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Valproic acid-induced deregulation in vitro of genes associated in vivo with neural tube defects
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Lennart Dencker)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Lennart Dencker)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Lennart Dencker)
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2009 (English)In: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 108, no 1, 132-148 p.Article in journal (Refereed) Published
Abstract [en]

The utility of an in vitro system to search for molecular targets and markers of developmental toxicity was explored, using microarrays to detect genes susceptible to deregulation by the teratogen valproic acid (VPA) in the pluripotent mouse embryonal carcinoma cell line P19. Total RNA extracted from P19 cells cultured in the absence or presence of 1, 2.5, or 10mM VPA for 1.5, 6, or 24 h was subjected to replicated microarray analysis, using CodeLink UniSet I Mouse 20K Expression Bioarrays. A moderated F-test revealed a significant VPA response for 2972 (p < 10(-3)) array probes (19.4% of the filtered gene list), 421 of which were significant across all time points. In a core subset of VPA target genes whose expression was downregulated (68 genes) or upregulated (125 genes) with high probability (p < 10(-7)) after both 1.5 and 6 h of VPA exposure, there was a significant enrichment of the biological process Gene Ontology term transcriptional regulation among downregulated genes, and apoptosis among upregulated, and two of the downregulated genes (Folr1 and Gtf2i) have a knockout phenotype comprising exencephaly, the major malformation induced by VPA in mice. The VPA-induced gene expression response in P19 cells indicated that approximately 30% of the approximately 200 genes known from genetic mouse models to be associated with neural tube defects may be potential VPA targets, suggestive of a combined deregulation of multiple genes as a possible mechanism of VPA teratogenicity. Gene expression responses related to other known effects of VPA (histone deacetylase inhibition, G(1)-phase cell cycle arrest, induction of apoptosis) were also identified. This study indicates that toxicogenomic responses to a teratogenic compound in vitro may correlate with known in vitro and in vivo effects, and that short-time (< or =6 h) exposures in such an in vitro system could provide a useful component in mechanistic studies and screening tests in developmental toxicology.

Place, publisher, year, edition, pages
2009. Vol. 108, no 1, 132-148 p.
Keyword [en]
alternative methods, embryonal carcinoma cells, exencephaly, histone deacetylase inhibitor, in vitro toxicology, microarray, neural tube defects, teratogen, toxicogenomics, valproic acid
National Category
Pharmacology and Toxicology
Research subject
Toxicology
Identifiers
URN: urn:nbn:se:uu:diva-104322DOI: 10.1093/toxsci/kfp002ISI: 000263606600014PubMedID: 19136453OAI: oai:DiVA.org:uu-104322DiVA: diva2:219639
Available from: 2009-05-28 Created: 2009-05-28 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Pluripotent Stem Cells of Embryonic Origin: Applications in Developmental Toxicology
Open this publication in new window or tab >>Pluripotent Stem Cells of Embryonic Origin: Applications in Developmental Toxicology
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

General toxicity evaluation and risk assessment for human exposure is essential when developing new pharmaceuticals and chemicals. Developmental toxicology is an important part of this risk assessment which consumes large resources and many laboratory animals. The prediction of developmental toxicity could potentially be assessed in vitro using embryo-derived pluripotent stem cells for lead characterization and optimization.

This thesis explored the potential of short-time assays with pluripotent stem cells of embryonic origin using toxicogenomics. Three established pluripotent stem cell lines; P19 mouse embryonal carcinoma (EC) cells, R1 mouse embryonic stem (mES) cells, and SA002 human embryonic stem (hES) cells were used in the studies.

Valproic acid (VPA), an antiepileptic drug which can cause the neural tube defects spina bifida in human and exencephaly in mouse, was used together with microarrays to investigate the global transcriptional response in pluripotent stem cells using short-time exposures (1.5 - 24 h). In addition to VPA, three closely related VPA analogs were tested, one of which was not teratogenic in mice. These analogs also differed in their ability to inhibit histone deacetylase (HDAC) allowing this potential mechanism of VPA teratogenicity to be investigated. The results in EC cells indicated a large number of genes to be putative VPA targets, many of which are known to be involved in neural tube morphogenesis. When compared with data generated in mouse embryos, a number of genes emerged as candidate in vitro markers of VPA-induced teratogenicity. VPA and its teratogenic HDAC inhibiting analog induced major and often overlapping deregulation of genes in mES cells and hES cells. On the other hand, the two non-HDAC inhibiting analogs (one teratogenic and one not) had only minor effects on gene expression. This indicated that HDAC inhibition is likely to be the major mechanism of gene deregulation induced by VPA. In addition, a comparison between human and mouse ES cells revealed an overlap of deregulated genes as well as species specific deregulated genes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 116
Keyword
Embryonic stem cell, Microarray, Toxicogenomics, Valproic acid, Developmental toxicology, Teratogenicity, Neural tube defects, Histone deacetylase inhibitor, In vitro toxicology
National Category
Pharmacology and Toxicology
Research subject
Toxicology
Identifiers
urn:nbn:se:uu:diva-109946 (URN)978-91-554-7660-1 (ISBN)
Public defence
2009-12-18, B21, BMC, Husargatan 3, Uppsala, Uppsala, 09:15 (English)
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Available from: 2009-11-27 Created: 2009-11-01 Last updated: 2011-05-11Bibliographically approved

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