- 1 Event Title
- 2 Key Event Overview
- 3 How this Key Event works
- 4 How it is Measured or Detected
- 5 Evidence Supporting Taxonomic Applicability
- 6 Evidence for Chemical Initiation of this Molecular Initiating Event
- 7 References
Key Event Overview
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AOPs Including This Key Event
The following are chemical initiators that operate directly through this Event:
- Ethylene thiourea
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Level of Biological Organization
How this Key Event works
Thyroperoxidase (TPO) is a heme-containing apical membrane protein within the follicular lumen of thyrocytes that acts as the enzymatic catalyst for thyroid hormone (TH) synthesis. TPO catalyzes several reactions in the thyroid gland, including the oxidation of iodide, nonspecific iodination of tyrosyl residues of thyroglobulin (Tg), and the coupling of iodotyrosyls to produce Tg-bound monoiodotyrosine (MIT) and diiodotyrosine (DIT) (Divi et al., 1997; Kessler et al., 2008; Ruf et al., 2006; Taurog et al., 1996). The outcome of TPO inhibition is decreased synthesis of thyroxine (T4) and triiodothyronine (T3), a decrease in release of these hormones from the gland into circulation, and unless compensated, a consequent decline in systemic concentrations of T4, and possibly T3. The primary product of TPO-catalyzed TH synthesis is T4 (Taurog et al., 1996; Zoeller et al., 2007) that would be peripherally or centrally deiodinated to T3.
The figure below illustrates the enzymatic and nonenzymatic reactions mediated by TPO.
Inhibition of TPO can be reversible, with transient interaction between the enzyme and the chemical, or irreversible, whereby suicide substrates permanently inactivate the enzyme. Reversible and irreversible TPO inhibition may be determined by the chemical structure, may be concentration dependent, or may be influenced by other conditions, including the availability of iodine (Doerge and Chang, 2002).
The ontogeny of TPO has been determined using both direct and indirect evidence, including development of TPO itself, as well as the ontogeny of Tg, and measurements of serum TH concentrations. Together this points to around the 11th to 12th fetal week as the beginning of functional TPO in humans. In rodents, TPO function begins late in the second fetal week with the first evidence of T4 secretion on gestational day 17 (Remy et al., 1980).Thyroid specific genes appear according to a specific temporal pattern – thyroglobulin (Tg), TPO (Tpo), and TSH receptor (Tshr) genes are expressed by gestational day 14, and the sodium iodide symporter, NIS (Nis) by gestational day 16. Maturation to adult function is thought to occur within a few weeks after parturition in rats and mice, and within the first few months in neonatal humans (Santisteban and Bernal, 2005). Tg is first detected in human starting at 5th week of gestation and rises throughout gestation (Thorpe-Beeston et al., 1992), but iodine trapping and T4 production does not occur until around 10-12 weeks. Also, the dimerization of Tg, characteristic of adults, is not found until much later in gestation (Pintar, 2000). In rats, Tg immunoreactivity does not appear until day 15 of gestation. The vast majority of research and knowledge on Tg is from mammals, although genomic orthologs are known for a varieity of other species (Holzer et al., 2016).
How it is Measured or Detected
There are no approved OECD or EPA guideline study protocols for measurement of TPO inhibition. From the early 1960's, microsomal fractions prepared from porcine thyroid glands and isolated porcine follicles were used as a source of TPO for inhibition experiments. Limited information has been published more recently using microsomes from human goiter samples (Vickers et al., 2012) and rat thyroid glands (Paul et al., 2013; 2014). TPO activity has been measured for decades via indirect assessment by kinetic measurement of the oxidation of guaiacol (Chang & Doerge 2000; Hornung et al., 2010; Schmutzler et al., 2007). This method is a low-throughput assay due to the very rapid kinetics of the guaiacol oxidation reaction. More recently, higher-throughput methods have been developed that use commercial fluorescent and luminescent substrates with rodent, porcine and human microsomal TPO and have been utilized (Vickers et al., 2012; Paul et al., 2013; 2014; Kaczur et al., 1997). This assay substitutes a pre-fluorescent substrate (AmplexRed) for guaiacol that when incubated with a source of peroxidase and excess hydrogen peroxidase results in a stable product for estimation of TPO inhibition., (Vickers et al., 2012). The stability of the product allows this assay to be used in a higher throughput format.
