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Event: 1002

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Inhibition, Deiodinase 2

Short name
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Inhibition, Deiodinase 2
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Biological Context

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Level of Biological Organization
Molecular

Cell term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Organ term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor).  Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description.  To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons.  If a desired term does not exist, a new term request may be made via Term Requests.  Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Process Object Action
catalytic activity type II iodothyronine deiodinase decreased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
DIO2i posterior swim bladder MolecularInitiatingEvent Dries Knapen (send email) Under Development: Contributions and Comments Welcome WPHA/WNT Endorsed
DIO2i anterior swim bladder MolecularInitiatingEvent Dries Knapen (send email) Under Development: Contributions and Comments Welcome WPHA/WNT Endorsed
DIO2 inhib alters metamorphosis MolecularInitiatingEvent Jonathan Haselman (send email) Under Development: Contributions and Comments Welcome

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 KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
rat Rattus norvegicus Moderate NCBI
human Homo sapiens High NCBI
pigs Sus scrofa Moderate NCBI
Oreochromis niloticus Oreochromis niloticus Moderate NCBI
zebrafish Danio rerio Moderate NCBI
fathead minnow Pimephales promelas Moderate NCBI
African clawed frog Xenopus laevis NCBI

Life Stages

An indication of the the relevant life stage(s) for this KE. More help
Life stage Evidence
All life stages Moderate

Sex Applicability

An indication of the the relevant sex for this KE. More help
Term Evidence
Unspecific Moderate

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

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. 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 outer, phenolic ring of T4 to form T3, while inactivation occurs via inner ring deiodination (IRD), i.e. removing iodine from the inner tyrosol ring of T4 or T3.

Three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates that activate or inactivate THs and are therefore important mediators of TH action. All deiodinases are integral membrane proteins of the thioredoxin superfamily that contain selenocysteine in their catalytic centre. Type I deiodinase is capable to convert T4 into T3, as well as to convert reverse T3 (rT3) to 3,3'-Diiodothyronine (3,3’ T2), through outer ring deiodination. rT3, rather than T4, is the preferred substrate for DIO1. furthermore, DIO1 has a very high Km (µM range, compared to nM range for DIO2) (Darras and Van Herck, 2012). Type II deiodinase (DIO2) is only capable of ORD activity with T4 as a preferred substrate (i.e., activation of T4 to T3). DIO3 can inner ring deiodinate T4 and T3 to the inactive forms of THs,  rT3 and 3,3’-T2 respectively. DIO2 is a transmembrane protein anchored to the endoplasmic reticulum and the active site faces the perinuclear cytosol. The relative contribution of the DIOs to thyroid hormone levels varies amongst species, developmental stages and tissues.

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

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. In vitro assays can be used to examine inhibition of deiodinase 2 (DIO2) activity upon exposure to thyroid disrupting compounds.

Several methods for deiodinase activity measurements are 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 (Forhead et al., 2006; Pavelka, 2010; Houbrechts et al., 2016; Stinckens et al., 2018). Each of these assays requires a source of deiodinase which can be obtained for example using unexposed pig liver tissue (available from slaughterhouses) or rat liver tissue. Olker et al. (2019) on the other hand used an adenovirus expression system to produce the DIO2 enzyme and developed an assay for nonradioactive measurement of iodide released using the Sandell-Kolthoff method, a photometric method based on Ce4+ reduction (Renko et al., 2012). This assay was then used to screen the ToxCast Phase 1 chemical library. The specific synthesis of DIO2 through the adenovirus expression system provides an important advantage over other methods where activity of the different deiodinase isoforms needs to be distinguished in other ways, such as based on differences in enzyme kinetics.

Measurements of in vivo deiodinase activity in tissues collected from animal experiments are scarce. Noyes et al. (2011) showed decreased rate of outer ring deiodination (mediated by DIO1 and DIO2) in whole fish microsomes after exposure to BDE-209. After incubation with the substrate, thyroid hormone levels were measured using LC-MS/MS. Houbrechts et al. (2016) confirmed DIO2 deiodination activity in a DIO1-DIO2 knockdown zebrafish at the ages of 3 and 7 days post fertilization. Decreased T3 levels are often used as evidence of DIO inhibition, for example after exposure to iopanoic acid, in fish species such as zebrafish (Stinckens et al., 2020) and fathead minnow (Cavallin et al., 2017). It should be noted that it is difficult to make the distinction between decreased T3 levels caused by outer ring deiodination mediated by DIO2 inhibition or DIO1 inhibition.

