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Developmental Neurotoxicity Testing Using In vitro Approaches
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There is a great concern about children’s health as the developing brain in foetuses and children is much more vulnerable to injury caused by different classes of chemicals than the adult brain. This vulnerability is partly due to the fact that the adult brain is well protected against chemicals by the blood brain barrier (BBB) and children have increased absorption rates and diminished ability to detoxify many exogenous compounds, in comparison to that of adults. Moreover, the development of the central nervous system (CNS) is a very complex process involving several different important events, e.g. proliferation, migration and differentiation of cells. These events are occurring within a strictly controlled time frame and therefore create different windows of vulnerability. Furthermore, the brain consists of numerous different cell types (neuronal, glial and endothelial cells) that have specific functions. The development of each cell type occurs within a specific time window and is therefore susceptible to environmental disturbances at different time periods.

Evidence indicates that exposure to industrial chemicals, pesticides or drugs, contributes to the increasing incidence of neurodevelopment disorders. However, due to lack of studies only a few industrial chemicals have been identified as developmental neurotoxicants so far. The current developmental neurotoxicity (DNT) guidelines (OECD TG 426 and US EPA 712-C-98-239) are based entirely on in vivo studies that are time consuming, complex, costly and not suitable for the testing of a high number of chemicals. Applying alternative approaches such as in silico, in vitro and non-mammalian models as a part of an integrated test strategy, could speed up the process of DNT evaluation and reduce and refine animal usage. Both in vitro and non-mammalian test systems offer the possibility of providing an early screening for a large number of chemicals, and could be particularly useful in characterising the compound-induced mechanism of toxicity of various developmental processes.

This thesis has characterised two primary neuronal cultures (cerebellar granule cells (CGCs) and cortical neuronal cultures) and identified them as relevant models for DNT testing, since the key processes of brain development are present, such as cell proliferation, migration and neuronal/glial differentiation. Furthermore, two emerging technologies (gene expression and electrical activity) have been evaluated and were identified as promising tools for in vitro DNT assessment. In combination with other assays they could be included into a DNT intelligent testing strategy to speed up the process of DNT evaluation mainly by prioritising chemicals with DNT potential for further testing.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Gren Institute, Stockholm University , 2009. , 64 p.
National Category
Pharmacology and Toxicology
Research subject
Toxicology
Identifiers
URN: urn:nbn:se:su:diva-30056ISBN: 978-91-7155-941-8 (print)OAI: oai:DiVA.org:su-30056DiVA: diva2:241016
Public defence
2009-10-30, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
The work of this thesis was performed at ECVAM, European Commission, Italy. At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: In progress. Paper 4: In progress. Available from: 2009-10-08 Created: 2009-09-30 Last updated: 2009-10-01Bibliographically approved
List of papers
1. Gene expression as a sensitive endpoint to evaluate cell differentiation andmaturation of the developing central nervous system in primary cultures of ratcerebellar granule cells (CGCs) exposed to pesticides
Open this publication in new window or tab >>Gene expression as a sensitive endpoint to evaluate cell differentiation andmaturation of the developing central nervous system in primary cultures of ratcerebellar granule cells (CGCs) exposed to pesticides
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2009 (English)In: Toxicology and Applied Pharmacology, ISSN 0041-008X, Vol. 235, 268-286 p.Article in journal (Refereed) Published
Abstract [en]

