A systematic approach to metabolic characterization of thyroid-disrupting chemicals and their in vitro biotransformants based on pre diction-assiste d metabolomic analysis

초록

Thyroid-disrupting compounds (TDCs) are chemicals that modify thyroid gland function and disrupt hor-monal homeostasis. Like other endocrine-disrupting chemicals (EDCs), TDCs often show altered activities following post-metabolic modification via endogenous enzymatic reaction. Hence, we developed evaluation system consisting of (1) in vitro metabolic reaction module, (2) high -resolution mass-spectrometry, and (3) human cell-based reporter gene assay. We developed the reac-tion module using rat S9 fraction where levothyroxine (T4) as a model compound, was subjected to phase-I or phase-I + II biotransformation. The metabolic profiles of the biotransformants were systemat-ically configured based on in-silico prediction of potential products and experimental validation using liquid-chromatography Orbitrap mass-spectrometry. Thyroid agonistic activities of the biotransformants were evaluated by thyroid receptor-mediated stably transfected transcriptional activation assay using hTRE_HeLa cells. Indeed, we detected the increased activities following metabolic conversion of T4 in a dose-dependent manner. Note that the activity by phase-I + II reaction was much greater than by phase-I reaction (3.8-fold increase). Subsequently, we explored metabolic signatures, which potentially contributed to the hyperac-tivity by phase-I + II reaction. A total of 77 metabolic features were annotated based on the in-silico pre-diction, which included biotransformants with deiodination and conjugation. The glucuronide-conjugated form was found at the highest fold-increase (970-fold increase) whereas marginal increases were deter-mined in the deiodinized forms (1.6-fold increase in T3 and 2.0-fold increase in rT3). Further, the sys-tematic approach was evaluated and comparably analyzed by the metabolic profiles of bithionol, which is structurally related to T4. Our current result suggested the potential application of in vitro evaluation system to risk assessment of thyroid-disrupting activity. (c) 2021 Elsevier B.V. All rights reserved.

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