Direct measurement by HPLC of MIT, DIT, and iodothyronine hormone (T4 and T3) formation has been measured in a reaction mixture containing TPO, or a surrogate enzyme such as lactoperoxidase (Divi & Doerge 1994). The tools and reagents for this method are all available. However, HPLC or other analytical chemistry techniques make this a low-medium throughput assay depending on the level of automation. A primary advantage of this method is that it directly informs hypotheses regarding how a chemical may impact thyroid hormone synthesis in vitro.
Evidence Supporting Taxonomic Applicability
TPO inhibition is conserved across taxa. Data from experimental models and human clinical testing have demonstrated TPO inhibition across species. This is a function of high degree of similarity of protein sequences in the catalytic domain of mammalian peroxidases (Taurog, 1999). Ample data available for human, rat, and porcine TPO inhibition demonstrate qualitative concordance across these species. A comparison of rat TPO and pig TPO, bovine lactoperoxidase, and human TPO inhibition by genistein demonstrated good qualitative and quantitative (40–66%) inhibition across species, as indicated by quantification of MIT and DIT production (Doerge and Chang, 2002). Ealey et al. (1984) demonstrated peroxidase activity in guinea pig thyroid tissue using 3,3'-diaminobenzidine tetrahydrochloride (DAB) as a substrate that is oxidized by the peroxidase to form a brown reaction product insoluble reaction product. Formation of this reaction product was inhibited by 3-amino-1,2,4-triazole and the TPO inhibitor, methimazole (MMI). A comparative analysis of MMI between rat and human thyroid suggested concordance of qualitative response. Data also suggests an increased quantitative sensitivity to MMI in rat compared to human (Vickers et al., 2012). Paul et al. (2013) tested 12 chemicals using the guaiacol assay with both porcine and rat thyroid microsomes. The authors concluded that there was an excellent qualitative concordance between rat and porcine TPO inhibition, as all chemicals that inhibited TPO in porcine thyroid microsomes also inhibited TPO in rat thyroid microsomes.
Evidence for Chemical Initiation of this Molecular Initiating Event
There is a wealth of information on the inhibition of TPO by drugs such as MMI and PTU, as well as environmental xenobiotics. In the landmark paper on thyroid disruption by environmental chemicals, Brucker-Davis (1998) identified environmental chemicals that depressed TH synthesis by inhibiting TPO. Hurley (1998) listed TPO as a major target for thyroid tumor inducing pesticides. More recent work has tested over 1000 chemicals (Paul-Friedman et al., 2016).
Brucker-Davis F. 1998. Effects of environmental synthetic chemicals on thyroid function. Thyroid : official journal of the American Thyroid Association 8:827-856.
Chang, H. C. and D. R. Doerge (2000) Dietary genistein inactivates rat thyroid peroxidase in vivo without an apparent hypothyroid effect. Toxicology and applied pharmacology 168, 244-252.
Divi, R. L., & Doerge, D. R. (1994). Mechanism-based inactivation of lactoperoxidase and thyroid peroxidase by resorcinol derivatives. Biochemistry, 33(32), 9668–74.
Divi, R. L., Chang, H. C., & Doerge, D. R. (1997). Anti-Thyroid Isoflavones from Soybean. Biochemical Pharmacology, 54(10), 1087–1096. doi:10.1016/S0006-2952(97)00301-8.
Doerge DR, Chang HC. Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo. J Chromatogr B Analy Technol Biomed Life Sci. 2002 Sep 25;777(1-2):269-79.
Ealey PA, Henderson B, Loveridge N.A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland. Histochem J. 1984 Feb;16(2):111-22.
Fukiishi Y, Harauchi T, Yoshizaki T, Hasegawa Y, Eguchi Y. Ontogeny of thyroid peroxidase activity in perinatal rats. Acta Endocrinol (Copenh). 1982 Nov;101(3):397-402.
Holzer G, Morishita Y, Fini JB, Lorin T, Gillet B, Hughes S, Tohmé M, Deléage G, Demeneix B, Arvan P, Laudet V. Thyroglobulin Represents a Novel Molecular Architecture of Vertebrates. J Biol Chem. 2016 Jun 16.
