Ändra sökning
RefereraExporteraLänk till posten
Permanent länk

Direktlänk
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Cell models for evaluation of adult and developmental neurotoxicity: Focus on acrylamide
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholm University. (Anna Forsby)ORCID-id: 0000-0002-6611-0785
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: urn:nbn:se:su:diva-168196ISBN: 978-91-7797-642-4 (tryckt)ISBN: 978-91-7797-643-1 (digital)OAI: oai:DiVA.org:su-168196DiVA, id: diva2:1306709
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: 2020-05-15Bibliografiskt granskad
Delarbeten
1. Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y
Öppna denna publikation i ny flik eller fönster >>Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y
Visa övriga...
2016 (Engelska)Ingår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 35, s. 100-111Artikel i tidskrift (Refereegranskat) 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.

Nyckelord
Developmental neurotoxicity, Acrylamide, Neural progenitor cells, SH-SY5Y, C17.2, Differentiation
Nationell ämneskategori
Immunologi inom det medicinska området Cell- och molekylärbiologi
Forskningsämne
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-133367 (URN)10.1016/j.tiv.2016.05.014 (DOI)000380603700013 ()27241584 (PubMedID)
Tillgänglig från: 2016-09-12 Skapad: 2016-09-06 Senast uppdaterad: 2020-03-05Bibliografiskt granskad
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
Öppna denna publikation i ny flik eller fönster >>Whole genome microarray analysis of neural progenitor C17.2 cells during differentiation and validation of 30 neural mRNA biomarkers for estimation of developmental neurotoxicity
Visa övriga...
2017 (Engelska)Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, nr 12, artikel-id e0190066Artikel i tidskrift (Refereegranskat) 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.

Nyckelord
C17.2 neurotoxicology
Nationell ämneskategori
Biologiska vetenskaper
Forskningsämne
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-151628 (URN)10.1371/journal.pone.0190066 (DOI)000418564200086 ()29261810 (PubMedID)
Forskningsfinansiär
Forskningsrådet FormasKnut och Alice Wallenbergs StiftelseVetenskapsrådet, K2013-79X-21373-05-3
Tillgänglig från: 2018-01-16 Skapad: 2018-01-16 Senast uppdaterad: 2019-12-16Bibliografiskt granskad
3. Acrylamide alters CREB and retinoic acid signaling pathways during differentiation of the human neuroblastoma SH-SY5Y cell line
Öppna denna publikation i ny flik eller fönster >>Acrylamide alters CREB and retinoic acid signaling pathways during differentiation of the human neuroblastoma SH-SY5Y cell line
Visa övriga...
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
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.

Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-168195 (URN)
Tillgänglig från: 2019-04-24 Skapad: 2019-04-24 Senast uppdaterad: 2019-12-16Bibliografiskt granskad
4. Altered mRNA Expression and Cell Membrane Potential in the Differentiated C17.2 Cell Model as Indicators of Acute Neurotoxicity
Öppna denna publikation i ny flik eller fönster >>Altered mRNA Expression and Cell Membrane Potential in the Differentiated C17.2 Cell Model as Indicators of Acute Neurotoxicity
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.

Nyckelord
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:nbn:se:su:diva-143229 (URN)10.1089/aivt.2016.0022 (DOI)
Forskningsfinansiär
Vetenskapsrådet, 521-2010-2804
Tillgänglig från: 2017-05-19 Skapad: 2017-05-19 Senast uppdaterad: 2019-12-16Bibliografiskt granskad

Open Access i DiVA

Cell models for evaluation of adult and developmental neurotoxicity(13873 kB)84 nedladdningar
Filinformation
Filnamn FULLTEXT01.pdfFilstorlek 13873 kBChecksumma SHA-512
9c1cf59cfae96f52a7f0fc14df923a9a44e11dbe47d6448248f6805c7ef0ec88a4a5834f35cd5a334d02634326bba858626beb9ec053f737f9c77de0f61d5370
Typ fulltextMimetyp application/pdf

Sök vidare i DiVA

Av författaren/redaktören
Attoff, Kristina
Av organisationen
Institutionen för biokemi och biofysik
Biokemi och molekylärbiologi

Sök vidare utanför DiVA

GoogleGoogle Scholar
Totalt: 84 nedladdningar
Antalet nedladdningar är summan av nedladdningar för alla fulltexter. Det kan inkludera t.ex tidigare versioner som nu inte längre är tillgängliga.

isbn
urn-nbn

Altmetricpoäng

isbn
urn-nbn
Totalt: 1297 träffar
RefereraExporteraLänk till posten
Permanent länk

Direktlänk
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf