Disruption of the thyroid hormone (TH) system by environmental chemicals poses significant risks to human and wildlife health. Reliable in vitro assays are essential for assessing thyroid peroxidase (TPO) inhibition, which disrupts a key step in thyroid hormone (TH) synthesis, while adhering to 3R principles. This study aimed to compare the sensitivity and specificity of two types of in vitro assays, tyrosine iodination (Tyr-I) detected by HPLC-ICP-MS and Amplex UltraRed (AUR) and its modifications, using human HEK-TPOA7 cells and rat thyroid microsomes. It involved the evaluation of TPO inhibition by 21 chemicals (concentration range 0.002-200 µM) from diverse use categories, including industrial pollutants, pesticides, and pharmaceuticals. TPO-inhibition potential was indicated for 14 compounds. The Tyr-I assay, which measures the conversion of L-tyrosine to monoiodotyrosine, demonstrated superior sensitivity by capturing both the peroxidation and iodination steps of TPO activity, with effective concentrations for some human exposure-relevant chemicals (benzophenone 2, resorcinol) in the 15-74 nM range confirmed with both human and rat TPO sources. In contrast, the AUR assay detects only the peroxidation step, limiting its ability to fully assess TPO inhibition. The inclusion of sodium iodide (NaI) in the AUR assay significantly enhanced its sensitivity (though it was still lower than in Tyr-I assay), while adding L-tyrosine together with NaI did not. The study documents the advantages and limitations, as well as the application and interpretation potential of the different assays´ variants. It provides valuable information and scientifically sound methodology to support the development of efficient testing strategies for the assessment of thyroid hormone system-disrupting chemicals.

Faculty of Science RECETOX Masaryk University Kamenice 753 5 pavilion A29 625 00 Brno Czech Republic

Zobrazit více v PubMed

Abas L, Luschnig C (2010) Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation. Anal Biochem 401(2):217–227. https://doi.org/10.1016/j.ab.2010.02.030 PubMed DOI PMC

Bernasconi C, Bartnicka J, Asturiol D, Bowe G, Langezaal I, Coecke S, Kienzler A, Liska R, Milcamps A, Munoz Pineiro A, Pistollato F, Whelan M, & European Commission. Joint Research Centre (2023) Validation of a battery of mechanistic methods relevant for the detection of chemicals that can disrupt the thyroid hormone system. https://doi.org/10.2760/862948

Cambrai-Erb A, Denis F, Pons R, Radauceanu A, Ndaw S, Grova N (2025) Development of a novel HPLC-HRMS method for quantitative analysis of resorcinol in urine: application to hairdressers’ occupational exposure. J Chromatogr B Analyt Technol Biomed Life Sci 1253:124472. https://doi.org/10.1016/j.jchromb.2025.124472 PubMed DOI

Doerge DR, Takazawa RS (1990) Mechanism of thyroid peroxidase inhibition by ethylenethiourea. Chem Res Toxicol 3(2):98–101. https://doi.org/10.1021/tx00014a003 PubMed DOI

Dong H, Friedman KP, Filiatreault A, Thomson EM, Wade MG (2024) A high throughput screening assay for human Thyroperoxidase inhibitors. Toxicol in Vitro 101:105946. https://doi.org/10.1016/j.tiv.2024.105946 PubMed DOI

Dong H, Godlewska M, Wade MG (2020) A rapid assay of human thyroid peroxidase activity. Toxicol in Vitro 62:104662. https://doi.org/10.1016/j.tiv.2019.104662 PubMed DOI

Freyberger A, Ahr H-J (2006) Studies on the goitrogenic mechanism of action of N,N, N′, N′-tetramethylthiourea. Toxicology 217(2):169–175. https://doi.org/10.1016/j.tox.2005.09.005 PubMed DOI

Friedman KP, Watt ED, Hornung MW, Hedge JM, Judson RS, Crofton KM, Houck KA, Simmons SO, 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(1):160–180. https://doi.org/10.1093/toxsci/kfw034 DOI

