Disruption of the thyroid hormone (TH) system is connected with diverse adverse health outcomes in wildlife and humans. It is crucial to develop and validate suitable in vitro assays capable of measuring the disruption of the thyroid hormone (TH) system. These assays are also essential to comply with the 3R principles, aiming to replace the ex vivo tests often utilised in the chemical assessment. We compared the two commonly used assays applicable for high throughput screening [Luminol and Amplex UltraRed (AUR)] for the assessment of inhibition of thyroid peroxidase (TPO, a crucial enzyme in TH synthesis) using several cell lines and 21 compounds from different use categories. As the investigated cell lines derived from human and rat thyroid showed low or undetectable TPO expression, we developed a series of novel cell lines overexpressing human TPO protein. The HEK-TPOA7 model was prioritised for further research based on the high and stable TPO gene and protein expression. Notably, the Luminol assay detected significant peroxidase activity and signal inhibition even in Nthy-ori 3-1 and HEK293T cell lines without TPO expression, revealing its lack of specificity. Conversely, the AUR assay was specific to TPO activity. Nevertheless, despite the different specificity, both assays identified similar peroxidation inhibitors. Over half of the tested chemicals with diverse structures and from different use groups caused TPO inhibition, including some widespread environmental contaminants suggesting a potential impact of environmental chemicals on TH synthesis. Furthermore, in silico SeqAPASS analysis confirmed the high similarity of human TPO across mammals and other vertebrate classes, suggesting the applicability of HEK-TPOA7 model findings to other vertebrates.

• Klíčová slova
• Amplex UltraRed, Cross-species, In vitro, Luminol, Peroxidation, Thyroid,
• MeSH
• autoantigeny metabolismus   MeSH
• buněčné linie MeSH
• endokrinní disruptory toxicita   MeSH
• HEK293 buňky MeSH
• jodidperoxidasa * antagonisté a inhibitory   metabolismus   genetika   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• luminol MeSH
• oxaziny MeSH
• proteiny vázající železo metabolismus   MeSH
• rychlé screeningové testy metody   MeSH
• štítná žláza účinky léků   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• Amplex Red MeSH Prohlížeč
• autoantigeny MeSH
• endokrinní disruptory MeSH
• jodidperoxidasa * MeSH
• luminol MeSH
• oxaziny MeSH
• proteiny vázající železo MeSH
• TPO protein, human MeSH Prohlížeč

Monitoring methodologies reflecting the long-term quality and contamination of surface waters are needed to obtain a representative picture of pollution and identify risk drivers. This study sets a baseline for characterizing chemical pollution in the Danube River using an innovative approach, combining continuous three-months use of passive sampling technology with comprehensive chemical (747 chemicals) and bioanalytical (seven in vitro bioassays) assessment during the Joint Danube Survey (JDS4). This is one of the world's largest investigative surface-water monitoring efforts in the longest river in the European Union, which water after riverbank filtration is broadly used for drinking water production. Two types of passive samplers, silicone rubber (SR) sheets for hydrophobic compounds and AttractSPETM HLB disks for hydrophilic compounds, were deployed at nine sites for approximately 100 days. The Danube River pollution was dominated by industrial compounds in SR samplers and by industrial compounds together with pharmaceuticals and personal care products in HLB samplers. Comparison of the Estimated Environmental Concentrations with Predicted No-Effect Concentrations revealed that at the studied sites, at least one (SR) and 4-7 (HLB) compound(s) exceeded the risk quotient of 1. We also detected AhR-mediated activity, oxidative stress response, peroxisome proliferator-activated receptor gamma-mediated activity, estrogenic, androgenic, and anti-androgenic activities using in vitro bioassays. A significant portion of the AhR-mediated and estrogenic activities could be explained by detected analytes at several sites, while for the other bioassays and other sites, much of the activity remained unexplained. The effect-based trigger values for estrogenic and anti-androgenic activities were exceeded at some sites. The identified drivers of mixture in vitro effects deserve further attention in ecotoxicological and environmental pollution research. This novel approach using long-term passive sampling provides a representative benchmark of pollution and effect potentials of chemical mixtures for future water quality monitoring of the Danube River and other large water bodies.

• Klíčová slova
• Bioanalytical equivalent, Concentration addition, Iceberg modelling, Mixture effects, Passive sampling, Risk prioritization,
• MeSH
• antagonisté androgenů MeSH
• chemické látky znečišťující vodu * toxicita   analýza   MeSH
• ekotoxikologie MeSH
• estron MeSH
• kvalita vody * MeSH
• monitorování životního prostředí metody   MeSH
• řeky chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antagonisté androgenů MeSH
• chemické látky znečišťující vodu * MeSH
• estron MeSH