Object Action
catalytic activity type II iodothyronine deiodinase decreased

Stressors

Name
iopanoic acid
PERFLUOROOCTANOIC ACID

Biological Context

Level of Biological Organization
Molecular

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
zebrafish Danio rerio NCBI
fathead minnow Pimephales promelas NCBI

Deiodination by DIO enzymes is known to exist in a wide range of vertebrates and invertebrates.


Key Event Description

Disruption of the thyroid hormone system is increasingly being recognized as an important toxicity pathway, as it can cause many adverse outcomes. Thyroid hormones do not only play an important role in the adult individual, but they are also critical during embryonic development. Thyroid hormones (THs) play an important role in a wide range of biological processes in vertebrates including growth, development, reproduction, cardiac function, thermoregulation, response to injury, tissue repair and homeostasis. Numerous chemicals are known to disturb thyroid function, for example by inhibiting thyroperoxidase (TPO) or deiodinase (DIO), upregulating excretion pathways or modifying gene expression. The two major thyroid hormones are triiodothyronine (T3) and thyroxine (T4), both iodinated derivatives of tyrosine. The synthesis of the thyroid hormones is a process that involves several steps. Thyroglobulin, the thyroid hormone precursor, is produced by the thyroid epithelial cells and transported to the lumen via exocytosis. Then thyroperoxidase (TPO) plays an essential role in the production of mainly T4. The prohormone T4 is then released in the circulation under the influence of thyroid stimulating hormone (TSH), in order to be transported to the various tissues, including the liver, the kidneys and the heart. Most TH actions depend on the binding of T3 to its nuclear receptors. 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 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 to convert T4 into T3, as well as to convert rT3 to the inactive thyroid hormone 3,3’ T2, through outer ring deiodination. rT3 is the preferred substrate for DIO1 (Hennemann G, Visser TJ 1997). Type II deiodinase (DIO2) is only capable of ORD activity with T4 as a preferred substrate. DIO3 can inner ring deiodinate T4 and T3 to the inactive forms of THs, reverse T3, (rT3) and 3,3’-T2 respectively.


How it is Measured or Detected

At this time, there are no approved OECD or EPA guideline protocols for measurement of DIO inhibition. Deiodination is the major pathway regulating T3 bioavailability in mammalian tissues. The objective of this in vitro assay is to examine inhibition of deiodinase 2 (DIO2) activity upon exposure to thyroid disrupting compounds, using unexposed pig liver tissue. There are three types of deiodinase measurements available. A first in vitro assay measures deiodinase activities by quantifying the radioactive iodine release from iodine-labelled substrates, depending on the preferred substrates of the isoforms of deiodinases. A second assay is a chromatography-based method coupled to mass spectroscopy to measure products of thyroxin by deiodinase type-1 activity (Butt et al., 2010). Finally, a colorimetric method was developed (Renko et al., 2012) that measures the release of iodine from T4.

Although the radioactive based assays uses radioactivity to measure deiodinase activity, they provide a good balance between specificity and resources needed. The chromatography-based assay has a high sensitivity and specificity to measure all thyroid hormones metabolites, but a high degree of technical expertise and expensive instrumentation is required. Although the colorimetric method is a promising alternative, the sensitivity of this assay is still limited.

For all the reasons above, we chose to use the radioactive method. Since DIO1 and DIO2 prefer a different substrate to deiodinate, i.e. rT3 and T4 respectively, it is possible to quantify outer-ring deiodination using the specific enzymkinetics of both enzymes. This assay measures the amount of radioactive iodine that is released from 125I-labelled substrates by conversion of one of the substrates by the DIO enzymes. We used a pig liver homogenate preparation and reaction buffers containing DTT as co-substrate. Furthermore, a concentration range of potential thyroid-disrupting chemicals can be added to measure the inhibitory potencies of the chemicals the inhibit DIO enzyme activity. Enzym activity is expressed as picomoles or femtomoles of released radioactive iodine per minute per mg protein and if inhibition occurs, the half maximal inhibitory concentration (IC50) was determined.


References

Visser, T.J., Van Overmeeren, E., Fekkes, D., Docter, R., Hennemann, G. 1979. Inhibition of iodothyronine 5'-deiodinase by thioureylenes: structure-activity relationship. FEBS Letters, 103, 2.

Butt, C.M., Wang, D., Stapleton, H.M. 2011. Halogenated phenolic contaminants inhibit the in vitro activity of the thyroid-regulating deiodinases in human liver. Toxicological sciences 124: 339-347.

Renko, K., Hoefie, C.S., Hiller, F., Schomburg, L., Köhrle, J. 2012. Identification of Iopanoic acid as substrate of type 1 deiodinase by a novel nonradioactive iodide-release assay. Endocrinology, 153: 2506-2513.


List of Key Events in the AOP

Event: 1003: Decreased, Triiodothyronine (T3) in serum

Short Name: Decreased, Triiodothyronine (T3) in serum

Key Event Component

Process Object Action
abnormal circulating hormone level decreased

Biological Context

Level of Biological Organization
Tissue

Organ term

Organ term
serum

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
zebrafish Danio rerio NCBI
fathead minnow Pimephales promelas NCBI

The overall evidence supporting taxonomic applicability is strong. With few exceptions vertebrate species have circulating T3 and T4 that are bound to transport proteins in blood. Clear species differences exist in transport proteins (Yamauchi and Isihara, 2009). Specifically, the majority of supporting data for TH decreases in serum come from rat studies, and the predominant iodothyronine binding protein in rat serum is transthyretin (TT4). TT4 demonstrates a reduced binding affinity for T4 when compared with thyroxine binding globulin (TBG), the predominant serum binding protein for T4 in humans. This difference in serum binding protein affinity for THs is thought to modulate serum half-life for T4; the half-life of T4 in rats is 12-24 hr, wherease the half-life in humans is 5-9 days (Capen, 1997). While these species differences impact hormone half-life, possibly regulatory feedback mechanisms, and quantitative dose-response relationships, measurement of serum THs is still regarded as a measurable key event causatively linked to downstream adverse outcomes.

THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in amphibian and larbean metamorphoses is well established (Manzon and Youson, 1997; Yaoita and Brown, 1990). Their existence and importance has been also described in many differrent animal and plant kingdoms (Eales, 1997; Heyland and Moroz, 2005), while their role as environmental messenger via exogenous routes in echinoderms confirms the hypothesis that these molecules are widely distributed among the living organisms (Heyland and Hodin, 2004). However, the role of TH in the different species may differ depending on the expression or function of specific proteins (e.g receptors or enzymes) that are related to TH function, and therefore extrapolation between species should be done with cautious.


Key Event Description

There are two biological active thyroid hormones (THs), triiodothyronine (T3) and thyroxine (T4), and a few inactive iodothyronines (rT3, 3,5-T2), which are all derived from the modification of tyrosine molecules (Hulbert, 2000). However, the plasma concentrations of the other iodothyronines are significantly lower than those of T3 and T4. The different iodothyronines are formed by the sequential outer or inner ring monodeiodination of T4 by the deiodinating enzymes, Dio1, Dio2, and Dio3 (Gereben et al., 2008). Deiodinase structure is considered to be unique, as THs are the only molecules in the body that incorporate iodide.

The circulatory system serves as the major transport and delivery system for THs from synthesis in the gland to delivery to tissues. The majority of THs in the blood are bound to transport proteins (Bartalena and Robbins, 1993). In humans, the major transport proteins are TBG (thyroxine binding globulin), TTR (transthyretin) and albumin. The percent bound to these proteins in adult humans is about 75, 15 and 10 percent, respectively (Schussler 2000). Unbound (free) hormones are approximately 0.03 and 0.3 percent for T4 and T3, respectively. In serum, it is the free form of the hormone that is active.

There are major species differences in the predominant binding proteins and their affinities for THs (see section below on Taxonomic applicability). However, there is broad agreement that changes in serum concentrations of THs is diagnostic of thyroid disease or chemical-induced disruption of thyroid homeostasis (Zoeller et al., 2007).

It is notable that the changes measured in the TH concentration reflect mainly the changes in the serum transport proteins rather than changes in the thyroid status. These thyroid-binding proteins serve as hormonal store which ensure their even and constant distribution in the different tissues, while they protect the most sensitive ones in the case of severe changes in thyroid availability, like in thyroidectomies (Obregon et al., 1981). Until recently, it was believed that all of the effects of TH were mediated by the binding of T3 to the thyroid nuclear receptors (TRa and TRb), a notion which is now questionable due to the increasing evidence that support the non-genomic action of TH (Davis et al., 2010, Moeller et al., 2006). Many non-nuclear TH binding sites have been identified to date and they usually lead to rapid cellular response in TH-effects (Bassett et al., 2003), but the specific pathways that are activated in this regard need to be elucidated.

The production of THs in the thyroid gland and the circulation levels in the bloodstream are self-controlled by an efficiently regulated feedback mechanism across the Hypothalamus-Pituitary-Thyroid (HPT) axis. One of the most unique characteristics of TH is their ability to regulate their own concentration, not only in the plasma level, but also in the individual cell level, to maintain their homeostasis. This is succeed by the efficient regulatory mechanism of the thyroid hormone axis which consists of the following: (1) the hypothalamic secretion of the thyrotropin-releasing hormone (TRH), (2) the thyroid-stimulating hormone (TSH) secretion from the anterior pituitary, (3) hormonal transport by the plasma binding proteins, (4) cellular uptake mechanisms in the cell level, (5) intracellular control of TH concentration by the deiodinating mechanism (6) transcriptional function of the nuclear thyroid hormone receptor and (7) in the fetus, the transplacental passage of T4 and T3 (Cheng et al., 2010).

In regards to the brain, the TH concentration involves also an additional level of regulation, namely the hormonal transport through the Blood Brain Barrier (BBB) (Williams, 2008). The TRH and the TSH are actually regulating the production of pro-hormone T4 and in a lesser extent of T3, which is the biologically active TH. The rest of the required amount of T3 is produced by outer ring deiodination of T4 by the deiodinating enzymes D1 and D2 (Bianco et al., 2006), a process which takes place mainly in liver and kidneys but also in other target organs such as in the brain, the anterior pituitary, brown adipose tissue, thyroid and skeletal muscle (Gereben et al., 2008; Larsen, 2009). Both hormones exert their action in almost all tissues of mammals and they are acting intracellularly, and thus the uptake of T3 and T4 by the target cells is a crucial step of the overall pathway. The trans-membrane transport of TH is performed mainly through transporters that differ depending on the cell type (Hennemann et al., 2001; Friesema et al., 2005; Visser et al., 2008). Many transporter proteins have been identified up to date but the monocarboxylate transporters (Mct8, Mct10) and the anion-transporting polypeptide (OATP1c1) show the highest degree of affinity towards TH (Jansen et al., 2005).

T3 and T4 have significant effects on normal development, neural differentiation, growth rate and metabolism (Yen, 2001; Brent, 2012; Williams, 2008), with the most prominent ones to occur during the fetal development and early childhood. The clinical features of hypothyroidism and hyperthyroidism emphasize the pleiotropic effects of these hormones on many different pathways and target organs. The thyroidal actions though are not only restricted to mammals, as their high significance has been identified also for other vertebrates, with the most well-studied to be the amphibian metamorphosis (Furlow and Neff, 2006). The importance of the thyroid-regulated pathways becomes more apparent in iodine deficient areas of the world, where a higher rate of cretinism and growth retardation has been observed and linked to decreased TH levels (Gilbert et al., 2012). Another very common cause of severe hypothyroidism in human is the congenital hypothyroidism, but the manifestation of these effects is only detectable in the lack of adequate treatment and is mainly related to neurological impairment and growth retardation (Glinoer, 2001), emphasizing the role of TH in neurodevelopment in all above cases. In adults, the thyroid-related effects are mainly linked to metabolic activities, such as deficiencies in oxygen consumption, and in the metabolism of the vitamin, proteins, lipids and carbohydrates, but these defects are subtle and reversible (Oetting and Yen, 2007). Blood tests to detect the amount of thyroid hormone (T4) and thyroid stimulating hormone (TSH) are routinely done for newborn babies for the diagnosis of congenital hypothyroidism at the earliest stage possible.


