Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Neuroblastoma SH-SY5Y and neural progenitor C17.2 cell lines as models for neurotoxicological studies​
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-6662-0868
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
Keywords [en]
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: urn:nbn:se:su:diva-151654ISBN: 978-91-7797-108-5 (print)ISBN: 978-91-7797-109-2 (electronic)OAI: oai:DiVA.org:su-151654DiVA, id: diva2:1175360
Public defence
2018-03-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2018-02-07 Created: 2018-01-17 Last updated: 2018-02-05Bibliographically approved
List of papers
1. Neurofunctional endpoints assessed in human neuroblastoma SH-SY5Y cells for estimation of acute systemic toxicity
Open this publication in new window or tab >>Neurofunctional endpoints assessed in human neuroblastoma SH-SY5Y cells for estimation of acute systemic toxicity
Show others...
2010 (English)In: Toxicology and Applied Pharmacology, ISSN 0041-008X, E-ISSN 1096-0333, Vol. 245, no 2, p. 191-202Article in journal (Refereed) Published
Abstract [en]

The objective of the EU-funded integrated project ACuteTox is to develop a strategy in which general cytotoxicity, together with organ-specific toxicity and biokinetic features, are used for the estimation of human acute systemic toxicity. Our role in the project is to characterise the effect of reference chemicals with regard to neurotoxicity. We studied cell membrane potential (CMP), noradrenalin (NA) uptake, acetylcholine esterase (AChE) activity, acetylcholine receptor (AChR) signalling and voltage-operated calcium channel (VOCC) function in human neuroblastoma SH-SY5Y cells after exposure to 23 pharmaceuticals, pesticides or industrial chemicals. Neurotoxic alert chemicals were identified by comparing the obtained data with cytotoxicity data from the neutral red uptake assay in 3T3 mouse fibroblasts. Furthermore, neurotoxic concentrations were correlated with estimated human lethal blood concentrations (LC50). The CMP assay was the most sensitive assay, identifying eight chemicals as neurotoxic alerts and improving the LC50 correlation for nicotine, lindane, atropine and methadone. The NA uptake assay identified five neurotoxic alert chemicals and improved the LC50 correlation for atropine, diazepam, verapamil and methadone. The AChE, AChR and VOCC assays showed limited potential for detection of acute toxicity. The CMP assay was further evaluated by testing 36 additional reference chemicals. Five neurotoxic alert chemicals were generated and orphendrine and amitriptyline showed improved LC50 correlation. Due to the high sensitivity and the simplicity of the test protocol, the CMP assay constitutes a good candidate assay to be included in an in vitro test strategy for prediction of acute systemic toxicity.

Keywords
ACuteTox, Acute toxicity, Neurotoxicity In vitro, SH-SY5Y
National Category
Biological Sciences Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-50346 (URN)10.1016/j.taap.2010.02.018 (DOI)000278083100006 ()20211194 (PubMedID)
Funder
EU, European Research Council, LSHB-CT-2004-512051
Note

authorCount :6

Available from: 2010-12-22 Created: 2010-12-22 Last updated: 2018-01-22Bibliographically approved
2. Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests
Open this publication in new window or tab >>Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests
Show others...
2013 (English)In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 27, no 5, p. 1565-1569Article in journal (Refereed) Published
Abstract [en]

Here we present a multipotent neuronal progenitor cell line for toxicity testing as an alternative to primary cultures of mixed cell types from brain tissue. The v-myc immortalised C17.2 cell line, originally cloned from mouse cerebellar neural stem cells, were maintained as monolayer in cell culture dishes in DMEM supplemented with fetal calf serum, horse serum and antibiotics. Different media and exposure scenarios were used to induce differentiation. The optimal condition which generated mixed cultures of neurons and astrocytes included serum-free DMEM:F12 medium with N2 supplements, brain-derived neurotrophic factor and nerve growth factor. The medium was changed every 3rd or 4th day to fresh N2 medium with supplements. After 7days, the culture contained two different morphological cell types, assumed to be neurons and glia cells. The presence of astrocytes and neurons in the culture was confirmed by RT-PCR and Western blot analyses, indicating increased mRNA and protein levels of the specific biomarkers glial fibrillary acidic protein (GFAP) and βIII-tubulin, respectively. Concomitantly, the expression of the neural progenitor cell marker nestin was down-regulated.

