Cell models for evaluation of adult and developmental neurotoxicity

94 Pages·2013·13.55 MB·English

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ISBN 978-91-7797-642-4
Department of Biochemistry and Biophysics
Doctoral Thesis in Neurochemistry with Molecular Neurobiology at Stockholm University, Sweden 2019
Cell models for evaluation of adult and
developmental neurotoxicity
Focus on acryalmide
Kristina Attoff
Academic dissertation for the Degree of Doctor of Philosophy in Neurochemistry with
Molecular Neurobiology at Stockholm University to be publicly defended on Friday 14 June
2019 at 10.00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B.
Abstract
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.
Stockholm 2019
http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-168196
ISBN 978-91-7797-642-4
ISBN 978-91-7797-643-1
Department of Biochemistry and Biophysics
Stockholm University, 106 91 Stockholm

CELL MODELS FOR EVALUATION OF ADULT AND
DEVELOPMENTAL NEUROTOXICITY

Kristina Attoff

Cell models for evaluation of
adult and developmental
neurotoxicity

Focus on acryalmide

Kristina Attoff
©Kristina Attoff, Stockholm University 2019

ISBN print 978-91-7797-642-4
ISBN PDF 978-91-7797-643-1

Cover photo: SH-SY5Y cells differentiated for 9 days, taken by Kristina Attoff
Printed in Sweden by Universitetsservice US-AB, Stockholm 2019
Sho till hela min familj,
utan er jag ingenting.

List of publications

I. Attoff K., Kertika D., Lundqvist J., Oredsson S. and Forsby A
Acrylamide affects proliferation and differentiation of the neural progenitor
cell line C17.2 and the neuroblastoma cell line SH-SY5Y
Toxicology in Vitro, 2016, 35, 100–111

II. Attoff K., Gliga A., Lundqvist J., Norinder U. and Forsby A
Whole genome microarray analysis of neural progenitor C17.2 cells during
differentiation and validation of 30 neural mRNA biomarkers for estimation
of developmental neurotoxicity
PLoS One, 2017, 12(12), 1-29

III. Attoff K., Gliga A., Johansson Y., Cediel Ulloa A., Lundqvist J and Forsby A
Acrylamide alters CREB and retinoic acid signaling pathways during
differentiation of the human neuroblastoma SH-SY5Y cell line
Manuscript

IV. Lundqvist J., Svensson C., Attoff K. and Forsby A
Altered mRNA expression and cell membrane potential in the differentiated
C17.2 cell model as indicators of acute neurotoxicity
Applied In Vitro Toxicology, 2016, 3(2), 154-162

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