How it is Measured or Detected

T3 and T4 can be measured as free (unbound) or total (bound + unbound). Free hormone are considered more direct indicators of T4 and T3 activities in the body. The majority of T3 and T4 measurements are made using either RIA or ELISA kits. In animal studies, total T3 and T4 are typically measured as the concentrations of free hormone are very low and difficult to detect. Historically, the most widely used method in toxicology is RIA. The method is routinely used in rodent endocrine and toxicity studies. The ELISA method has become more routine in rodent studies. The ELISA method is a commonly used as a human clinical test method. Least common is analytical determination of iodothyronines (T3, T4, rT3, T2) and their conjugates, though methods employing HLPC and mass spectrometry (DeVito et al., 1999; Miller et al., 2009).

Any of these measurements should be evaluated for fit-for-purpose, relationship to the actual endpoint of interest, repeatability, and reproducibility. All three of the methods summarized above would be fit-for-purpose, depending on the number of samples to be evaluated and the associated costs of each method. Both RIA and ELISA measure THs by a an indirect methodology, whereas analytical determination is the most direct measurement available. All of these methods, particularly RIA, are repeatable and reproducible.


References

  • Bartalena L, Robbins J.Thyroid hormone transport proteins.Clin Lab Med. 1993 Sep;13(3):583-98.
  • Bassett JH, Harvey CB, Williams GR. (2003). Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol Cell Endocrinol. 213:1-11.
  • Bianco AC, Kim BW. (2006). Deiodinases: implications of the local control of thyroid hormone action. J Clin Invest. 116: 2571–2579.
  • Brent GA. (2012). Mechanisms of thyroid hormone action. J Clin Invest. 122: 3035-3043.
  • Cheng SY, Leonard JL, Davis PJ. (2010).Molecular aspects of thyroid hormone actions. Endocr Rev. 31:139–170.
  • Davis PJ, Zhou M, Davis FB, Lansing L, Mousa SA, Lin HY. (2010). Mini-review: Cell surface receptor for thyroid hormone and nongenomic regulation of ion fluxes in excitable cells. Physiol Behav. 99:237–239.
  • DeVito M, Biegel L, Brouwer A, Brown S, Brucker-Davis F, Cheek AO, Christensen R, Colborn T, Cooke P, Crissman J, Crofton K, Doerge D, Gray E, Hauser P, Hurley P, Kohn M, Lazar J, McMaster S, McClain M, McConnell E, *Meier C, Miller R, Tietge J, Tyl R. (1999). Screening methods for thyroid hormone disruptors. Environ Health Perspect. 107:407-415.
  • Eales JG. (1997). Iodine metabolism and thyroid related functions in organisms lacking thyroid follicles: Are thyroid hormones also vitamins? Proc Soc Exp Biol Med. 214:302-317.
  • Friesema EC, Jansen J, Milici C, Visser TJ. (2005). Thyroid hormone transporters. Vitam Horm. 70: 137–167.
  • Furlow JD, Neff ES. (2006). A developmental switch induced by thyroid hormone: Xenopus laevis metamorphosis. Trends Endocrinol Metab. 17:40–47.
  • Gereben B, Zavacki AM, Ribich S, Kim BW, Huang SA, Simonides WS, Zeöld A, Bianco AC. (2008). Cellular and molecular basis of deiodinase-regulated thyroid hormone signalling. Endocr Rev. 29:898–938.
  • Gilbert ME, Rovet J, Chen Z, Koibuchi N. (2012).Developmental thyroid hormone disruption: prevalence, environmental contaminants and neurodevelopmental consequences. Neurotoxicology. 33: 842-852.
  • Glinoer D. (2001).Potential consequences of maternal hypothyroidism on the offspring: evidence and implications. Horm Res. 55:109-114.
  • Hennemann G, Docter R, Friesema EC, de Jong M, Krenning EP, Visser TJ. (2001). Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocr Rev. 22:451-476.
  • Heyland A, Hodin J. (2004). Heterochronic developmental shift caused by thyroid hormone in larval sand dollars and its implications for phenotypic plasticity and the evolution of non-feeding development. Evolution. 58: 524-538.
  • Heyland A, Moroz LL. (2005). Cross-kingdom hormonal signaling: an insight from thyroid hormone functions in marine larvae. J Exp Biol. 208:4355-4361.
  • Hulbert A J. (2000). Thyroid hormones and their effects: A new perspective. Biol Rev. 75: 519-631.
  • Jansen J, Friesema EC, Milici C, Visser TJ. (2005). Thyroid hormone transporters in health and disease. Thyroid. 15: 757-768.
  • Larsen PR. (2009).Type 2 iodothyronine deiodinase in human skeletal muscle: new insights into its physiological role and regulation. J Clin Endocrinol Metab. 94:1893-1895.
  • Manzon RG, Youson JH. (1997). The effects of exogenous thyroxine (T4) or triiodothyronine (T3), in the presence and absence of potassium perchlorate, on the incidence of metamorphosis and on serum T4 and T3 concentrations in larval sea lampreys (Petromyzon marinus L.). Gen Comp Endocrinol. 106:211-220.
  • Miller MD, Crofton KM, Rice DC, Zoeller RT. (2009).Thyroid-disrupting chemicals: interpreting upstream biomarkers of adverse outcomes. Environ Health Perspect. 117:1033-1041.
  • Moeller LC, Dumitrescu AM, Seo H, Refetoff S. (2006). Thyroid hormone mediated changes in gene expression can be initiated by cytosolic action of the thyroid hormone receptor β through the phosphatidylinositol 3-kinase pathway. NRS. 4:1-4.
  • Obregon MJ, Mallol J, Escobar del Rey F, Morreale de Escobar G. (1981). Presence of l-thyroxine and 3,5,3-triiodo-l-thyronine in tissues from thyroidectomised rats. Endocrinology 109:908-913.
  • Oetting A, Yen PM. (2007). New insights into thyroid hormone action. Best Pract Res Clin Endocrinol Metab. 21:193–208.
  • Schussler, G.C. (2000). The thyroxine-binding proteins. Thyroid 10:141–149.
  • Visser WE, Friesema EC, Jansen J, Visser TJ. (2008). Thyroid hormone transport in and out of cells. Trends Endocrinol Metab. 19:50-56.
  • Williams GR. (2008). Neurodevelopmental and neurophysiological actions of thyroid hormone. J Neuroendocrinol. 20:784–794.
  • Yamauchi K1, Ishihara A. Evolutionary changes to transthyretin: developmentally regulated and tissue-specific gene expression.FEBS J. 2009 Oct;276(19):5357-66.
  • Yaoita Y, Brown DD. (1990). A correlation of thyroid hormone receptor gene expression with amphibian metamorphosis. Genes Dev. 4:1917-1924.
  • Yen PM. (2001). Physiological and molecular basis of thyroid hormone action. Physiol Rev. 81:1097-1142.
  • Zoeller RT, Tan SW, Tyl RW. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Crit Rev Toxicol. 2007 Jan-Feb;37(1-2):11-53