Hornung, M. W., Degitz, S. J., Korte, L. M., Olson, J. M., Kosian, P. a, Linnum, A. L., & Tietge, J. E. (2010). Inhibition of thyroid hormone release from cultured amphibian thyroid glands by methimazole, 6-propylthiouracil, and perchlorate. Toxicological Sciences, 118(1), 42–51. doi:10.1093/toxsci/kfq166
Hurley PM. 1998. Mode of carcinogenic action of pesticides inducing thyroid follicular cell tumors in rodents. Environ Health Perspect 106:437-445.
Kaczur, V., Vereb, G., Molnár, I., Krajczár, G., Kiss, E., Farid, N. R., & Balázs, C. (1997). Effect of anti-thyroid peroxidase (TPO) antibodies on TPO activity measured by chemiluminescence assay. Clinical Chemistry, 43(8 Pt 1), 1392–6.
Kessler, J., Obinger, C., Eales, G., 2008. Factors influencing the study of peroxidase- generated iodine species and implications for thyroglobulin synthesis. Thyroid 18, 769–774.
Paul KB, Hedge JM, Macherla C, Filer DL, Burgess E, Simmons SO, Crofton KM, Hornung MW. Cross-species analysis of thyroperoxidase inhibition by xenobiotics demonstrates conservation of response between pig and rat.Toxicology. 2013 Oct 4;312:97-107
Paul, K.B., Hedge, J.M., Rotroff, D.M., Hornung, M.W., Crofton, K.M., Simmons, S.O. 2014. Development of a thyroperoxidase inhibition assay for high-throughput screening. Chemical Research in Toxicology. 27(3), 387-399.
Paul-Friedman K, Watt ED, Hornung MW, Hedge JM, Judson RS, Crofton KM, Houck KA, Simmons SO. 2016. Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors Within the ToxCast Phase I and II Chemical Libraries. Toxicol Sci. 151:160-80.
Pintar, J.E. (2000) Normal development of the hypothalamic-pituitary-thryoid axis. In. Werner & Ingbar’s The Thyroid. (8th ed), Braverman. L.E. and Utiger,R.D. (eds) Lippincott Williams and Wilkins, Philadelphia.
Remy L, Michel-Bechet M, Athouel-Haon AM, Magre S. Critical study of endogenous peroxidase activity: its role in the morphofunctional setting of the thyroid follicle in the rat fetus.Acta Histochem. 1980;67(2):159-72.
Ruf, J., & Carayon, P. (2006). Structural and functional aspects of thyroid peroxidase. Archives of Biochemistry and Biophysics, 445(2), 269–77. doi:10.1016/j.abb.2005.06.023
Santisteban P, Bernal J. Thyroid development and effect on the nervous system. Rev Endocr Metab Disord. 2005 Aug;6(3):217-28.
Schmutzler, C., Bacinski, A., Gotthardt, I., Huhne, K., Ambrugger, P., Klammer, H., Schlecht, C., Hoang-Vu, C., Gruters, A., Wuttke, W., Jarry, H., Kohrle, J., 2007a. The ultraviolet filter benzophenone 2 interferes with the thyroid hormone axis in rats and is a potent in vitro inhibitor of human recombinant thyroid peroxidase. Endocrinology 148, 2835–2844.
Taurog, a, Dorris, M. L., & Doerge, D. R. (1996). Mechanism of simultaneous iodination and coupling catalyzed by thyroid peroxidase. Archives of Biochemistry and Biophysics, Taurog A. Molecular evolution of thyroid peroxidase.Biochimie. 1999 May;81(5):557-62
Thorpe-Beeston JG, Nicolaides KH, McGregor AM. Fetal thyroid function. Thyroid. 1992 Fall;2(3):207-17. Review.
Vickers AE, Heale J, Sinclair JR, Morris S, Rowe JM, Fisher RL. Thyroid organotypic rat and human cultures used to investigate drug effects on thyroid function, hormone synthesis and release pathways. Toxicol Appl Pharmacol. 2012 Apr 1;260(1):81-8.
Zoeller, R. T., Tan, S. W., & Tyl, R. W. (2007). General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical Reviews in Toxicology, 37(1-2), 11–53.