Developing an in vitro assay for thyroid hormone sulphation in primary human hepatocytes

The Ministry of Infrastructure and Water Management – Directorate-General for the Environment and International Affairs aims to increase the availability of accepted test methods for characterizing endocrine-disrupting substances. An important focus in this regard is the development and refinement of animal-free methods that provide insight into potential disruption of the endocrine system indicating potential developmental neurotoxicity. In the present study, an in vitro method was explored that may be suitable for testing disturbances in thyroid hormone metabolism. Primary human hepatocytes were used to investigate the induction of thyroid hormone sulphation, with a specific focus on testing positive controls as a first step toward validation. For this purpose, an analytical method was first developed to quantify the thyroid hormones thyroxine (T4) and triiodothyronine (T3) and their metabolites, T4-sulphate, T4-glucuronide, and T3-sulphate. Subsequently, the usability of the hepatocytes was assessed by examining cell viability and the sulphation activity of T4 and T3 under non-stimulated conditions. Finally, a set of candidate compounds was included as positive controls to induce sulphation activity in the hepatocytes. Although the hepatocytes demonstrated sulphation activity for T4 and T3, the selected candidate compounds were not able to significantly increase this activity. In the future, further investigation of candidate compounds for positive controls is required, with particular attention to improving the experimental conditions through the use of Matrigel.