Surface and treated wastewater are contaminated with highly complex mixtures of micropollutants, which may cause numerous adverse effects, often mediated by endocrine disruption. However, there is limited knowledge regarding some important modes of action, such as interference with thyroid hormone (TH) regulation, and the compounds driving these effects. This study describes an effective approach for the identification of compounds with the potential to bind to transthyretin (TTR; protein distributing TH to target tissues), based on their specific separation in a pull-down assay followed by non-target analysis (NTA). The method was optimized with known TTR ligands and applied to complex water samples. The specific separation of TTR ligands provided a substantial reduction of chromatographic features from the original samples. The applied NTA workflow resulted in the identification of 34 structures. Twelve compounds with available standards were quantified in the original extracts and their TH-displacement potency was confirmed. Eleven compounds were discovered as TTR binders for the first time and linear alkylbenzene sulfonates (LAS) were highlighted as contaminants of concern. Pull-down assay combined with NTA proved to be a well-functioning approach for the identification of unknown bioactive compounds in complex mixtures with great application potential across various biological targets and environmental compartments.

Cluster of Excellence EXC 2124 Controlling Microbes to Fight Infections University of Tübingen Tübingen Germany

Interfaculty Institute of Microbiology and Infection Medicine Tübingen Institute for Bioinformatics and Medical Informatics University of Tübingen Tübingen Germany

International Clinical Research Center St Anne's University Hospital Brno 601 77 Czech Republic

Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University Brno 601 77 Czech Republic

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

University of South Bohemia in České Budějovice Faculty of Fisheries and Protection of Waters Research Institute of Fish Culture and Hydrocenoses Zátiší 728 2 389 25 Vodňany Czech Republic

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