Key Event Title
|Level of Biological Organization|
Key Event Components
|catalytic activity||type I iodothyronine deiodinase||decreased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|DIO1i posterior swim bladder||MolecularInitiatingEvent|
|DIO1i anterior swim bladder||MolecularInitiatingEvent|
|DIO1 inhib alters metamorphosis||MolecularInitiatingEvent|
|Ovis orientalis aries||Ovis aries||Moderate||NCBI|
|fathead minnow||Pimephales promelas||Moderate||NCBI|
|gilthead bream||Sparus aurata||Moderate||NCBI|
|African clawed frog||Xenopus laevis||Moderate||NCBI|
|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 of both ORD and IRD, including the conversion of T4 into T3, as well as the conversion of rT3 to the inactive thyroid hormone 3,3’ T2. 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. DIO1 is a plasma membrane protein with its catalytic domain facing the 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 1 (DIO1) 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 (Ferreira et al., 2002; Forhead et al., 2006; Freyberger and Ahr, 2006; Pavelka, 2010; Stinckens et al., 2018). Another assay uses a chromatography-based method coupled to mass spectroscopy to measure products of thyroxin resulting from deiodinase type-1 activity (Butt et al., 2011). A colorimetric method (Renko et al., 2012), the Sandell-Kolthoff method, that measures the release of iodine from T4 is also available. 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. Hornung et al. (2018) and Olker et al. (2019) on the other hand used an adenovirus expression system to produce the DIO1 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 DIO1 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. Studies reporting DIO1 inhibition have used human liver (Kuiper et al., 2006), human recombinant DIO1 enzyme (Olker et al., 2019), rat (Rattus norvegicus) liver (Klaren et al., 2005; Freyberger and Ahr, 2006; Kuiper et al., 2006; Pavelka, 2010) and thyroid gland (Ferreira et al., 2002), mouse (Mus musculus) brain (hernandez et al., 2006), hog (Sus scrofa domesticus) liver (Stinckens et al., 2018), sheep (Ovis orientalis aries) fetal hepatic, renal and perirenal adipose tissue (Forhead et al., 2006), tadpole (Xenopus laevis) liver (Kuiper et al., 2006), fathead minnow (Pimephales promelas) whole fish (Noyes et al., 2011), Nile tilapia (Oreochromis niloticus) liver (Walpita et al., 2007), Gilthead Seabream (Sparus aurata) kidney (Klaren et al., 2005), and killifish (Fundulus heteroclitus) liver (Orozco et al., 2003) among others. The latter teleostean DIO1 enzymes as well as amphibian enzymes differ from other vertebrate DIO1 enzymes in their lower sensitivity to propylthiouracil (PTU), a typical DIO1 inhibitor in mammals.
Deiodinase 1 in liver is the main supplier of T3 to circulation in mammals (Marsili et al., 2011), and the same appears to be true for birds. By contrast, DIO1 function in teleostean and amphibian T3 plasma regulation is less clear (Finnson et al. 1999, Kuiper et al. 2006). The presence of DIO1 in the liver of teleosts has been a controversial issue, and both the high level of DIO2 activity and its expression in the liver of teleosts are unique among vertebrates (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. However, the role of DIO1 and DIO2 seems to be distinct. The fact that DIO1 knockdown during zebrafish development only causes developmental defects when combined with DIO2 knockdown (Walpita et al., 2010), suggests that DIO1 is only important in cases of increased TH need during specific stages of development, as supported by increased expression during such stages (Vergauwen et al., 2018), and in cases of thyroid hormone depletion in fish.
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Propylthiouracil (PTU) is the prototypical DIO1 inhibitor in mammals, although teleostean and amphibian DIO1 enzymes are less sensitive to inhibition by PTU (Orozco et al., 2003; Kuiper et al., 2006). Olker et al. (2019) identified 22 DIO1-specific inhibitors using a human recombinant DIO1 enzyme (e.g., genistein, 6-methyl-2-thiouracil, sulfasalazine). Another well-known inhibitor of DIO1 (and DIO2 and 3) is iopanoic acid (IOP). Renko et al. (2003, 2015) pointed out that IOP is actually a substrate of DIO1 (and DIO2 and 3) which is in line with its action as a competitive inhibotor. In fact, many compounds inhibit all three DIO isoforms. Olker et al. (2019) identified 93 compounds that inhibit DIOs 1, 2 and 3.
Butt, C.M., Wang, D.L., 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.
Darras, V.M., Van Herck, S.L.J., 2012. Iodothyronine deiodinase structure and function: from ascidians to humans. Journal of Endocrinology 215, 189-206.
Ferreira, A.C.F., Lisboa, P.C., Oliveira, K.J., Lima, L.P., Barros, I.A., Carvalho, D.P., 2002. Inhibition of thyroid type 1 deiodinase activity by flavonoids. Food and Chemical Toxicology 40, 913-917.
Finnson, K.W., McLeese, J.M., Eales, J.G., 1999. Deiodination and deconjugation of thyroid hormone conjugates and type I deiodination in liver of rainbow trout, Oncorhynchus mykiss. General and Comparative Endocrinology 115, 387-397.
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.
Freyberger, A., Ahr, H.J., 2006. Studies on the goitrogenic mechanism of action of N,N,N',N'-tetramethylthiourea. Toxicology 217, 169-175.
Hernandez, A., Martinez, M.E., Fiering, S., Galton, V.A., St Germain, D., 2006. Type 3 deiodinase is critical for the maturation and function of the thyroid axis. J Clin Invest 116, 476-484.
Hornung, M.W., Korte, J.J., Olker, J.H., Denny, J.S., Knutsen, C., Hartig, P.C., Cardon, M.C., Degitz, S.J., 2018. Screening the ToxCast Phase 1 Chemical Library for Inhibition of Deiodinase Type 1 Activity. Toxicological Sciences 162, 570-581.
Klaren, P.H.M., Haasdijk, R., Metz, J.R., Nitsch, L.M.C., Darras, V.M., Van der Geyten, S., Flik, G., 2005. Characterization of an iodothyronine 5 '-deiodinase in gilthead seabream (Sparus auratus) that is inhibited by dithiothreitol. Endocrinology 146, 5621-5630.
Kuiper, G., Klootwijk, W., Dubois, G.M., Destree, O., Darras, V.M., Van der Geyten, S., Demeneix, B., Visser, T.J., 2006. Characterization of recombinant Xenopus laevis type I iodothyronine deiodinase: substitution of a proline residue in the catalytic center by serine (Pro132Ser) restores sensitivity to 6-propyl-2-thiouracil. Endocrinology 147, 3519-3529.
Marsili, A., Zavacki, A.M., Harney, J.W., Larsen, P.R., 2011. Physiological role and regulation of iodothyronine deiodinases: A 2011 update. Journal of Endocrinological Investigation 34, 395-407.
Noyes, P.D., Hinton, D.E., Stapleton, H.M., 2011. Accumulation and Debromination of Decabromodiphenyl Ether (BDE-209) in Juvenile Fathead Minnows (Pimephales promelas) Induces Thyroid Disruption and Liver Alterations. Toxicological Sciences 122, 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.
Orozco, A., Villalobos, P., Jeziorski, M.C., Valverde, C., 2003. The liver of Fundulus heteroclitus expresses deiodinase type 1 mRNA. General and Comparative Endocrinology 130, 84-91.
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.
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.
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.
Walpita, C.N., Crawford, A.D., Darras, V.M., 2010. Combined antisense knockdown of type 1 and type 2 iodothyronine deiodinases disrupts embryonic development in zebrafish (Danio rerio). Gen Comp Endocrinol 166, 134-141.
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.