The major advantage of primary neuronal cultures for developmental neurotoxicity (DNT) testing is their ability to replicate the crucial stages of neurodevelopment. In our studies using primary culture of cerebellar granule cells (CGCs) we have evaluated whether the gene expression relevant to the most critical developmental processes such as neuronal differentiation (NF-68 and NF-200) and functional maturation (NMDA and GABA presence of neural precursor cells (nestin and Sox10) could be used as an endpoint for in vitro DNT. The expression of these genes was assessed after exposure to various pesticides (paraquat parathion, dichlorvos, pentachlorophenol and cycloheximide) that could induce developmental neurotoxicity through different mechanisms. All studied pesticides signi different stages of neuronal and/or glial cell development and maturation. The most signi observed after exposure to paraquat and parathion (i.e. down-regulation of mRNA expression of NF-68 and NF-200, NMDA and GABA expression of NF-68 and GABA as signi astrocyte marker (S100 multiple pathways of neurodevelopment can be identi in different stages of cell development and maturation, and that gene expression could be used as a sensitive endpoint for initial screening to identify the compounds with the potential to cause developmental neurotoxicity. A receptors), proliferation and differentiation of astrocytes (GFAP and S100β) as well as theficantly modified the expression of selected genes, related to theficant changes wereA receptors). Similarly, dichlorvos affected mainly neurons (decreased mRNAA receptors) whereas cycloheximide had an effect on neurons and astrocytes,ficant decreases in the mRNA expression of both neurofilaments (NF-68 and NF-200) and theβ) were observed. Our results suggest that toxicity induced by pesticides that targetfied by studying expression of genes that are involved

Identifiers
urn:nbn:se:su:diva-30071 (URN)10.1016/j.taap.2008.12.014 (DOI)000264207900002 ()
Available from: 2009-10-01 Created: 2009-10-01 Last updated: 2009-10-01Bibliographically approved
2. mRNA Expression is a Relevant Tool to Identify Developmental Neurotoxicants Using an In Vitro Approach
Open this publication in new window or tab >>mRNA Expression is a Relevant Tool to Identify Developmental Neurotoxicants Using an In Vitro Approach
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2009 (English)In: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 113, no 1, 95-115 p.Article in journal (Refereed) Published
Abstract [en]

So far, only a few industrial chemicals have been identified as developmental neurotoxicants. Because the current developmental neurotoxicity (DNT) guideline (Organisation for Economic Cooperation and Development TG 426) is based entirely on in vivo studies that are both time consuming and costly, there is a need to develop alternative in vitro methods for initial screening to prioritize chemicals for further DNT testing. In this study, gene expression at the mRNA level was evaluated to determine whether this could be a suitable endpoint to detect potential developmental neurotoxicants. Primary cultures of rat cerebellar granule cells (CGCs) were exposed to well known (developmental) neurotoxicants (methyl mercury chloride, lead chloride, valproic acid, and tri-methyl tin chloride) for different time periods. A significant downregulation of the mRNA level for the neuronal markers (NF- 68, NF-200, N-methyl D-aspartate glutamate receptor, and gamma amino butyric acid receptor) was observed after exposure to methyl mercury chloride, valproic acid, and tri-methyl tin chloride. Moreover, a significant increase of the neural precursor marker nestin mRNA was also observed. The mRNA expression of the astrocytic markers (glial fibrillary acidic protein [GFAP] and S100b) was unchanged. In contrast, exposure to lead chloride significantly decreased the mRNA level of the astrocytic marker GFAP, whereas the neuronal markers were less affected. These results suggest that gene expression could be used as a sensitive tool for the initial identification of DNT effects induced by different mechanisms of toxicity in both cell types (neuronal and glial) and at various stages of cell development and maturation.

