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Short-Time Gene Expression Response to Valproic Acid and Valproic Acid analogs in Mouse Embryonic Stem Cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Toxicology. (Lennart)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
Center for Food Science, Department of Food Toxicology, Veterinary University, Hannover.
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2011 (English)In: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 121, no 2, 328-342 p.Article in journal (Refereed) Published
Abstract [en]

The prediction of potential developmental toxicity in vitro could be based ontoxicogenomic endpoints a short time after exposure in cultured embryo-derived celllines. Our previous microarray studies in P19 mouse embryonal carcinoma cells andmouse embryos have indicated that the teratogen valproic acid (VPA), an inducerof neural tube defects, deregulates the expression of a large number of genes, manyof which have critical roles in neural tube formation and closure. In this study weexposed undifferentiated R1 mouse embryonic stem (ES) cells to VPA and VPA analogto define genes whose expression responses may be related to teratogenic potential.After 6 h of exposure, RNA samples were subjected to microarray analysis usingCodeLinkTM Mouse Whole Genome Bioarrays. VPA (1 mM) and the teratogenic VPAanalog (S)-2-pentyl-4-pentynoic (0.25 mM or 0.5 mM) deregulate a large numberof genes, whereas for the non-teratogenic (and potentially pharmacologically active)analog 2-ethyl-4-methyl-pentanoic acid (1 mM) the expression of only a few geneswas affected. Biological process ontology groups related to embryonic development,morphogenesis, and cell behavior were overrepresented among the affected teratogentarget genes. Multivariate analysis indicated that as few as five genes (out of ~2500array probes correlating with the separation) could separate the data set accordingto teratogenicity. Genes deregulated by the two teratogens showed a substantialoverlap with genes previously found to be deregulated by VPA in P19 cells and mouseembryos. A panel of candidate genes was defined as potential markers predictiveof teratogenicity and evaluated through TaqMan low density array analysis. Theteratogens butyrate and trichostatin A, which like VPA and (S)-2-pentyl-4-pentynoicacid are known histone deacetylase (HDAC) inhibitors, induced similar responsesas these two teratogens for a large subset of markers. This indicates that HDACinhibition may be a major mechanism by which VPA induces gene deregulation andpossibly teratogenicity. Other teratogenic compounds tested had no effect on thepanel of selected markers, indicating that they may not be predicitive of teratogenicityfor compounds acting through other mechanisms than VPA.

Place, publisher, year, edition, pages
2011. Vol. 121, no 2, 328-342 p.
Keyword [en]
developmental/teratology, embryonic stem cells, in vitro and alternatives to animal testing, microarray, predictive toxicology, toxicogenomics
National Category
Pharmacology and Toxicology
Research subject
URN: urn:nbn:se:uu:diva-110247DOI: 10.1093/toxsci/kfr070ISI: 000290931000011PubMedID: 21427059OAI: oai:DiVA.org:uu-110247DiVA: diva2:275578
Available from: 2009-11-06 Created: 2009-11-05 Last updated: 2011-10-13Bibliographically 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 116
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
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)
Available from: 2009-11-27 Created: 2009-11-01 Last updated: 2011-05-11Bibliographically approved

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ToxicologyDepartment of Medical Biochemistry and MicrobiologyThe Linnaeus Centre for Bioinformatics
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