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Whole genome microarray analysis of neural progenitor C17.2 cells during differentiation and validation of 30 neural mRNA biomarkers for estimation of developmental neurotoxicity
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0002-6611-0785
Stockholm University, Faculty of Science, Department of Neurochemistry. Swetox, Karolinska Institutet, Sweden.ORCID iD: 0000-0001-6662-0868
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2017 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 12, article id e0190066Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2017. Vol. 12, no 12, article id e0190066
Keyword [en]
C17.2 neurotoxicology
National Category
Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-151628DOI: 10.1371/journal.pone.0190066ISI: 000418564200086PubMedID: 29261810OAI: oai:DiVA.org:su-151628DiVA, id: diva2:1174695
Funder
Swedish Research Council FormasKnut and Alice Wallenberg FoundationSwedish Research Council, K2013-79X-21373-05-3
Available from: 2018-01-16 Created: 2018-01-16 Last updated: 2018-02-05Bibliographically approved
In thesis
1. Neuroblastoma SH-SY5Y and neural progenitor C17.2 cell lines as models for neurotoxicological studies​
Open this publication in new window or tab >>Neuroblastoma SH-SY5Y and neural progenitor C17.2 cell lines as models for neurotoxicological studies​
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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. 

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2018. p. 84
Keyword
SH-SY5Y, C17.2, in vitro neurotoxicity, cell culture conditions, biomarkers, in vitro developmental neurotoxicity, whole genome microarray
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-151654 (URN)978-91-7797-108-5 (ISBN)978-91-7797-109-2 (ISBN)
Public defence
2018-03-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Available from: 2018-02-07 Created: 2018-01-17 Last updated: 2018-02-05Bibliographically approved

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