 


Event: 1004: Reduced, Posterior swim bladder inflation

Short Name: Reduced, Posterior swim bladder inflation

Key Event Component

Process Object Action
swim bladder inflation posterior chamber swim bladder decreased

Biological Context

Level of Biological Organization
Organ

Organ term

Organ term
swim bladder

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
zebrafish Danio rerio NCBI
fathead minnow Pimephales promelas NCBI

The evidence for impaired posterior chamber of the swim bladder currently comes from work on zebrafish and fathead minnow.


Key Event Description

The swim bladder of bony fish is evolutionary homologous to the lung (Zheng et al., 2011). The teleost swim bladder is a gas-filled structure that consists of two chambers, the posterior and anterior chamber. In zebrafish, the posterior chamber inflates around 96 h post fertilization (hpf) which is 2 days post hatch, and the anterior chamber inflates around 21 dpf. In fathead minnow, the posterior and anterior chamber inflate around 6 and 14 dpf respectively.

The posterior chamber is formed from a bud originating from the foregut endoderm (Winata et al., 2009). The posterior chamber operates as a hydrostatic organ. The volume of gas in the adult swim bladder is continuously adjusted to regulate body density and buoyancy.

Many amphibians and frogs go through an embryo-larval transition phase marking the switch from endogenous feeding (from the yolk) to exogenous feeding. In zebrafish, embryonic-to-larval transition takes place around 96 hours post fertilization (hpf). As in amphibians, the transition between the different developmental phases includes maturation and inflation of the swim bladder (Liu and Chan, 2002).

