Identification of hydrocarbon sulfonates as previously overlooked transthyretin ligands in the environment

21 December 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Incidences of thyroid disease, which has long been hypothesized to be partially caused by exposure to thyroid hormone disrupting chemicals (TDCs), have rapidly increased in recent years. However, only ~1% of the binding activity of human transthyretin (hTTR), an important thyroid hormone transporter protein, can be explained by known TDCs. In this study, we aimed to identify the major hTTR ligands in Canadian indoor dust and sewage sludge by employing protein-guided nontargeted analysis. hTTR binding activities were detected in all 11 indoor dust and 9 out of 10 sewage sludge samples (median 458 and 1134 μg T4/g in dust and sludge, respectively) by the FITC-T4 displacement assay. Through employing protein Affinity Purification with Nontargeted Analysis (APNA), 31 putative hTTR ligands were detected including perfluorooctane sulfonate (PFOS). Two of the most abundant ligands were identified as hydrocarbon surfactants (e.g., dodecyl benzenesulfonate), which were confirmed by authentic chemical standards. Structure-activity relationships (SAR) of hydrocarbon surfactants were explored by investigating the binding activity of 11 hydrocarbon surfactants to hTTR. Optimal carbon chain length (C12-14) was found to achieve a high binding affinity. By employing de novo nontargeted analysis, another abundant ligand was surprisingly identified as a di-sulfonate fluorescent brightener, 4,4'-Bis(2-sulfostyryl)biphenyl sodium (CBS). CBS was validated as a nM-affinity hTTR ligand with an IC50 of 345 nM. In total, hydrocarbon surfactants and fluorescent brightener could explain 1.92-17.0% and 5.74-54.3% of hTTR binding activities in dust and sludge samples, respectively, whereas PFOS only contributed <0.0001% to the activity. Our study revealed for the first time that hydrocarbon sulfonates are previously overlooked hTTR ligands in the environment.

Keywords

TTR
Hydrocarbon surfactants
CBS
Nontargeted analyses
Bioanalytical equivalent concentration

Supplementary materials

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Supporting Information
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The supporting information provides text, tables, and figures addressing: (1) Supplementary materials and methods; (2) Validation of the FITC-T4 probe; (3) Validation of the displacement assay; (4) hTTR binding activities of indoor dust and sewage sludge samples; (5) Correlation between hTTR and PPARγ LBD activities; (6) Verification of the recombinant His-tagged hTTR protein; (7) Benchmarking of the APNA method; (8) hTTR binding activities of hydrocarbon surfactants; (9) Relationship between carbon chain length and hTTR activity; (10) Molecular docking; (11) Isotopic distributions of the doubly charged ions; (12) List of standards; (13) IC50 and BEQbio values of environmental samples; (14) List of pulled-out LC-MS features; (15) IC50 and REP values of tested chemicals; (16) Environmental concentrations of CBS and hydrocarbon surfactants; (17) Contributions of identified chemicals to the total hTTR binding activities.
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