T. Nguyen-Jones, A. Salminen1, E. Rogers, J. Willoughby, D. Austin, K. Wolf, T. Stone, S. Presnell, J.Chen, J. McKim, E. LeCluyse

LifeNet Health LifeSciences, Virginia Beach, VA

Abstract

Purpose: Thyroid hormone dysregulation affects organ growth, metabolism, and neurodevelopmental processes. Disruptions in thyroxine (T4) synthesis, particularly during fetal and neonatal development, can lead to severe cognitive and skeletal abnormalities. With rising chemical exposure, regulatory agencies seek better models to identify endocrine-disrupting chemicals (EDCs) that affect thyroid function. Traditional in vitro models often fail to replicate the physiological conditions of the human thyroid. This study evaluates primary human thyrocytes in a 3D thyroid microtissue model as a new approach methodology (NAM) for assessing thyroid disruption.

Methods: Cryopreserved primary human thyrocytes were cultured on Matrigel-coated 96-well plates to form 3D thyroid microtissues. Cells were stimulated with bovine thyroid-stimulating hormone on day 2 of culture, and media were exchanged every other day thereafter. Microtissues were maintained in culture until day 9, at which point they were exposed to known thyroid peroxidase (TPO) inhibitors (methimazole, catechin, kaempferol, resorcinol, triclosan, and others), sodium/iodide symporter (NIS) inhibitors (potassium hexafluorophosphate), and thyroid-stimulating hormone receptor (TSHR) inhibitors (K1-70 recombinant antibody) across a range of concentrations. T4 levels were quantified via ELISA, while cell viability was assessed using CellTiter-Glo (ATP). The half-maximal inhibitory concentration (IC50) for each compound was calculated using GraphPad Prism software.

Results: Human thyroid microtissues formed uniform follicular structures and exhibited increased T4 production in response to bTSH, as compared to untreated controls. Eight TPO inhibitors reduced T4 synthesis dose-dependently, with IC50 values ranging from 0.08 μM (methimazole) to 42.08 μM (epigallocatechin gallate). The NIS inhibitor potassium hexafluorophosphate had an IC50 of 2.15 μM, inhibiting T4 synthesis by more than 85%. The TSHR inhibitor K1-70 recombinant antibody reduced T4 levels by 70% at the highest tested concentrations, though complete inhibition curves were not obtained. High K1-70 doses also decreased cell viability. Triclosan exhibited significant ATP reduction, highlighting the importance in distinguishing direct thyroid disruption from cytotoxicity.

Conclusions: The 3D thyroid microtissue model successfully mimics native follicular structure, sustains T4 synthesis, and responds to both small molecule and antibody-based inhibitors. This NAM provides a valuable tool for toxicology, environmental screening, and thyroid-targeted drug development, enhancing next-generation risk assessment for thyroid-disrupting chemicals.

Introduction

Chemicals that disrupt thyroid function can cause pronounced effects on thyroid hormone (TH) homeostasis resulting in significant adverse effects in humans, including neurodevelopmental impairment and preterm birth. In recent years, different government agencies across the world have increased efforts to develop programs to identify, screen, and assess the potential effects of chemicals found in the environment on thyroid hormone synthesis. These efforts include United States Environmental Protection Agency (US-EPA)’s Endocrine Disruptor Screening Program (EDSP) and the European Chemical Agency (ECHA)’s guidance on endocrine disruptors. Thyroid hormone production in vivo is controlled through the hypothalamus-pituitary-thyroid axis (HPT). Perturbations in systemic TH levels can occur when one or more pathways involved in synthesis or degradation are impacted. Currently, many of the high-throughput in-vitro models used for this purpose do not possess all phases of TH synthesis, and the use of animals for toxicity testing is time consuming and expensive. Here, we describe a fully human in vitro 3D microtissue system that retains the capacity to synthesize and secrete T4 through activation of the TSH receptor, with sufficient dynamic range and stability to enable detection of perturbations over a reasonable chemical exposure window. The model was subsequently challenged with thyroid disrupting compounds (TDCs) known to have adverse impacts on the production of T4.