Reduced inflation of the posterior chamber may manifest itself as either a complete failure to inflate the chamber or a reduced size of the chamber.


How it is Measured or Detected

In several fish species, inflation of the posterior chamber can easily be observed using a stereomicroscope because the larvae are still transparent during those early developmental stages. This is for example true for zebrafish and fathead minnow. Posterior chamber size can then be measured based on photographs with a calibrator.


References

Zheng, W., Wang, Z., Collins, J.E., Andrews, R.M., Stemple, D., Gong, Z., 2011.Comparative transcriptome analyses indicate molecular homology of zebrafishswimbladder and mammalian lung. PLoS One 6, http://dx.doi.org/10.1371/journal.pone.0024019.

Winata, C.L., Korzh, S., Kondrychyn, I., Zheng, W., Korzh, V., Gong, Z., 2009.Development of zebrafish swimbladder: the requirement of Hedgehogsignaling 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.

Liu, Y.W., Chan, W.K., 2002. Thyroid hormones are important for embryonic tolarval transitory phase in zebrafish. Differentiation 70, 36–45, http://dx.doi.org/10.1046/j.1432-0436.2002.700104.x.


Event: 1005: Reduced, Swimming performance

Short Name: Reduced, Swimming performance

Key Event Component

Process Object Action
aquatic locomotion decreased

Biological Context

Level of Biological Organization
Individual

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
zebrafish Danio rerio NCBI

Importance of swimming performance for natural behaviour is generally applicable to fish.


Key Event Description

Adequate swimming performance in fish is essential for behaviour such as foraging, predator avoidance and reproduction.


How it is Measured or Detected

For fish larvae, automated observation and tracking systems are commercially available and increasingly used for measuring swimming performance including distance travelled, duration of movements, swimming speed, etc. This kind of measurements is often included in publications describing effects of chemicals in zebrafish larvae (Hagenaars et al., 2014; Stinckens et al., 2016; Vergauwen et al., 2015).


References

Hagenaars, A., Stinckens, E., Vergauwen, L., Bervoets, L., Knapen, D., 2014. PFOSaffects posterior swim bladder chamber inflation and swimming performanceof zebrafish larvae. Aquat. Toxicol. 157, 225–235.

Stinckens, E., Vergauwen, L., Schroeder, A.L., Maho, W., Blackwell, B., Witter, H.,Blust, R., Ankley, G.T., Covaci, A., Villenueve, D.L., Knapen, D., 2016. Disruption of thyroid hormone balance after 2-mercaptobenzothiazole exposure causes swim bladder inflation impairment—part II: zebrafish. Aquat. Toxicol. 173:204-17.

Vergauwen, Lucia; Nørgaard Schmidt, Stine; Maho, Walid; Stickens, Evelyn; Hagenaars, An; Blust, Ronny; Mayer, Philipp; Covaci, Adrian; Knapen, Dries. 2014. A high throughput passive dosing format for the Fish Embryo Acute Toxicity test. Chemosphere. 139: 9-17.


Event: 1006: Reduced, Young of year survival

Short Name: Reduced, Young of year survival

Key Event Component

Process Object Action
survival decreased

Biological Context

Level of Biological Organization
Individual

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
zebrafish Danio rerio NCBI

Survival is important for all species.


Key Event Description

Young of year refers to young animals (usually fish) produced in one reproductive year, which have not yet reached one year of age. Small fish, hatched from eggs spawned in the current year, are considered young of year.

Young of year survival directly impacts population structure, growth and fitness. Maintenance of sustainable fish and wildlife populations is an accepted regulatory goal upon which risk assessments and risk management decisions are based.


How it is Measured or Detected

Young of year survival can be measured:

  • in the lab by recording survival during prolonged exposure experiments
  • in dedicated mesocosms, or in drainable ponds
  • in the field, for example by determining age structure after one capture, or by capture-tag-recapture efforts

Event: 1007: Reduced, Anterior swim bladder inflation

Short Name: Reduced, Anterior swim bladder inflation

Key Event Component

Process Object Action
swim bladder inflation anterior chamber swim bladder decreased

Biological Context

Level of Biological Organization
Organ

Organ term

Organ term
swim bladder

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
zebrafish Danio rerio NCBI
fathead minnow Pimephales promelas NCBI

The evidence for impaired inflation of the anterior chamber of the swim bladder currently comes from work on zebrafish and fathead minnow.


