Abstract
Despite its high relevance, developmental neurotoxicity (DNT) is one of the least studied forms of toxicity. Current guidelines for DNT testing are based on in vivo testing and they require extensive resources. Transcriptomic approaches using relevant in vitro models have been suggested as a useful tool for identifying possible DNT-generating compounds. In this study, we performed whole genome microarray analysis on the murine progenitor cell line C17.2 following 5 and 10 days of differentiation. We identified 30 genes that are strongly associated with neural differentiation. The C17.2 cell line can be differentiated into a co-culture of both neurons and neuroglial cells, giving a more relevant picture of the brain than using neuronal cells alone. Among the most highly upregulated genes were genes involved in neurogenesis (CHRDL1), axonal guidance (BMP4), neuronal connectivity (PLXDC2), axonogenesis (RTN4R) and astrocyte differentiation (S100B). The 30 biomarkers were further validated by exposure to non-cytotoxic concentrations of two DNT-inducing compounds (valproic acid and methylmercury) and one neurotoxic chemical possessing a possible DNT activity (acrylamide). Twenty-eight of the 30 biomarkers were altered by at least one of the neurotoxic substances, proving the importance of these biomarkers during differentiation. These results suggest that gene expression profiling using a predefined set of biomarkers could be used as a sensitive tool for initial DNT screening of chemicals. Using a predefined set of mRNA biomarkers, instead of the whole genome, makes this model affordable and high-throughput. The use of such models could help speed up the initial screening of substances, possibly indicating alerts that need to be further studied in more sophisticated models.
Highlights
During the last 3 decades, there has been an increase in the number of children diagnosed with learning and neurodevelopmental disorders
Results from the microarray showed that markers for neural stem cells were downregulated during differentiation (e.g. SOX1/3, NES and MKI67), the results were not validated with Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) (Gene Expression Omnibus GSE97337)
One approach for analysis of microarray data is by using principal component analysis (PCA)
Summary
During the last 3 decades, there has been an increase in the number of children diagnosed with learning and neurodevelopmental disorders. This alarming trend has given rise to an emerging need for good models and methods to evaluate possible developmental neurotoxicity (DNT) induced by exposure to different chemicals [1]. The current approved guidelines for toxicity testing rely solely on in vivo models using endpoints such as behavior, sexual maturation, brain weight and neuropathology, which are broad and unspecific. According to the Toxicity testing in the 21st century paradigm, there is a call for reliable in vitro methods that can provide rapid, high-throughput screening of chemicals [5]. Due to the various uncertainties and problems mentioned for the current guidelines for DNT testing, there is a great need of DNT screening specific alternative methods [6]
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