Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|TPO Inhibition and Altered Neurodevelopment||KeyEvent|
|NIS inhibition and learning and memory impairment||KeyEvent|
|Nuclear receptor induced TH Catabolism and Developmental Hearing Loss||KeyEvent|
|NIS and Neurodevelopment||KeyEvent|
|NIS and Cognitive Dysfunction||KeyEvent|
|TPOi anterior swim bladder||KeyEvent|
|TPO inhib alters metamorphosis||KeyEvent|
|NIS inhib alters metamorphosis||KeyEvent|
|Hepatic nuclear receptor activation alters metamorphosis||KeyEvent|
|a to y||KeyEvent|
|thyroperoxidase alter metamorphosis||KeyEvent|
|123 to 345||KeyEvent|
|Xenopus laevis||Xenopus laevis||High||NCBI|
|zebra fish||Danio rerio||Moderate||NCBI|
|All life stages||High|
Key Event Description
All iodothyronines are derived from the modification of tyrosine molecules (Taurog, 2000). There are two biologically active thyroid hormones (THs) in serum, triiodothyronine (T3) and T4, and a few inactive iodothyronines (rT3, 3,5-T2). T4 is the predominant TH in circulation, comprising approximately 80% of the TH excreted from the thyroid gland and is the pool from which the majority of T3 in serum is generated (Zoeller et al., 2007). As such, serum T4 changes usually precede changes in other serum THs. Decreased thyroxine (T4) in serum results result from one or more MIEs upstream and is considered a key biomarker of altered TH homeostasis (DeVito et al., 1999).
Serum T4 is used as a biomarker of TH status because the circulatory system serves as the major transport and delivery system for TH delivery to tissues. The majority of THs in the blood are bound to transport proteins (Bartalena and Robbins, 1993). In serum, it is the unbound, or ‘free’ form of the hormone that thought to be available for transport into tissues. Free hormones are approximately 0.03 and 0.3 percent for T4 and T3, respectively. There are major species differences in the predominant binding proteins and their affinities for THs (see below). However, there is broad agreement that changes in serum concentrations of THs is diagnostic of thyroid disease or chemical-induced disruption of thyroid homeostasis (DeVito et al., 1999; Miller et al., 2009; Zoeller et al., 2007).
Normal serum T4 reference ranges can be species and lifestage specific. In rodents, serum THs are low in the fetal circulation, increasing as the fetal thyroid gland becomes functional on gestational day 17, just a few days prior to birth. After birth serum hormones increase steadily, peaking at two weeks, and falling slightly to adult levels by postnatal day 21 (Walker et al., 1980; Harris et al., 1978; Goldely et al., 1995; Lau et al., 2003). Similarly, in humans, adult reference ranges for THs do not reflect the normal ranges for children at different developmental stages, with TH concentrations highest in infants, still increased in childhood, prior to a decline to adult levels coincident with pubertal development (Corcoran et al. 1977; Kapelari et al., 2008). In some frog species, there is an analogous peak in thyroid hormones in tadpoles that starts around NF stage 56, peaks at Stage 62 and the declines to lower levels by Stage 56 (Sternberg et al., 2011; Leloup and Buscaglia, 1977).
How It Is Measured or Detected
Serum T3 and T4 can be measured as free (unbound) or total (bound + unbound). Free hormone concentrations are clinically considered more direct indicators of T4 and T3 activities in the body, but in animal studies, total T3 and T4 are typically measured. Historically, the most widely used method in toxicology is radioimmunoassay (RIA). The method is routinely used in rodent endocrine and toxicity 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 exist (Hornung et al., 2015; DeVito et al., 1999; Spencer, 2013).
Any of these measurements should be evaluated for the relationship to the actual endpoint of interest, repeatability, reproducibility, and lower limits of quantification using a fit-for-purpose approach (i.e., different regulatory needs will require different levels of confidence in the AOP). 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 an indirect methodology, whereas analytical determination is the most direct measurement available. All these methods, particularly RIA, are repeatable and reproducible.
Thyroxine-immunofluorescence quantitative disruption test (TIQDT) was designed to provide a simple, rapid, alternative bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function. Thyroid gland functionality was evaluated by
measuring IT4C in 4768 120 hpf zebrafish eleutheroembryos, following the TIQDT protocol (Thienpont et al., 2011).This study demonstrated that zebrafish eleutheroembryos provided a suitable vertebrate model, not only for screening the potential thyroid disrupting effect of molecules, but also for estimating the potential hazards associated with exposure to chemicals directly impairing thyroxine (T4) synthesis. Amitrole, potassium perchlorate, potassium thiocyanate, methimazole (MMI), phloroglucinol, 6-propyl-2-thiouracil, ethylenethiourea, benzophenone-2, resorcinol, pyrazole, sulfamethoxazole, sodium bromide, mancozeb, and genistein were classified as thyroid gland function disruptors. Concordance between TIQDT on zebrafish and mammalian published data was very high. TIQDT performed on zebrafish eleutheroembryos is an alternative whole-organism screening assay that provides relevant information for environmental and human risk assessments.
