Valproic Acid–Induced Deregulation In Vitro of Genes Associated In Vivo with Neural Tube Defects

Abstract

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.