Giulivo M, de Lopez Alda M, Capri E, Barceló D (2016) Human exposure to endocrine disrupting compounds: their role in reproductive systems, metabolic syndrome, and breast cancer. a review. Environ Res 151:251–264. https://doi.org/10.1016/j.envres.2016.07.011 PubMed DOI

Godlewska M, Krasuska W, Czarnocka B (2018) Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines. PLoS ONE 13(3):e0193624. https://doi.org/10.1371/JOURNAL.PONE.0193624 PubMed DOI PMC

Haigis A-C, Vergauwen L, LaLone CA, Villeneuve DL, O’Brien JM, Knapen D (2023) Cross-species applicability of an adverse outcome pathway network for thyroid hormone system disruption. Toxicol Sci off J Soc Toxicol 195(1):1–27. https://doi.org/10.1093/toxsci/kfad063 DOI

Holbech H, Matthiessen P, Hansen M, Schüürmann G, Knapen D, Reuver M, Flamant F, Sachs L, Kloas W, Hilscherova K, Leonard M, Arning J, Strauss V, Iguchi T, Baumann L (2020) ERGO: breaking down the wall between human health and environmental testing of endocrine disrupters. Int J Mol Sci 21(8):2954. https://doi.org/10.3390/ijms21082954 PubMed DOI PMC

Hornung MW, Kosian PA, Haselman JT, Korte JJ, Challis K, Macherla C, Nevalainen E, Degitz SJ (2015) In vitro, ex vivo, and in vivo determination of thyroid hormone modulating activity of benzothiazoles. Toxicol Sci 146(2):254–264. https://doi.org/10.1093/toxsci/kfv090 PubMed DOI

Huwiler M, Bürgi U, Kohler H (1985) Mechanism of enzymatic and non-enzymatic tyrosine iodination inhibition by excess hydrogen peroxide and/or iodide. Eur J Biochem 147(3):469–476. https://doi.org/10.1111/j.0014-2956.1985.00469.x PubMed DOI

Jomaa B, De Haan LHJ, Peijnenburg AACM, Bovee TFH, Aarts JMMJG, Rietjens IMCM (2015) Simple and rapid in vitro assay for detecting human thyroid peroxidase disruption. Altex 32(3):191–200. https://doi.org/10.14573/altex.1412201 PubMed DOI

Kakked G, Bhatt N, Lakhani J, Prakash S (2013) Electromyographic evaluation of blink reflex as a tool for early diagnosis of neurological dysfunction in patients of hypothyroidism. Ann Neurosci 20(3):95. https://doi.org/10.5214/ANS.0972.7531.200304 PubMed DOI PMC

Keane KA, Parker GA, Regan KS, Picut C, Dixon D, Creasy D, Giri D, Hukkanen RR (2015) Scientific and regulatory policy committee (SRPC) points to consider. Toxicol Pathol 43(8):1047–1063. https://doi.org/10.1177/0192623315579943 PubMed DOI PMC

Knapen D, Stinckens E, Cavallin JE, Ankley GT, Holbech H, Villeneuve DL, Vergauwen L (2020) Toward an AOP network-based tiered testing strategy for the assessment of thyroid hormone disruption. Environ Sci Technol 54(14):8491–8499. https://doi.org/10.1021/acs.est.9b07205 PubMed DOI PMC

Leusch FDL, Aneck-Hahn NH, Cavanagh JAE, Du Pasquier D, Hamers T, Hebert A, Neale PA, Scheurer M, Simmons SO, Schriks M (2018) Comparison of in vitro and in vivo bioassays to measure thyroid hormone disrupting activity in water extracts. Chemosphere 191:868–875. https://doi.org/10.1016/j.chemosphere.2017.10.109 PubMed DOI

Li Z-T, Zhai R, Liu H-M, Wang M, Pan D-M (2020) Iodine concentration and content measured by dual-source computed tomography are correlated to thyroid hormone levels in euthyroid patients: A cross-sectional study in China. BMC Med Imaging 20:10. https://doi.org/10.1186/s12880-020-0411-8 PubMed DOI PMC