Domain of Applicability
The overall evidence supporting taxonomic applicability is strong. THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in zebra fish (Thienpont et al., 2011), amphibian and lamprey metamorphoses is well established (Manzon and Youson, 1997; Yaoita and Brown, 1990; Furlow and Neff, 2006). Their existence and importance has also been described in many different 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 depends on the expression and function of specific proteins (e.g receptors or enzymes) under TH control and may vary across species and tissues. As such extrapolation regarding TH action across species should be done with caution.
With few exceptions, vertebrate species have circulating T4 (and T3) that are bound to transport proteins in blood. Clear species differences exist in serum transport proteins (Dohler et al., 1979; Yamauchi and Isihara, 2009). There are three major transport proteins in mammals; thyroid binding globulin (TBG), transthyretin (TTR), and albumin. In adult humans, the percent bound to these proteins is about 75, 15 and 10 percent, respectively (Schussler 2000). In contrast, in adult rats the majority of THs are bound to TTR. Thyroid binding proteins are developmentally regulated in rats. TBG is expressed in rats until approximately postnatal day (PND) 60, with peak expression occurring during weaning (Savu et al., 1989). However, low levels of TBG persist into adult ages in rats and can be experimentally induced by hypothyroidism, malnutrition, or caloric restriction (Rouaze-Romet et al., 1992). While these species differences impact TH half-life (Capen, 1997) and possibly regulatory feedback mechanisms, there is little information on quantitative dose-response relationships of binding proteins and serum hormones during development across different species. Serum THs are still regarded as the most robust measurable key event causally linked to downstream adverse outcomes.
Evidence for Perturbation by Stressor
6-n-propylthouracil is a classic positive control for inhibition of TPO
Classic positive control
Axelrad DA, Baetcke K, Dockins C, Griffiths CW, Hill RN, Murphy PA, Owens N, Simon NB, Teuschler LK. Risk assessment for benefits analysis: framework for analysis of a thyroid-disrupting chemical. J Toxicol Environ Health A. 2005 68(11-12):837-55.
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.
Capen CC. Mechanistic data and risk assessment of selected toxic end points of the thyroid gland. Toxicol Pathol. 1997 25(1):39-48.
Cope RB, Kacew S, Dourson M. A reproductive, developmental and neurobehavioral study following oral exposure of tetrabromobisphenol A on Sprague-Dawley rats. Toxicology. 2015 329:49-59.
Corcoran JM, Eastman CJ, Carter JN, Lazarus L. (1977). Circulating thyroid hormone levels in children. Arch Dis Child. 52: 716-720.
Crofton KM. Developmental disruption of thyroid hormone: correlations with hearing dysfunction in rats. Risk Anal. 2004 Dec;24(6):1665-71.
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.
Döhler KD, Wong CC, von zur Mühlen A (1979). The rat as model for the study of drug effects on thyroid function: consideration of methodological problems. Pharmacol Ther B. 5:305-18.
Eales JG. (1997). Iodine metabolism and thyroid related functions in organisms lacking thyroid follicles: Are thyroid hormones also vitaminsProc Soc Exp Biol Med. 214:302-317.
Furlow JD, Neff ES. (2006). A developmental switch induced by thyroid hormone: Xenopus laevis metamorphosis. Trends Endocrinol Metab. 17:40–47.
Goldey ES, Crofton KM. Thyroxine replacement attenuates hypothyroxinemia, hearing loss, and motor deficits following developmental exposure to Aroclor 1254 in rats. Toxicol Sci. 1998 45(1):94-10
Goldey ES, Kehn LS, Lau C, Rehnberg GL, Crofton KM. Developmental exposure to polychlorinated biphenyls (Aroclor 1254) reduces circulating thyroid hormone concentrations and causes hearing deficits in rats. Tox Appl Pharmacol. 1995 135(1):77-88.
Harris AR, Fang SL, Prosky J, Braverman LE, Vagenakis AG. Decreased outer ring monodeiodination of thyroxine and reverse triiodothyronine in the fetal and neonatal rat. Endocrinology. 1978 Dec;103(6):2216-22
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.
Hill RN, Crisp TM, Hurley PM, Rosenthal SL, Singh DV. Risk assessment of thyroid follicular cell tumors. Environ Health Perspect. 1998 Aug;106(8):447-57.
Hornung MW, Kosian P, Haselman J, Korte J, Challis K, Macherla C, Nevalainen E, Degitz S (2015) In vitro, ex vivo and in vivo determination of thyroid hormone modulating activity of benzothiazoles. Toxicol Sci 146:254-264.
