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Altered mRNA Expression and Cell Membrane Potential in the Differentiated C17.2 Cell Model as Indicators of Acute Neurotoxicity
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.ORCID-id: 0000-0001-6662-0868
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.ORCID-id: 0000-0002-6611-0785
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Swetox, Karolinska Institutet, Sweden.ORCID-id: 0000-0001-6298-201X
2017 (Engelska)Ingår i: Applied In Vitro Toxicology, ISSN 2332-1539, Vol. 3, nr 2, s. 154-162Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2017. Vol. 3, nr 2, s. 154-162
Nyckelord [en]
acute neurotoxicity, astrocytes, C17.2 cells, cell membrane potential, HSP32, mRNA biomarkers, neuronal in vitro model, neurons
Nationell ämneskategori
Biologiska vetenskaper Kemi
Forskningsämne
neurokemi med molekylär neurobiologi
Identifikatorer
URN: urn:nbn:se:su:diva-143229DOI: 10.1089/aivt.2016.0022OAI: oai:DiVA.org:su-143229DiVA, id: diva2:1096815
Forskningsfinansiär
Vetenskapsrådet, 521-2010-2804Tillgänglig från: 2017-05-19 Skapad: 2017-05-19 Senast uppdaterad: 2019-12-16Bibliografiskt granskad
Ingår i avhandling
1. Estimation of acute toxicity by using the differentiated neuronal progenitor C17.2 cell model
Öppna denna publikation i ny flik eller fönster >>Estimation of acute toxicity by using the differentiated neuronal progenitor C17.2 cell model
2017 (Engelska)Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

The authorities in Europe and United States request information regarding possible toxicity for substances that are produced in one tonne or more per year. Estimation of acute systemic toxicity is conducted in vivo using mice or rats. These tests can be time consuming, costly, unethical, and in some cases irrelevant due to the lack of similarity between humans and rodents. It has been proposed that determining general cytotoxicity together with more tissue-specific effects assessed by using in vitro test systems, e.g. reflecting adverse structure or function in the nervous system, can be an alternative approach to the in vivo tests. Neurotoxicity studies in vitro can be performed by using primary cell cultures from fresh tissue or established cell lines, the latter being often preferred as they are beneficial both economically and ethically.

Here, I present a murine neural progenitor cell line called C17.2 with the potential to differentiate to a mixed culture of both neurons and astrocytes. The differentiation process was examined using 3 different media compositions and 3 different exposures, totally 9 different scenarios. After 7 days in culture with DMEM/F-12 medium containing N2 supplements and 10 ng/mL nerve growth factor and 10 ng/mL brain derived neurotrophic factor, the culture contained two morphological distinguishable cell types, assumed to be neurons and astrocytes. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Western blot analyses were performed which confirmed the presence of neurons and astrocytes in the differentiated cultures. The mRNA and protein levels of the neuronal marker bIII-tubulin and the astrocyte marker glial fibrillary acidic protein (GFAP) were up-regulated during differentiation, while the progenitor cell marker nestin was down-regulated.

To further investigate how this differentiated neural cell model responded to neurotoxic and non-neurotoxic substances, cell membrane potential (CMP) and mRNA expression of bIII-tubulin, GFAP and heat shock protein-32 were examined after exposure to nicotine, atropine, strychnine, ethanol, digoxin, and acetylsalicylic acid. The concentrations that induced effects on the CMP and biomarker expression were compared to general cytotoxicity (Inhibitory Concentration 50%) determined by the neutral red uptake assay in a mouse fibroblast 3T3 cell line, i.e. the 3T3/NRU assay. The CMP assay showed that nicotine, atropine and strychnine exposure induced depolarisation of the cell membrane. However, no effect on the CMP was seen when the cells were treated with acetylsalicylic acid, digoxin, and ethanol at non-cytotoxic concentrations. Alternation in the mRNA expression levels for one of the three biomarkers was seen at non-cytotoxic concentrations for all the compounds that induced acute toxicity by neuronal modes of action, i.e. nicotine, atropine and strychnine. No significant alteration was seen in any of the biomarker mRNA levels when the differentiated C17.2 cells were exposed to compounds that do not induce acute toxicity by neuronal modes of action, i.e. digoxin and acetylsalicylic acid and ethanol.

In conclusion, acute toxicity, which could be induced by neuronal modes of action, may be detected in the differentiated C17.2 cell model by using CMP and gene expression biomarkers as endpoints. The simple cell culture requirements for culturing and differentiating the C17.2 cells into a mixed culture of neurons and astrocytes, the robustness in toxicity read-out, and the cost-effectiveness of the assay make the C17.2 cell line attractive as a model for acute neurotoxicity studies.

Ort, förlag, år, upplaga, sidor
Department of Neurochemistry, Stockholm University, 2017. s. 58
Nyckelord
Neurotoxicity, C17.2, cell membrane potential, Optimisation culture condition
Nationell ämneskategori
Kemi
Forskningsämne
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-143247 (URN)978-91-7649-884-2 (ISBN)
Presentation
2017-06-13, Heilbronnsalen, Svante Arrhenius väg 16B, Stockholm, 13:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2017-05-30 Skapad: 2017-05-30 Senast uppdaterad: 2017-05-22
2. Neuroblastoma SH-SY5Y and neural progenitor C17.2 cell lines as models for neurotoxicological studies​
Öppna denna publikation i ny flik eller fönster >>Neuroblastoma SH-SY5Y and neural progenitor C17.2 cell lines as models for neurotoxicological studies​
2018 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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. 

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Neurochemistry, Stockholm University, 2018. s. 84
Nyckelord
SH-SY5Y, C17.2, in vitro neurotoxicity, cell culture conditions, biomarkers, in vitro developmental neurotoxicity, whole genome microarray
Nationell ämneskategori
Kemi
Forskningsämne
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-151654 (URN)978-91-7797-108-5 (ISBN)978-91-7797-109-2 (ISBN)
Disputation
2018-03-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2018-02-07 Skapad: 2018-01-17 Senast uppdaterad: 2018-02-05Bibliografiskt granskad
3. Cell models for evaluation of adult and developmental neurotoxicity: Focus on acrylamide
Öppna denna publikation i ny flik eller fönster >>Cell models for evaluation of adult and developmental neurotoxicity: Focus on acrylamide
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. s. 77
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-168196 (URN)978-91-7797-642-4 (ISBN)978-91-7797-643-1 (ISBN)
Disputation
2019-06-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

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

Tillgänglig från: 2019-05-22 Skapad: 2019-04-24 Senast uppdaterad: 2019-12-16Bibliografiskt granskad

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Lundqvist, JessicaKristina, AttoffForsby, Anna
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