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  More help

Taxonomic: Deiodination by DIO enzymes is known to exist in a wide range of vertebrates and invertebrates. This KE is plausibly applicable across vertebrates. Reports of inhibition of DIO2 activity are relatively scarce compared to DIO1. Studies reporting DIO2 inhibition have used human recombinant DIO2 enzyme (Olker et al., 2019), primary human astrocytes (Roberts et al., 2015), rat pituitary (Li et al., 2012), pig liver (Stinckens et al., 2018), Nile tilapia (Oreochromis niloticus) liver (Walpita et al., 2007). Evidence for fish (e.g., zebrafish and fathead minnow) is mostly indirect since DIO enzyme activity is usually not measured in chemical exposure experiments. Houbrechts et al. (2016) showed decreased DIO2 activity in a DIO1-DIO2 knockdown zebrafish at the ages of 3 and 7 days post fertilization together with impaired swim bladder inflation, showing that the enzyme is present, the activity is measurable and impairing its activity has negative effects. Noyes confirmed decreased outer ring deiodination activity in fathead minnows exposed to decabromodiphenyl ether (BDE-209). Walpita et al. (2007) showed decreased DIO2 activity in the liver of Nile tilapia injected with dexamethasone. Stinckens et al. (2018) showed that chemicals with DIO inhibitory potential in pig liver impaired swim bladder inflation in zebrafish, a thyroid hormone regulated process. Six out of seven DIO1 inhibitors impaired posterior chamber inflation, but almost all of these compounds also inhibit DIO2. TCBPA, the only compound that inhibits DIO1 and not DIO2, had no effect on the posterior swim bladder. Based on these results, DIO2 seemed to be more important than DIO1.  

In mammals, DIO2 is thought to control the intracellular concentration of T3, while DIO1 is thought to be more important in determining systemic T3 levels. The cells that express DIO2 locally produce T3 that can more rapidly access the thyroid receptors in the nucleus than T3 from plasma (Bianco et al., 2002). For example, DIO2 is highly expressed in the mammalian brain. However, this hypothesis has been challenged. For example, Maia et al. (2005) determined that in a normal physiological situation in humans the contribution of DIO2 to plasma T3 levels is twice that of DIO1. Only in a hyperthyroid state was the contribution of DIO1 higher than that of DIO2. A DIO1 knockout mouse showed normal T3 levels and a normal general phenotype and DIO1 was rather found to play a role in limiting the detrimental effects of conditions that alter normal thyroid function, including hyperthyroidism and iodine deficiency (Schneider et al., 2006). van der Spek et al. concluded that the primary role of DIO1 in vivo is to degrade inactivated TH (van der Spek et al., 2017).

The presence of DIO1 in the liver of teleosts has been a controversial issue and DIO1 function in teleostean and amphibian T3 plasma regulation is unclear (Finnson et al., 1999; Kuiper et al., 2006). In teleosts, DIO2 has a markedly higher activity level compared to other vertebrates and it is expressed in liver (Orozco and Valverde, 2005), suggesting its importance in determining systemic thyroid hormone levels. This could explain why DIO2 inhibition seems to be more important than DIO1 inhibition in determining the adverse outcome in zebrafish (Stinckens et al., 2018). 

Life stage: Deiodinase activity is important for all vertebrate life stages. Already during early embryonic development, deiodinase activity is needed to regulate thyroid hormone concentrations and coordinate developmental processes. DIO2 shows more marked changes in expression around the time of the embryo-larval and larval-to-juvenile transition periods during zebrafish development, highlighting its importance for early life stages (Vergauwen et al., 2018).

Sex: This KE is plausibly applicable to both sexes. Deiodinases are important for TH homeostasis and identical in both sexes. Therefore inhibition of deiodinases is not expected to be sex-specific.

References

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

Bianco, A.C., Salvatore, D., Gereben, B., Berry, M.J., Larsen, P.R., 2002. Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocrine Reviews 23, 38-89.