Hulbert AJ. Thyroid hormones and their effects: a new perspective. Biol Rev Camb Philos Soc. 2000 Nov;75(4):519-631. Review.
Kapelari K, Kirchlechner C, Högler W, Schweitzer K, Virgolini I, Moncayo R. 2008. Pediatric reference intervals for thyroid hormone levels from birth to adulthood: a retrospective study. BMC Endocr Disord. 8: 15.
Lau C, Thibodeaux JR, Hanson RG, Rogers JM, Grey BE, Stanton ME, Butenhoff JL, Stevenson LA. Exposure to perfluorooctane sulfonate during pregnancy in rat and mouse. II: postnatal evaluation. Toxicol Sci. 2003 Aug;74(2):382-92.
Leloup, J., and M. Buscaglia. La triiodothyronine: hormone de la métamorphose des amphibiens. CR Acad Sci 284 (1977): 2261-2263.
Liu J, Liu Y, Barter RA, Klaassen CD.: Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: a dose-response study. J Pharmacol Exp Ther 273, 977-85, 1994
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.
McClain RM. Mechanistic considerations for the relevance of animal data on thyroid neoplasia to human risk assessment. Mutat Res. 1995 Dec;333(1-2):131-42
Miller MD, Crofton KM, Rice DC, Zoeller RT. Thyroid-disrupting chemicals: interpreting upstream biomarkers of adverse outcomes. Environ Health Perspect. 2009 117(7):1033-41
Morse DC, Wehler EK, Wesseling W, Koeman JH, Brouwer A. Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to polychlorinated biphenyls (Aroclor 1254). Toxicol Appl Pharmacol. 1996 Feb;136(2):269-79.
NTP National Toxicology Program.: NTP toxicology and carcinogenesis studies of 3,3'-dimethylbenzidine dihydrochloride (CAS no. 612-82-8) in F344/N rats (drinking water studies). Natl Toxicol Program Tech Rep Ser 390, 1-238, 1991.
O'Connor, J. C., J. C. Cook, et al. (1998). "An ongoing validation of a Tier I screening battery for detecting endocrine-active compounds (EACs)." Toxicol Sci 46(1): 45-60.
O'Connor, J. C., L. G. Davis, et al. (2000). "Detection of dopaminergic modulators in a tier I screening battery for identifying endocrine-active compounds (EACs)." Reprod Toxicol 14(3): 193-205.
Rouaze-Romet M, Savu L, Vranckx R, Bleiberg-Daniel F, Le Moullac B, Gouache P, Nunez EA. 1992. Reexpression of thyroxine-binding globulin in postweaning rats during protein or energy malnutrition. Acta Endocrinol (Copenh).127:441-448.
Savu L, Vranckx R, Maya M, Gripois D, Blouquit MF, Nunez EA. 1989. Thyroxine-binding globulin and thyroxinebinding prealbumin in hypothyroid and hyperthyroid developing rats. BiochimBiophys Acta. 992:379-384.
Schneider S, Kaufmann W, Strauss V, van Ravenzwaay B. Vinclozolin: a feasibility and sensitivity study of the ILSI-HESI F1-extended one-generation rat reproduction protocol. Regul Toxicol Pharmacol. 2011 Feb;59(1):91-100.
Schussler, G.C. (2000). The thyroxine-binding proteins. Thyroid 10:141–149.
Spencer, CA. (2013). Assay of thyroid hormone and related substances. In De Groot, LJ et al. (Eds). Endotext. South Dartmouth, MA
Sternberg RM, Thoemke KR, Korte JJ, Moen SM, Olson JM, Korte L, Tietge JE, Degitz SJ Jr. Control of pituitary thyroid-stimulating hormone synthesis and secretion by thyroid hormones during Xenopus metamorphosis. Gen Comp Endocrinol. 2011. 173(3):428-37
Taurog A. 2005. Hormone synthesis. In: Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text (Braverman LE, Utiger RD, eds). Philadelphia:Lippincott, Williams and Wilkins, 47–81Walker P, Dubois JD, Dussault JH. Free thyroid hormone concentrations during postnatal development in the rat. Pediatr Res. 1980 Mar;14(3):247-9.
Thienpont B, Tingaud-Sequeira A, Prats E, Barata C, Babin PJ, Raldúa D. Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis. Environ Sci Technol. 2011 Sep 1;45(17):7525-32.
Yamauchi K1, Ishihara A. Evolutionary changes to transthyretin: developmentally regulated and tissue-specific gene expression. FEBS J. 2009. 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, R. T., R. Bansal, et al. (2005). "Bisphenol-A, an environmental contaminant that acts as a thyroid hormone receptor antagonist in vitro, increases serum thyroxine, and alters RC3/neurogranin expression in the developing rat brain." Endocrinology 146(2): 607-612.
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