Place, publisher, year, edition, pages
Oxford university press, 2009
Keyword
gene expressiondevelopmental neurotoxicityprimary cell culture
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:su:diva-30072 (URN)10.1093/toxsci/kfp175 (DOI)
Available from: 2009-10-01 Created: 2009-10-01 Last updated: 2010-12-22Bibliographically approved
3. Comparative toxicity induced by domoic acid in immature and mature mixed primarycultures of cerebellar granule cells: involvement of AMPA and NMDA receptors
Open this publication in new window or tab >>Comparative toxicity induced by domoic acid in immature and mature mixed primarycultures of cerebellar granule cells: involvement of AMPA and NMDA receptors
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Domoic acid is a naturally occurring shellfish toxin that can induce brain damage in mammalians. The toxic effects is thought to be mediated through activation of the AMPA/KA receptor, which induces increased levels of intracellular Ca2+ which in turn cause glutamate release and activates the NMDA receptor. Most studies have been performed in adult animals but neonates have been shown to be more sensitivity to domoic acid per body weight than adults. Prenatal exposure to domoic acid has been associated with damage to neurons in different brain regions, decreased brain GABA levels and increased glutamate levels. In this study we evaluated domoic acid induced toxicity in immature and mature cultures of primary rat cerebellar granule cells (CGCs) by measuring the mRNA levels of selected genes identified as specific glial and neuronal markers. Moreover, we assessed if the induced effects were mediated by activation of the AMPA/KA and/or the NMDA receptor. In addition the influence of the neurotransmitter GABA on domoic acid toxicity was evaluated. The mRNA levels of all the neuronal markers (NF-68, NF-200, NMDA receptor and GABAA receptor) were down-regulated after domoic acid exposure in both immature and mature cultures. However, the mature cultures seemed to be more sensitive to the treatment as the effects were observed at lower concentrations and at an earlier time point than for the immature ones. This could be due to lower expression in young cultures of the receptors that are mediating the toxicity. Indeed, the domoic acid effect could be prevented by the antagonist of the AMPA/KA receptor (NBQX) indicating that this receptor is involved, in contrast to the antagonist for the NMDA receptor (APV) that did not induced any effects. Interestingly, the astrocytic markers (GFAP and S100β) and the neural precursor marker (nestin) were only affected in the mature cultures. These effects could partly be prevented by NBQX, APV and the neurotransmitter GABA, indicating that domoic acid induced toxicity by different mechanisms in astrocytes compared to neurons.

Identifiers
urn:nbn:se:su:diva-30073 (URN)
Available from: 2009-10-01 Created: 2009-10-01 Last updated: 2010-01-14Bibliographically approved
4. Application of micro electrode arrays (MEAs) as an emerging technology for domoic acid- induced developmental neurotoxicity evaluation in primary cultures of rat cortical neurons
Open this publication in new window or tab >>Application of micro electrode arrays (MEAs) as an emerging technology for domoic acid- induced developmental neurotoxicity evaluation in primary cultures of rat cortical neurons
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Due to lack of knowledge only a few industrial chemicals have been identified as developmental neurotoxicants. Current developmental neurotoxicity (DNT) guidelines (OECD TG 426 and EPA712-C-98-239) are based entirely on in vivo studies that are both time consuming and costly. Consequently, there is a high demand to develop alternative in vitro methods for initial screening to prioritise chemicals for further DNT testing. One of the most promising tools for neurotoxicity assessment is the measurement of electrical activity using micro electrode arrays (MEA) that provides a functional and neuronal specific endpoint that until now mainly has been used to detect acute neurotoxicity. Here, electrical activity measurements were evaluated to be a suitable endpoint for the detection of potential developmental neurotoxicants. Initially, primary cortical neurons grown on MEA were characterized for different cell markers (neural precursor cells, neurons and astrocytes) over time using immunocytochemistry to evaluate if the model could be suitable for DNT testing. Indeed, our results show that primary cortical neurons could be a promising in vitro model for DNT testing since some of the most critical neurodevelopment processes such as progenitor cell commitment, proliferation and differentiation of astrocytes and maturation of neurons are present. To evaluate if electrical activity could be a suitable endpoint to detect chemicals with DNT effects primary cortical neurons grown on MEA were exposed to domoic acid (DA), a potential developmental neurotoxicant for up to 4 weeks. Long term exposure to a low concentration (50 nM) of DA increased the basal spontaneous electrical activity as measured by spike and burst rates, as compared to the control cultures. Moreover, the effect induced by the GABAA receptor antagonist bicuculline was significantly lower in the DA treated cultures than in the untreated ones. Obtained data indicates that electrical activity measurements can be used as a tool to detect chemicals with DNT potential. However, more DNT chemicals as well as non-neurotoxic chemicals (negative controls) should be tested to confirm the use of electrical activity measurements for initial DNT screening purposes.

Identifiers
urn:nbn:se:su:diva-30077 (URN)
Available from: 2009-10-01 Created: 2009-10-01 Last updated: 2010-01-14Bibliographically approved

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