Testing Using In vitro Approaches

There is a great concern about children’s health as the developing brain in foetuses and children is much more vulnerable to injury caused by different classes of chemicals than the adult brain. This vulnerability is partly due to the fact that the adult brain is well protected against chemicals by the blood brain barrier (BBB) and children have increased absorption rates and diminished ability to detoxify many exogenous compounds, in comparison to that of adults. Moreover, the development of the central nervous system (CNS) is a very complex process involving several different important events, e.g. proliferation, migration and differentiation of cells. These events are occurring within a strictly controlled time frame and therefore create different windows of vulnerability. Furthermore, the brain consists of numerous different cell types (neuronal, glial and endothelial cells) that have specific functions. The development of each cell type occurs within a specific time window and is therefore susceptible to environmental disturbances at different time periods. Evidence indicates that exposure to industrial chemicals, pesticides or drugs, contributes to the increasing incidence of neurodevelopment disorders. However, due to lack of studies only a few industrial chemicals have been identified as developmental neurotoxicants so far. The current developmental neurotoxicity (DNT) guidelines (OECD TG 426 and US EPA 712-C-98-239) are based entirely on in vivo studies that are time consuming, complex, costly and not suitable for the testing of a high number of chemicals. Applying alternative approaches such as in silico, in vitro and non-mammalian models as a part of an integrated test strategy, could speed up the process of DNT evaluation and reduce and refine animal usage. Both in vitro and non-mammalian test systems offer the possibility of providing an early screening for a large number of chemicals, and could be particularly useful in characterising the compound-induced mechanism of toxicity of various developmental processes. This thesis has characterised two primary neuronal cultures (cerebellar granule cells (CGCs) and cortical neuronal cultures) and identified them as relevant models for DNT testing, since the key processes of brain development are present, such as cell proliferation, migration and neuronal/glial differentiation. Furthermore, two emerging technologies (gene expression and electrical activity) have been evaluated and were identified as promising tools for in vitro DNT assessment. In combination with other assays they could be included into a DNT intelligent testing strategy to speed up the process of DNT evaluation mainly by prioritising chemicals with DNT potential for further testing. This thesis is based on the following papers, which are referred to in the text by their respective Roman numerals. I. Hogberg, H. T. , Kinsner-Ovaskainen, A. , Hartung, T. , Coecke, S. , and Bal-Price, A. K. (2009) Gene expression as a sensitive endpoint to evaluate cell differentiation and maturation of the developing central nervous system in primary cultures of rat cerebellar granule cells (CGCs) exposed to pesticides. Toxicology and Applied Pharmacology 235, 268-286. II. Hogberg, H. T. , Kinsner-Ovaskainen, A. , Coecke, S. , Hartung, T. , and Bal-Price, A. K. (2009) mRNA expression is a relevant tool to identify developmental neurotoxicants using an in vitro approach. Toxicological Sciences (in press) III. Hogberg, H.T. , and Bal-Price, A.K. (2009) Comparative toxicity induced by domoic acid in immature and mature mixed primary cultures of cerebellar granule cells: involvement of AMPA and NMDA receptors. Manuscript IV. Hogberg, H.T. , Sobanski, T. , Novellino, A. , Whelan, M. , and Bal-Price, A. K. (2009) Application of micro electrode arrays (MEAs) as an emerging technology for domoic acid induced developmental neurotoxicity evaluation in primary cultures of rat cortical neurons. Manuscript It also refers to the following papers. Buzanska, L. , Sypecka, J., Molteni, S.N. , Compagnoni, A. , Hogberg, H.T. , Del Torchio, R. , Domanska-Janik, K. , Zimmer, J., and Coecke, S. (2009) A Human Stem Cell Based Model For Identifying Adverse Effects Of Organic And Inorganic Chemicals On The Developing Nervous System. Stem Cells (in press) Bal-Price, A.K. , Hogberg, H.T. , Buzanska, L. , and Coecke, S. (2009) Relevance of in vitro neurotoxicity testing for regulatory requirements: Challenges to be considered. Neurotoxicology and Teratology (in press)