Development of an in vitro test system for developmental neurotoxicity

Subject/Motivation/Background: Little is known about the effects on the human CNS development of most of the chemical substances we handle every day. Evidence is emerging that neurodegenerative diseases such as Alzheimer's disease might be correlated with exposure to toxicants in early life stages and early exposure e.g. to lead has affected the development of millions of children (Landrigan et al., 2005). Therefore we aim to establish a neurodevelopmental toxicity assay based on embryonic stem cells (ESC). Material and Methods: To differentiate mESCs into neurons a protocol published by Ying and Smith in Methods of Enzymology in 2003 was slightly modified. Successful differentiation was checked by RT-PCR and Immunofluorescence analysis. 5-Fluorouracil, CH sub(3)HgCl and PenicillinG were used as model substances to test the potential of our system to predict neurodevelopmental toxicity. Sublethal concentrations of these substances were applied to the culture for the last 6 days of differentiation. Effects were analyzed by RT-PCR and Western Blot. Results: Differentiation of Tubulin, III- beta mESC into neurons resulted in a percentage of 70% neurons (N-CAM), 15% nestin-positive precursor cells, and approximately 10% glial cells. The latter were located within rare and small islands within an even layer of tubulin positive cells to mature neurons was shown III- beta neurons. Maturation of by the presence of several markers such as, NeuN (postmitotic), SNAP25 (pre-synaptic), PSD95 (postsynaptic), SV2 (synaptic vesicles-2), NMDAR1 (postsynaptic), tyrosin hydroxylase (dopaminergic neurons), glutamic acid decarboxylase (GABAergic neurons). The staining pattern of the analyzed markers within ESC-derived neurons was similar to that of primary neurons (CGCs) isolated from mice. The whole differentiation process was monitored by RT-PCR and Western blot to determine the time window of the onset of neuronal differentiation. Toxic substances were added at that point in time to the culture and marker expression was analyzed. RT-PCR analysis showed a significant difference in the synaptophysin, N-CAM, nestin and MAP2 mRNA content of CH sub(3)HgCl assstreated cells compared to untreated cells. Glial markers like GFAP and S100 well as the endodermal marker GATA-4 and the mesodermal marker brachyury were not affected by the treatment with CH sub(3)HgCl. Treatment with the known strong teratogen 5-FU significantly reduced the amount of the endodermal marker GATA-4. Viabilit