Cavallin JE, Ankley GT, Blackwell BR, Blanksma CA, Fay KA, Jensen KM, Kahl MD, Knapen D, Kosian PA, Poole ST et al. 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry. 36(11):2942-2952.

Darras, V.M., Van Herck, S.L.J., 2012. Iodothyronine deiodinase structure and function: from ascidians to humans. Journal of Endocrinology 215, 189-206.

Forhead, A.J., Curtis, K., Kaptein, E., Visser, T.J., Fowden, A.L., 2006. Developmental control of iodothyronine deiodinases by cortisol in the ovine fetus and placenta near term. Endocrinology 147, 5988-5994.

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.

Li, N.N., Jiang, Y.Q., Shan, Z.Y., Teng, W.P., 2012. Prolonged high iodine intake is associated with inhibition of type 2 deiodinase activity in pituitary and elevation of serum thyrotropin levels. British Journal of Nutrition 107, 674-682.

Noyes PD, Hinton DE, Stapleton HM. 2011. Accumulation and debromination of decabromodiphenyl ether (bde-209) in juvenile fathead minnows (pimephales promelas) induces thyroid disruption and liver alterations. Toxicological Sciences. 122(2):265-274.

Olker, J.H., Korte, J.J., Denny, J.S., Hartig, P.C., Cardon, M.C., Knutsen, C.N., Kent, P.M., Christensen, J.P., Degitz, S.J., Hornung, M.W., 2019. Screening the ToxCast Phase 1, Phase 2, and e1k Chemical Libraries for Inhibitors of Iodothyronine Deiodinases. Toxicological Sciences 168, 430-442.

Orozco, A., Valverde, R.C., 2005. Thyroid hormone deiodination in fish. Thyroid 15, 799-813.

Pavelka, S., 2010. Radiometric enzyme assays: development of methods for extremely sensitive determination of types 1, 2 and 3 iodothyronine deiodinase enzyme activities. Journal of Radioanalytical and Nuclear Chemistry 286, 861-865.

Renko, K., Hoefig, C.S., Hiller, F., Schomburg, L., Kohrle, J., 2012. Identification of Iopanoic Acid as Substrate of Type 1 Deiodinase by a Novel Nonradioactive Iodide-Release Assay. Endocrinology 153, 2506-2513.

Renko, K., Schache, S., Hoefig, C.S., Welsink, T., Schwiebert, C., Braun, D., Becker, N.P., Kohrle, J., Schomburg, L., 2015. An Improved Nonradioactive Screening Method Identifies Genistein and Xanthohumol as Potent Inhibitors of Iodothyronine Deiodinases. Thyroid 25, 962-968.

Roberts, S.C., Bianco, A.C., Stapleton, H.M., 2015. Disruption of Type 2 Iodothyronine Deiodinase Activity in Cultured Human Glial Cells by Polybrominated Diphenyl Ethers. Chemical Research in Toxicology 28, 1265-1274.

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.

Stinckens E, Vergauwen L, Blackwell BR, Anldey GT, Villeneuve DL, Knapen D. 2020. Effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology. 54(10):6213-6223.

van der Spek, A.H., Fliers, E., Boelen, A., 2017. The classic pathways of thyroid hormone metabolism. Molecular and Cellular Endocrinology 458, 29-38.

Vergauwen, L., Cavallin, J.E., Ankley, G.T., Bars, C., Gabriels, I.J., Michiels, E.D.G., Fitzpatrick, K.R., Periz-Stanacev, J., Randolph, E.C., Robinson, S.L., Saari, T.W., Schroeder, A.L., Stinckens, E., Swintek, J., Van Cruchten, S.J., Verbueken, E., Villeneuve, D.L., Knapen, D., 2018. Gene transcription ontogeny of hypothalamic-pituitary-thyroid axis development in early-life stage fathead minnow and zebrafish. General and Comparative Endocrinology 266, 87-100.

Walpita, C.N., Grommen, S.V., Darras, V.M., Van der Geyten, S., 2007. The influence of stress on thyroid hormone production and peripheral deiodination in the Nile tilapia (Oreochromis niloticus). Gen Comp Endocrinol 150, 18-25.