Keywords
C17.2 cell line, Neural progenitor cells, Differentiation, Neurotrophic factors
National Category
Cell Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-83395 (URN)10.1016/j.tiv.2012.04.020 (DOI)000320686200018 ()22542584 (PubMedID)
Funder
Swedish Research Council, K2011-79X-21373-03-6
Available from: 2012-12-10 Created: 2012-12-10 Last updated: 2018-01-22Bibliographically approved
3. Whole genome microarray analysis of neural progenitor C17.2 cells during differentiation and validation of 30 neural mRNA biomarkers for estimation of developmental neurotoxicity
Open this publication in new window or tab >>Whole genome microarray analysis of neural progenitor C17.2 cells during differentiation and validation of 30 neural mRNA biomarkers for estimation of developmental neurotoxicity
Show others...
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.

Keywords
C17.2 neurotoxicology
National Category
Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-151628 (URN)10.1371/journal.pone.0190066 (DOI)000418564200086 ()29261810 (PubMedID)
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
4. Altered mRNA Expression and Cell Membrane Potential in the Differentiated C17.2 Cell Model as Indicators of Acute Neurotoxicity
Open this publication in new window or tab >>Altered mRNA Expression and Cell Membrane Potential in the Differentiated C17.2 Cell Model as Indicators of Acute Neurotoxicity
2017 (English)In: Applied In Vitro Toxicology, ISSN 2332-1539, Vol. 3, no 2, p. 154-162Article in journal (Refereed) Published
Abstract [en]

Using general cytotoxicity assays in combination with in vitro tests for organ-specific toxicity has been proposed as an alternative approach to animal tests for estimation of acute systemic toxicity. Here, we present the C17.2 neural progenitor cell line as an option for estimation of acute neurotoxicity. The C17.2 cells were differentiated for 6 days in serum-free N2 medium with brain-derived neurotrophic factor and nerve growth factor to a mixed culture of neurons and astrocytes. The cells were then exposed to noncytotoxic concentrations of acetylsalicylic acid, atropine, digoxin, ethanol, nicotine, or strychnine for 48 hours and the mRNA levels of glial fibrillary acidic protein, βIII-tubulin, and heat shock protein 32 were analyzed as biomarkers for astrocytes, neurons, and cellular stress respectively. As a functional endpoint, the cell membrane potential (CMP) was monitored after acute addition of each compound to the differentiated C17.2 cells, by using the fluorescent FLIPR® membrane potential assay. Nicotine [3.2E-04 M], atropine [1.2E-05 M], or strychnine [6.4E-05 M] resulted in altered gene expression of at least one biomarker for each compound, indicating alerts for neurotoxicity. The three compounds also induced depolarization of the CMP at the lowest observed effect concentrations 9.5E-05 M of nicotine, 1.5E-05 M of atropine, and 6.9E-07 M of strychnine. The non-neurotoxic compounds acetylsalicylic acid, ethanol, and digoxin did neither affect the mRNA levels, nor the CMP. This study showed that the differentiated C17.2 cells might be useful for estimation of acute neurotoxicity by analyzing expression of mRNA biomarkers and CMP alterations.

Keywords
acute neurotoxicity, astrocytes, C17.2 cells, cell membrane potential, HSP32, mRNA biomarkers, neuronal in vitro model, neurons
National Category
Biological Sciences Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-143229 (URN)10.1089/aivt.2016.0022 (DOI)
Funder
Swedish Research Council, 521-2010-2804
Available from: 2017-05-19 Created: 2017-05-19 Last updated: 2018-01-22Bibliographically approved

Open Access in DiVA

Neuroblastoma SH-SY5Y and neural progenitor C17.2 cell lines as models for neurotoxicological studies​(1371 kB)101 downloads
File information
File name FULLTEXT01.pdfFile size 1371 kBChecksum SHA-512
cb38569e877f0b2e05877faf8e22d62b8d2c6ea3506f226b28f08a5f2fde2158d94d1bf0a0d46ac4ad33272455c33c2a58df9fbf4e91925a9ce43ca1f0e35d6a
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Lundqvist, Jessica
By organisation
Department of Neurochemistry
Chemical Sciences

Search outside of DiVA

GoogleGoogle Scholar
Total: 101 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 313 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf