Evaluation of new alternative strategies to predict neurotoxicity with human based test systems

Abstract

Animal experiments are still the ‘gold standard’ in safety evaluation defined by the OECD (Organisation for Economic Co-operation and Development) or the US EPA (Environmental Protection Agency). Millions of animals are used each year to assess the risk of chemical toxicities for human health. But animal experiments are expensive, time-consuming and have a restricted prediction capacity regarding human toxicity. Hence the demand for validated alternative strategies is high. Validated differentiation protocols of embryonic stem cells or immortalized human organ specific cell lines provide the possibility to recapitulate human development and to study organ specific toxicity of different developmental stages (immature to mature) in vitro. In the framework of this doctoral thesis, we provide insights into the development and evaluation of test systems established specifically to assess neurodevelopmental toxicity as well as neurotoxicity in vitro.In a first step we evaluated an assay based on neurite outgrowth assessment to detect putative developmentally neurotoxic chemicals. This assay was based on a human mesencephalic neuronal precursor cell line, called LUHMES. In the study, the model has been challenged for its reliability and consistency using more than 50 compounds and combinations of them. We proved the applicability of the assay for screening, and suggest that the test has the potential to be used for identification and potency-ranking of putative developmental toxicants with regard to effects on neurite growth.In a second step we used different human stem cell-based test systems to mimic several stages of the early human neurodevelopment in vitro. We analysed the transcriptome changes of these test systems after exposure to two developmental toxicants, valproic acid and methylmercury. Both toxicants induced test system and compound specific transcriptome changes. A common toxicant specific signature of transcription factor binding sites was identified for the different test systems, which we suggest as classifier for compound grouping in future experiments.In a last step we used a well described model compound 1-methyl-4-phenylpyridinium (MPP+) to analyse the suitability of Omics combinations to monitor the MPP+ induced changes on LUHMES. We found early large adaptive metabolome and transcriptome changes which taken together lead to the identification of novel pathways involved in early MPP+ toxicity. The findings of this thesis contribute to alternative test-strategy development in neurotoxicity and disclose important considerations when developing in vitro test systems.