SAT-389 - A Human Multi‑Organ Platform for Mechanistic Thyroid Disruption Assessment

Thyroid hormones (TH) are essential regulators of energy balance by controlling cellular energy expenditure in each cell independent of systemic concentrations, which are controlled by the hypothalamic–pituitary–thyroid (HPT) axis. Environmental chemicals, including PCDD/Fs and PFAS, can disrupt TH homeostasis, and these disruptions in TH homeostasis are linked to neurodevelopmental and sensory deficits in humans. The established mechanism that PCDD/Fs activate the aryl hydrocarbon receptor (AHR) in hepatocytes to induce TH-glucuronidation and T4 clearance, causing TH imbalance, has been challenged by persistent TH disruption in glucuronidation-deficient rodents. This project aims to develop a human thyrocyte–hepatocyte co-culture model to study the mechanism(s) behind chemical-induced TH imbalance and support population-level risk assessment. To construct the model, hepatic metabolic endpoints were assessed in different human hepatocyte sources and formats. HepG2 cells using high-content confocal imaging, measuring fatty acid uptake (FAU), mitochondrial content, and mitochondrial membrane potential (MMP). TCDD reduced FAU and MMP dose-dependently (IC50: 0.9 and 0.09nM). Congeners 1,2,3,7,8-PCDF and 1,2,3,4,6,7,8-HCDF showed 10- and 100-fold lower IC50s. CYP1A1 induction mirrors these effects. PFOS increased MMP and FAU. However, HepG2 monolayers were unresponsive to TH. In 3D HepG2 spheroids, both TCDD and T4 elicited responses which TCDD reduced MMP (IC50 0.12nM), whereas PFOS effects were lost. The hepatocyte spheroid model demonstrates high predictive potential for low-nanomolar chemical disruptions. Having confirmed TH receptor responsiveness in hepatocytes, primary human thyrocytes were cultured, and an optimal seeding density supporting T4 secretion (~2.75 ng/10⁶ cells/2 days) was identified. The results demonstrate that human thyrocyte follicles are sensitive to TSH and respond to TPO and NIS inhibitors, as well as to drugs and environmental toxicants such as sodium perchlorate, propylthiouracil (PTU), and TCDD. These findings support the potential of a co-culture model as a platform to investigate mechanisms of chemical-induced TH imbalance.

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