Key Event Description

The swim bladder of bony fish is evolutionary homologous to the lung (Zheng et al., 2011). The teleost swim bladder is a gas-filled structure that consists of two chambers, the posterior and anterior chamber. In zebrafish, the posterior chamber inflates around 96 h post fertilization (hpf) which is 2 days post hatch, and the anterior chamber inflates around 21 dpf. In fathead minnow, the posterior and anterior chamber inflate around 6 and 14 dpf respectively.

The anterior chamber is formed by evagination from the cranial end of the posterior chamber (Robertson et al., 2007). Dumbarton et al. (2010) showed that the anterior chamber of zebrafish has particularly closely packed and highly organized bundles of muscle fibres, suggesting that contraction of these muscles would reduce swim bladder volume. While it had previously been suggested that the posterior chamber had a more important role as a hydrostatic organ, this implies high importance of the anterior chamber for buoyancy. The anterior chamber has an additional role in hearing (Bang et al., 2002). Weberian ossicles (the Weberian apparatus) connect the anterior chamber to the inner ear resulting in an amplification of sound waves. Reduced inflation of the anterior chamber may manifest itself as either a complete failure to inflate the chamber or reduced size of the chamber. Reduced size is often associated with a deviating morphology.


How it is Measured or Detected

In several fish species, inflation of the anterior chamber can be observed using a stereomicroscope because the larvae are still transparent during the larval stage. This is for example true for zebrafish and fathead minnow. Anterior chamber size can then be measured based on photographs with a calibrator.


References

Zheng, W., Wang, Z., Collins, J.E., Andrews, R.M., Stemple, D., Gong, Z. 2011. Comparative transcriptome analyses indicate molecular homology of zebrafish swim bladder and mammalian lung. PLoS One 6, http://dx.doi.org/10.1371/

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.

Dumbarton, T.C., Stoyek, M., Croll, R.P., Smith, F.M., 2010. Adrenergic control of swimbladder deflation in the zebrafish (Danio rerio). J. Exp. Biol. 213,2536–2546, http://dx.doi.org/10.1242/jeb.039792.

Bang, P.I., Yelick, P.C., Malicko, J.J., Sewell, W.F. 2002. High-throughput behavioral screening method for detecting auditory response defects in zebrafish. Journal of Neuroscience Methods. 118, 177-187.


Event: 1008: Reduced, Hearing

Short Name: Reduced, Hearing

Key Event Component

Process Object Action
sensory perception of sound decreased

Biological Context

Level of Biological Organization
Organ

Organ term

Organ term
ear

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
Vertebrates Vertebrates NCBI
Invertebrates Invertebrates NCBI
  • A sense of hearing is known to exist in a wide range of vertebrates and invertebrates, although the organs and structures involved vary widely.

Key Event Description

Hearing refers to the ability to perceive sound vibrations propagated as pressure changes through a medium such as air or water. Reduced hearing in the context of this key event can refer to reduction in the perceived volume of a sound relative to the amplitude of sound waves. Reduced hearing may also refer to a reduced range of frequencies that can be perceived.


How it is Measured or Detected

Hearing is generally measured behaviorally or electrophysiologically.

  • Common behavioral tests involve transmission of pure tones of defined amplitude and frequency using and audiometer or PC and using a behavioral response (e.g., clicking a button; startle response) to determine whether the tone is perceived.

Electrophysiological tests:

  • Auditory brainstem response (ABR): Uses electrodes placed on the head to detect auditory evoked potentials from background electrical activity in the brain.

Hearing tests in Fish:

  • Through the mid-late 1980s conditioning and behavioral tests were most commonly employed in testing fish hearing. Methods reviewed by Fay (1988)
  • A high throughput behavioral test for detecting auditory response in fish has been described (Bang et al. 2002).
  • Invasive electrophysiological methods involving surgical insertion of electrodes into the auditory nerves have been employed.
  • Non-invasive recording of Auditory Evoked Potentials (AEPs; synonymous with ABRs) are now the most common approach for measuring hearing in fish. AEPs can be recorded via electrodes attached cutaneously to the head (see review by Ladich and Fay, 2013).

