Endocrine Abstracts

Background: Over the last few decades, in vivo animal experiments have been the gold standard for the assessment of safety of chemicals and pharmaceuticals for human health. However, in vivo animal data has limited relevance for toxicity prediction in humans, especially considering species-specific issues of age, sex and exposure in different life-stages. Within the Virtual Human Platform for Safety Assessment (VHP4Safety) project we aim to develop an animal-free testing strategy to assess the safety of compounds for developmental neurotoxicity mediated by disruption of thyroid hormone signaling.

Method: We developed an Adverse Outcome Pathway (AOP) network on brain development based on available data of existing thyroid related AOPs and human physiology. By combining these data, critical molecular initiating events and key events have been identified and used as a basis for selecting parameters for an in vitro testing battery. The first part of the testing battery consists of human cell models representing neurons (SHSY-5Y and SK-N-AS), oligodendrocytes (MO3.13) and astrocytes (H4). Thyroid hormone uptake and metabolism by the cell lines was tested using radiolabeled thyroid hormone.

Results: The first critical events shown in the AOP are related to thyroid hormone availability in the brain via transport and metabolism. Time-dependent uptake of T3 and T4 was observed in all cell models, which was partially reduced by the MCT8 inhibitor silychristin. In vitro metabolism assays showed conversion of T4 into T3 by H4 cells, and conversion of T3 into T2 by MO3.13, SHSY-5Y and SK-N-AS cells. SHSY-5Y showed higher rates of T3 conversion than SK-N-AS cells.

Conclusion: The selected cell lines are suitable models to test the effect of compounds on uptake of thyroid hormones. Furthermore, deiodinase activity was as expected for the different cell types, with H4 cells showing D2 activity, and MO3.13, SHSY-5Y and SK-N-AS D3 activity. Future experiments will include testing (reference) compounds on uptake and in vivo metabolism to further validate the models. Furthermore, the cell models will be used to study whether developmental neurotoxic compounds disrupt the thyroid hormone balance in the developing brain. The VHP4Safety project NWA 1292.19.272 is part of the NWA research program ’Research along Routes by Consortia (ORC)’ and is funded by the Netherlands Organization for Scientific Research (NWO).