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.

• Keywords
• Amplex UltraRed, Cross-species, In vitro, Luminol, Peroxidation, Thyroid,
• MeSH
• Autoantigens metabolism   MeSH
• Cell Line MeSH
• Endocrine Disruptors toxicity   MeSH
• HEK293 Cells MeSH
• Iodide Peroxidase * antagonists & inhibitors   metabolism   genetics   MeSH
• Rats MeSH
• Humans MeSH
• Luminol MeSH
• Oxazines MeSH
• Iron-Binding Proteins metabolism   MeSH
• High-Throughput Screening Assays methods   MeSH
• Thyroid Gland drug effects   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Amplex Red MeSH Browser
• Autoantigens MeSH
• Endocrine Disruptors MeSH
• Iodide Peroxidase * MeSH
• Luminol MeSH
• Oxazines MeSH
• Iron-Binding Proteins MeSH
• TPO protein, human MeSH Browser

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.

• Keywords
• Bioanalytical equivalent, Concentration addition, Iceberg modelling, Mixture effects, Passive sampling, Risk prioritization,
• MeSH
• Androgen Antagonists MeSH
• Water Pollutants, Chemical * toxicity   analysis   MeSH
• Ecotoxicology MeSH
• Estrone MeSH
• Water Quality * MeSH
• Environmental Monitoring methods   MeSH
• Rivers chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Androgen Antagonists MeSH
• Water Pollutants, Chemical * MeSH
• Estrone MeSH