Key Event Title
|Level of Biological Organization|
Key Event Components
|catalytic activity||type II iodothyronine deiodinase||decreased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|DIO2i posterior swim bladder||MolecularInitiatingEvent|
|DIO2i anterior swim bladder||MolecularInitiatingEvent|
|DIO2 inhib alters metamorphosis||MolecularInitiatingEvent|
|Oreochromis niloticus||Oreochromis niloticus||Moderate||NCBI|
|All life stages||Moderate|
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 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 rT3 to the inactive thyroid hormone 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 tot T3). DIO3 can inner ring deiodinate T4 and T3 to the inactive forms of THs, reverse T3, (rT3) and 3,3’-T2 respectively. DIO2 is a transmembrane protein anchored to the endoplasmic reticulum and the active site faces the perinuclear cytosol.
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. 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 in a 96-well plate format. 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.
Domain of Applicability
Deiodination by DIO enzymes is known to exist in a wide range of vertebrates and invertebrates. Reports of inibition 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 zebrafish is indirect since DIO enzyme activity is usually not measured in chemical exposure experiments using zebrafish. 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. Based on these results, DIO2 seemed to be more important than DIO1.
In mammals, DIO2 controls the intracellular concentration of T3. 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. In teleosts, DIO2 has a markedly higher activity level compared to other vertebrates and it is expressed in liver (Orozco and Valverde, 2005). 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).
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).
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Olker et al. (2019) identified 20 DIO2-specific inhibitors using a human recombinant DIO2 enzyme (e.g., tetramethrin, elzasonan). Another typical inhibitor of DIO2 (and DIO1 and 3) is iopanoic acid (IOP), which acts as a substrate of all three DIO isoforms (Renko et al., 2015). In fact, many compounds inhibit all three DIO isoforms. Olker et al. (2019) identified 93 compounds that inhibit DIOs 1, 2 and 3.
Stinckens et al. (2018)
Stinckens et al. (2018)
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.
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.
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., 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.
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.
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.