Pull-down assay coupled to non-target mass spectrometry analysis as a tool to identify unknown endocrine disruptive transthyretin ligands in waste and surface water

Surface and treated wastewaters are contaminated with highly complex mixtures of micropollutants, which may cause adverse effects on aquatic biota or humans, often mediated by endocrine disruption. However, there is very limited knowledge regarding some important modes of action, such as interference with thyroid hormone (TH) regulation and the compounds driving these effects. The effects of environmental samples observed in bioassays addressing various endpoints in the endocrine, namely thyroid hormone pathways, remain largely unexplained with known active chemicals. Transthyretin (TTR) is a serum transport protein distributing thyroid hormones to target tissues in vertebrates; its binding inhibition by xenobiotics may lead to adverse effects such as impaired (neuro)development. In this study, we describe a novel approach for the identification of compounds with the potential to bind to TTR, based on the specific separation of these compounds in a pull-down assay followed by non-target analysis (NTA). The pull-down assay using purified TTR protein was established and optimized with known TTR ligands. The method was applied to separate and identify compounds responsible for TH displacing activity in highly complex wastewater and surface water samples. The samples after the pull-down assay elicited TH displacing activity and the specific separation of TTR ligands provided a substantial reduction of chromatographic features from the original complex water extract. The applied non-target screening workflow resulted in the identification of 34 structures. Thirteen identified compounds with available analytical standards were quantified in the original water extracts and their TH-displacement potency was confirmed. Twelve compounds were discovered as TTR binders for the first time and linear alkylbenzene sulfonates (LAS) were highlighted as contaminants of concern regarding the TH-displacement activity. Pull-down assay combined with NTA proved to be a well-functioning approach for the identification of bioactive compounds in complex environmental mixtures with great application potential across various biological target endpoints and environmental compartments.