References

  • Fay RR (1988) Hearing in vertebrates: a psychophysics databook. Hill-Fay Associates, Winnetka, Ill
  • Ladich F, Fay RR. Auditory evoked potential audiometry in fish. Reviews in Fish Biology and Fisheries. 2013;23(3):317-364. doi:10.1007/s11160-012-9297-z.
  • Bang PI, Yelick PC, Malicki JJ, Sewell WF. High-throughput behavioral screening method for detecting auditory response defects in zebrafish. J Neurosci Methods. 2002 Aug 30;118(2):177-87. PubMed PMID: 12204308.

List of Adverse Outcomes in this AOP

Event: 360: Decrease, Population trajectory

Short Name: Decrease, Population trajectory

Key Event Component

Process Object Action
population growth rate decreased

AOPs Including This Key Event

AOP ID and Name Event Type
Aop:23 - Androgen receptor agonism leading to reproductive dysfunction (in repeat-spawning fish) AdverseOutcome
Aop:25 - Aromatase inhibition leading to reproductive dysfunction AdverseOutcome
Aop:29 - Estrogen receptor agonism leading to reproductive dysfunction AdverseOutcome
Aop:30 - Estrogen receptor antagonism leading to reproductive dysfunction AdverseOutcome
Aop:100 - Cyclooxygenase inhibition leading to reproductive dysfunction via inhibition of female spawning behavior AdverseOutcome
Aop:122 - Prolyl hydroxylase inhibition leading to reproductive dysfunction via increased HIF1 heterodimer formation AdverseOutcome
Aop:123 - Unknown MIE leading to reproductive dysfunction via increased HIF-1alpha transcription AdverseOutcome
Aop:155 - Deiodinase 2 inhibition leading to reduced young of year survival via posterior swim bladder inflation AdverseOutcome
Aop:156 - Deiodinase 2 inhibition leading to reduced young of year survival via anterior swim bladder inflation AdverseOutcome
Aop:157 - Deiodinase 1 inhibition leading to reduced young of year survival via posterior swim bladder inflation AdverseOutcome
Aop:158 - Deiodinase 1 inhibition leading to reduced young of year survival via anterior swim bladder inflation AdverseOutcome
Aop:159 - Thyroperoxidase inhibition leading to reduced young of year survival via anterior swim bladder inflation AdverseOutcome
Aop:101 - Cyclooxygenase inhibition leading to reproductive dysfunction via inhibition of pheromone release AdverseOutcome
Aop:102 - Cyclooxygenase inhibition leading to reproductive dysfunction via interference with meiotic prophase I /metaphase I transition AdverseOutcome
Aop:63 - Cyclooxygenase inhibition leading to reproductive dysfunction AdverseOutcome
Aop:103 - Cyclooxygenase inhibition leading to reproductive dysfunction via interference with spindle assembly checkpoint AdverseOutcome

Biological Context

Level of Biological Organization
Population

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
all species all species NCBI
Life Stage Applicability
Life Stage Evidence
All life stages Not Specified
Sex Applicability
Sex Evidence
Unspecific Not Specified

Consideration of population size and changes in population size over time is potentially relevant to all living organisms.


Key Event Description

Maintenance of sustainable fish and wildlife populations (i.e., adequate to ensure long-term delivery of valued ecosystem services) is an accepted regulatory goal upon which risk assessments and risk management decisions are based.


How it is Measured or Detected

Population trajectories, either hypothetical or site specific, can be estimated via population modeling based on measurements of vital rates or reasonable surrogates measured in laboratory studies. As an example, Miller and Ankley 2004 used measures of cumulative fecundity from laboratory studies with repeat spawning fish species to predict population-level consequences of continuous exposure.


Regulatory Significance of the AO

Maintenance of sustainable fish and wildlife populations (i.e., adequate to ensure long-term delivery of valued ecosystem services) is a widely accepted regulatory goal upon which risk assessments and risk management decisions are based.


References

  • Miller DH, Ankley GT. 2004. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17ß-trenbolone as a case study. Ecotoxicology and Environmental Safety 59: 1-9.

Appendix 2

List of Key Event Relationships in the AOP