Developmental neurotoxicity in a human model system

Abstract

There is an urgent scientific and regulatory need for development and implementation of new approach methods (NAM), such as cell culture test methods. NAM can give infor-mation about mechanisms of toxicity and about pathways disturbed after test compound exposure. Such knowledge plays an increasingly larger role in modern toxicology. Partic-ularly, in the field of developmental neurotoxicity (DNT), in vitro test methods can model different stages of neurodevelopment and thereby inform on stage-specific toxicities. However, there is not yet a suitable test system available for every stage of neurodevel-opment. This thesis addresses this gap in two different ways: first, by investigating mech-anisms of neurotoxicity at various stages of neurodevelopment, and second, by establish-ing new test methods for stages not covered so far. In the first part of this thesis, human pluripotent stem cells (hPSC) were differentiated into neuroepithelial precursors (NEP) to investigate transcriptome changes triggered by the well-known teratogen valproic acid and other histone deacetylase inhibitors (HDACi) during very early neurodevelopment. Short exposures (up to 24 h) altered histone acetylation and the transcriptome only tran-siently. Transcriptome changes after drug exposure both reflected the mode of action (MoA) of a compound and described an altered differentiation state. Altered differentia-tion was observed after longer HDACi exposures (at least three days) accompanied by altered histone methylation. This study showed that epigenetic modifications can have neuroteratogenic effects by changing the chromatin state. In the second part of this work, it was examined in how far neurotoxicology of complex toxins could be evaluated with human neuronal cultures. To explore the neurotoxicity hazard of microcystins (MC) in humans, two mature neuronal cell types (LUHMES and peripheral neurons) were exposed to MC-LF. Clear neurotoxic effects were identified at low μM concentrations. In this study, the hazard data obtained in vitro were combined with literature exposure data for a gen-eral risk assessment. In the final part of this thesis, a novel test system was developed to close an obvious gap in the available neurotoxicity test battery: identification of toxicants interfering with ionotropic glutamate receptors. An hPSC-based differentiation protocol was established yielding a mixed cortical culture (MCC) with about 50% N-methyl-D-as-partate receptor (NMDA-R) expressing neurons. Toxicants known to interfere with gluta-mate receptors, like ibotenic acid and domoic acid, triggered changes in intracellular free calcium ion concentration which could be inhibited by antagonists like ketamine and phencyclidine. Hence, MCC contribute to the currently available test systems for investi-gation of compounds affecting important neuronal receptors. The studies presented in this thesis led to the development of novel in vitro test systems that allow the study of mode of action (MoA) of toxicants during different stages of neurodevelopment and the screening of unknown compounds of potential neurotoxicity.