Liu R, Novák J, Hilscherová K (2024) In vitro assessment of thyroid peroxidase inhibition by chemical exposure: comparison of cell models and detection methods. Arch Toxicol 98(8):2631–2645. https://doi.org/10.1007/s00204-024-03766-7 PubMed DOI PMC

Mao JF, Li W, Ong CN, He Y, Jong M-C, Gin KY-H (2022) Assessment of human exposure to benzophenone-type UV filters: a review. Environ Int 167:107405. https://doi.org/10.1016/j.envint.2022.107405 PubMed DOI

Marinovich M, Guizzetti M, Ghilardi F, Viviani B, Corsini E, Galli CL (1997) Thyroid peroxidase as toxicity target for dithiocarbamates. Arch Toxicol 71(8):508–512. https://doi.org/10.1007/s002040050420 PubMed DOI

Murthy M, Murthy B (2012) Thyroid disruptors and their possible clinical implications. Ind J Pharmacol 44(4):542. https://doi.org/10.4103/0253-7613.99351 DOI

Novák J, Vrana B, Rusina T, Okonski K, Grabic R, Neale PA, Escher BI, Macová M, Ait-Aissa S, Creusot N, Allan I, Hilscherová K (2018) Effect-based monitoring of the Danube River using mobile passive sampling. Sci Total Environ 636:1608–1619. https://doi.org/10.1016/j.scitotenv.2018.02.201 PubMed DOI

Nováková Z, Novák J, Bittner M, Čupr P, Přibylová P, Kukučka P, Smutná M, Escher BI, Demirtepe H, Miralles-Marco A, Hilscherová K (2022) Toxicity to bronchial cells and endocrine disruptive potentials of indoor air and dust extracts and their association with multiple chemical classes. J Hazard Mater. https://doi.org/10.1016/j.jhazmat.2021.127306 PubMed DOI

Nováková Z, Novák J, Kitanovski Z, Kukučka P, Smutná M, Wietzoreck M, Lammel G, Hilscherová K (2020) Toxic potentials of particulate and gaseous air pollutant mixtures and the role of PAHs and their derivatives. Environ Int 139:105634. https://doi.org/10.1016/j.envint.2020.105634 PubMed DOI

Noyes PD, Friedman KP, Browne P, Haselman JT, Gilbert ME, Hornung MW, Barone S, Crofton KM, Laws SC, Stoker TE, Simmons SO, Tietge JE, Degitz SJ (2019) Evaluating chemicals for thyroid disruption: opportunities and challenges with in vitro testing and adverse outcome pathway approaches. Environ Health Perspect 127(9):095001. https://doi.org/10.1289/EHP5297 PubMed DOI PMC

Nugegoda D, Kibria G (2017) Effects of environmental chemicals on fish thyroid function: implications for fisheries and aquaculture in Australia. Gen Comp Endocrinol 244:40–53. https://doi.org/10.1016/J.YGCEN.2016.02.021 PubMed DOI

OECD (2014) New scoping document on in vitro and ex vivo assays for the identification of modulators of thyroid hormone signalling. https://doi.org/10.1787/9789264274716-EN

Paul KB, Hedge JM, Rotroff DM, Hornung MW, Crofton KM, Simmons SO (2014) Development of a thyroperoxidase inhibition assay for high- throughput screening. Chem Res Toxicol 27(3):387–399. https://doi.org/10.1021/tx400310w PubMed DOI

Pinto-Vidal FA, Novák J, Jílková SR, Rusina T, Vrana B, Melymuk L, Hilscherová K (2024) Endocrine disrupting potential of total and bioaccessible extracts of dust from seven different types of indoor environment. J Hazard Mater. https://doi.org/10.1016/j.jhazmat.2024.133778 PubMed DOI

Porras SP, Hartonen M, Ylinen K, Tornaeus J, Tuomi T, Santonen T (2018) Environmental and occupational exposure to resorcinol in Finland. Toxicol Lett 298:125–133. https://doi.org/10.1016/j.toxlet.2018.03.027 PubMed DOI

