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Cell models for evaluation of adult and developmental neurotoxicity: Focus on acrylamide
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University. (Anna Forsby)ORCID iD: 0000-0002-6611-0785
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2019. , p. 77
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-168196ISBN: 978-91-7797-642-4 (print)ISBN: 978-91-7797-643-1 (electronic)OAI: oai:DiVA.org:su-168196DiVA, id: diva2:1306709
Public defence
2019-06-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2019-05-22 Created: 2019-04-24 Last updated: 2019-06-17Bibliographically approved
List of papers
1. Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y
Open this publication in new window or tab >>Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y
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2016 (English)In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 35, p. 100-111Article in journal (Refereed) Published
Abstract [en]

Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10 fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10 pM. Acrylamide significantly reduced the number of neurons starting at 1 mu M and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide.

Keywords
Developmental neurotoxicity, Acrylamide, Neural progenitor cells, SH-SY5Y, C17.2, Differentiation
National Category
Immunology in the medical area Cell and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-133367 (URN)10.1016/j.tiv.2016.05.014 (DOI)000380603700013 ()27241584 (PubMedID)
Available from: 2016-09-12 Created: 2016-09-06 Last updated: 2019-05-08Bibliographically approved
2. 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
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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.

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: 2019-04-24Bibliographically approved
3. Acrylamide alters CREB and retinoic acid signaling pathways during differentiation of the human neuroblastoma SH-SY5Y cell line
Open this publication in new window or tab >>Acrylamide alters CREB and retinoic acid signaling pathways during differentiation of the human neuroblastoma SH-SY5Y cell line
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Acrylamide is a known neurotoxic compound that we get exposed to through food and through the environment. It can cross the placental barrier as well as the blood-brain barrier resulting in exposure of the fetus and the infant child. We used the human neuroblastoma cell line SH-SY5Y to study the effects of non-cytotoxic acrylamide exposure during 9 days of differentiation on two differentially important signaling pathways, i.e. the retinoic acid receptor (RAR) and cAMP response element-binding protein (CREB) signaling in neurons. Our results showed that exposure of non-cytotoxic concentrations of acrylamide during 9 days of differentiation induced altered expression of multiple genes that are part of the CREB and RAR activation pathways, e.g. cellular retinoic acid binding protein 1, retinol binding protein 7, CREB5 and fibroblast growth factor receptor 2. Other well-established neuronal markers such as brain-derived neurotrophic factor, syntaxin binding protein 2, transforming growth factor beta 1, the dopaminergic markers monoamine oxidase A and dopamine receptor D2 as wells as the cholinergic marker choline O-acetyltransferase were also significantly altered by acrylamide. Our results reveal that acrylamide interferes with crucial pathways involved in neuronal differentiation in vitro and raise concerns over the potential toxic outcomes in humans.

National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-168195 (URN)
Available from: 2019-04-24 Created: 2019-04-24 Last updated: 2019-05-02Bibliographically 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: 2019-04-24Bibliographically approved

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