We are surrounded by chemicals, thus understanding how exposure to these chemicals affect us during our life is of great social importance. In order to predict human acute toxicity of chemicals, cosmetics or drugs, development of novel in vitro test strategies is required. The overall aim of this thesis was to evaluate whether two different cell line models could be used to predict acute neurotoxicity or developmental neurotoxicity. In paper one, we identified changes in cell membrane potential (CMP) as the most sensitive indicator of toxicity in neuroblastoma SH-SY5Y cells.

In the following studies, we evaluated the capacity of the murine neural progenitor cell line C17.2 to differentiate into mixed cell cultures. Upon differentiation of the C17.2 cells we could identify two morphologically distinguishable cell types; astrocytes and neurons (Paper II). We then investigated how differentiated C17.2 cells responded to non-cytotoxic concentrations of three known neurotoxic and three non-neurotoxic substances. The neurotoxicants induced depolarisation of CMP and alteration in the mRNA expression of at least one of the three biomarkers studied, i.e. βIII-tubulin, glial fibrillary acidic protein or heat shock protein-32. In contrast, no significant effects were observed when exposed to non-neurotoxic compounds (Paper IV).

To further characterise the C17.2 cell model during differentiation, an mRNA microarray analysis of the whole genome was performed. The 30 most significantly altered biomarkers with association to neuronal development were identified. The mRNA expression of the 30 biomarkers were used as a panel to alert for developmental neurotoxicity by exposing C17.2 cells during differentiation to toxicants known to induce impaired nervous system development. All but two of the selected genes were significantly altered by at least one of the chemicals, but none of the 30 genes were affected when treated with the negative control (Paper III).  

In conclusion, the differentiated C17.2 neural progenitor cell line seems to be an attractive model for studying and predicting acute and developmental neurotoxicity. 

This thesis is aimed at summarizing some of the alternative in vitro methods and models that have been used to study both adult and developmental neurotoxicity (DNT), and also to pinpoint some of the important aspects of using alternative in vitro methods. The aim of the papers included in this thesis was to challenge the hypothesis that neurotoxicity and DNT of chemicals can be studied using robust endpoints for proliferation and neural differentiation, such as neurite outgrowth, mRNA expression and protein expression, in two different cell lines. The aim was also to characterize the two cell lines and identify marker genes important for differentiation and to evaluate if these markers could be used as indicators for DNT. The hypothesis being that any chemical that change the expression of important genes for the developmental process could possibly result in DNT for the cells. The current developmental neurotoxicity testing guidelines, using animal models, are time consuming, expensive, ethically questionable and have relatively low sensitivity. Because of this, there has been a paradigm shift towards developing and using alternative methods capable of testing and screening large number of substances. The next generation of developmental neurotoxicity testing is predicted to consist of both in silico and in vitro testing that have to be used in a combined fashion so that it will generate a more rapid and efficient toxicity testing. The idea is to use a battery of refined endpoint studies that identify the specific toxicity of a compound, discriminate between different neural subpopulations and the different stages of neural differentiation. The use of transcriptomic approaches has been suggested as an example of such an endpoint. In this thesis we have evaluated the human neuroblastoma cell line SH-SY5Y and the murine neural progenitor cell line C17.2 in their ability to detect neurotoxic and developmental neurotoxic compounds. We have evaluated this by using functional endpoints, such as neurite outgrowth, cell membrane potential and phenotype ratios. We have also studied the effect of selected chemicals on the levels of mRNA markers specific for different neural cell populations or for neural differentiation in general. We have performed whole genome gene expression on the two cell lines during differentiation and identified and selected a limited number of genes that have been evaluated for their ability to detect developmental neurotoxicity. Both cell lines showed that they have the capability to identify neurotoxic and developmental neurotoxic compounds and could possibly serve as an addition to the testing battery of neurotoxicity in the future. Some of the focus of this thesis has been directed towards the neurodevelopmental effects of the neurotoxic compound acrylamide. Most people get exposed to acrylamide through food consumption and from environmental pollution. Since acrylamide crosses the placental barrier, it creates a risk for developmental consequences. We found that acrylamide affected both cell proliferation and differentiation in both cell lines. Acrylamide affected both neuronal and the glial phenotypes in the C17.2 cell line. We also revealed that acrylamide attenuated neural differentiation at concentrations that were seven orders of magnitude lower than the estimated plasma concentration of free acrylamide in the fetus. Low concentrations of acrylamide altered the gene expression of several genes involved in the retinoic acid signaling as well as the CREB signaling pathways during retinoic acid driven differentiation in the SH-SY5Y cells. Since sub-micromolar concentrations seem to inhibit the differentiation process in both cell lines, developmental neurotoxicity induced by daily intake of acrylamide is a matter of concern. We found that the C17.2 cell line could function as a good model for detecting acute neurotoxicity by evaluating the cell membrane potential of the cells in combination with gene expression of neural and stress marker genes.