Reinen J, Rijk J, de Laat E, Toersche J, Freyberger A, Wenker M (2024) Development and standardization of an assay to evaluate the in vitro inhibition of thyroid peroxidase -catalyzed iodination using FTC-238-hrTPO cell homogenates. Appl in Vitro Toxicol 10(3):51–64. https://doi.org/10.1089/aivt.2024.0029 DOI

Romarís-Hortas V, Bermejo-Barrera P, Moreda-Piñeiro A (2013) Ultrasound-assisted enzymatic hydrolysis for iodinated amino acid extraction from edible seaweed before reversed-phase high performance liquid chromatography–inductively coupled plasma-mass spectrometry. J Chromatogr A 1309:33–40. https://doi.org/10.1016/j.chroma.2013.08.022 PubMed DOI

Ruf J, Carayon P (2006) Structural and functional aspects of thyroid peroxidase. Arch Biochem Biophys 445(2):269–277. https://doi.org/10.1016/j.abb.2005.06.023 PubMed DOI

Sauer P, Vrana B, Escher BI, Grabic R, Tou Z, Miku P, Krauss M, Ohe P CVD, Maria K, Brack W, & Proke R. (2023) Bioanalytical and chemical characterization of organic micropollutant mixtures in long-term exposed passive samplers from the Joint Danube Survey 4: setting a baseline for water quality monitoring. 178(April). https://doi.org/10.1016/j.envint.2023.107957

Schmutzler C, Bacinski A, Gotthardt I, Huhne K, Ambrugger P, Klammer H, Schlecht C, Hoang-Vu C, Grüters A, Wuttke W, Jarry H, Köhrle J (2007) 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(6):2835–2844. https://doi.org/10.1210/en.2006-1280 PubMed DOI

Stoica G, Lungu G, Xie X, Abbott LC, Stoica HM, Jaques JT (2007) Inherited tertiary hypothyroidism in Sprague-Dawley rats. Brain Res 1148:205–216. https://doi.org/10.1016/j.brainres.2007.02.042 PubMed DOI

Street M, Angelini S, Bernasconi S, Burgio E, Cassio A, Catellani C, Cirillo F, Deodati A, Fabbrizi E, Fanos V, Gargano G, Grossi E, Iughetti L, Lazzeroni P, Mantovani A, Migliore L, Palanza P, Panzica G, Papini A, Amarri S (2018) current knowledge on endocrine disrupting chemicals (EDCs) from animal biology to humans, from pregnancy to adulthood: highlights from a national italian meeting. Int J Mol Sci 19(6):1647. https://doi.org/10.3390/ijms19061647 PubMed DOI PMC

Tater A, Gupta A, Upadhyay G, Deshpande A, Date R, Tamboli IY (2021) In vitro assays for characterization of distinct multiple catalytic activities of thyroid peroxidase using LC-MS/MS. Curr Res Toxicol 2:19–29. https://doi.org/10.1016/j.crtox.2021.01.001 PubMed DOI PMC

US EPA. (2024). ToxCast CompTox chemicals dashboard Version 2.4.1. https://comptox.epa.gov/dashboard/

Vergauwen L, Bajard L, Tait S, Langezaal I, Sosnowska A, Roncaglioni A, Hessel E, Van Den Brand AD, Haigis A-C, Novák J, Hilscherová K, Buławska N, Papaioannou N, Renieri E, Spilioti E, Spyropoulou A, Gutleb AC, Holbech H, Nikolopoulou D, Knapen D (2024) A 2024 inventory of test methods relevant to thyroid hormone system disruption for human health and environmental regulatory hazard assessment. Open Res Europe 4:242. https://doi.org/10.12688/openreseurope.18739.1 DOI

Wilson RA, Yanes EG, Kemppainen RJ (2016) Iodine speciation in dog foods and treats by high performance liquid chromatography with inductively coupled plasma mass spectrometry detection. J Chromatogr B 1022:183–190. https://doi.org/10.1016/j.jchromb.2016.04.002 DOI