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.

Abnormal emotional learning in a rat model of autism exposed to valproic acid in utero

“…Various biochemical studies indicate that VPA can suppress neuronal activity by blocking sodium and calcium channels and enhance the functioning of the inhibitory neurotransmitter, GABA, in the brain (Kwan et al, 2001; Gould et al, 2004). Further, studies have demonstrated the ability of VPA to alter gene expression in vitro and in vivo (Jergil et al, 2009; Kultima et al, 2010; Cohen et al, 2013; Oguchi-Katayama et al, 2013) and these affects have been attributed to VPA's ability to inhibit histone deacetylase (HDAC) (Phiel et al, 2001; Eyal et al, 2004; Haberland et al, 2009). HDAC plays an important role in regulating transcription during fetal development (Shaked et al, 2008; Montgomery et al, 2009).…”

Valproic Acid Increases Formation of Reactive Oxygen Species and Induces Apoptosis in Postimplantation Embryos: A Role for Oxidative Stress in Valproic Acid-Induced Neural Tube Defects

“…Furthermore, studies have supported a role for HDAC inhibition as a mechanism of teratogenesis because analogs of VPA that lack HDAC inhibitory activity are less teratogenic (Gurvich et al, 2005). Gene microarray studies have also demonstrated that VPA targets genes regulated by HDAC, including Mt1 and Mt2, both of which are ROS-sensitive (Jergil et al, 2009). In addition, HDAC inhibitors have been shown to increase ROS production and induce apoptosis in several cancer cell lines (Carew et al, 2008).…”

Characterisation of a Neural Teratogenicity Assay Based on Human ESCs Differentiation Following Exposure to Valproic Acid

“…In our analysis two genes, EMX2 and FOXG1, strictly related to CNS development are among the top 50 upregulated genes in exposed neural rosettes, together with SNAP25 (synaptosomal-associated protein), GABRB2 (gammaaminobutiric acid A receptor), NEFL (neurofilament light polypepetide), that are also involved in the processes of axonogenesis and synaptic transmission (Comparative Toxicogenomic Database, [38,39]. It is still unclear if these genes are directly disregulated by VA or indirectly through an inhibition of histone deacetylases [19], but the final effect of their overexpression is the disruption of neuronal developmental pathways. EMX2 is also regulated by the histone deacetylase activity [40] during embryonic kidney development and is a key gene for brain development, arealization [41] and antero-posterior pattern specification (Comparative Toxicogenomic Database and [42] ).…”

“…Most of these effects refer to the inhibition of histone deacetylase that causes apoptosis through caspase activation and increases or suppresses the transcription of many developmentally important genes [12,16,17]. In fact, several microarray studies conducted in vivo or in vitro in mice [18][19][20] have demonstrated that numerous genes can be activated or repressed through the inhibition of the histone deacetylase pathway, including genes involved directly or indirectly with the process of neural tube formation and closure. The aim of this work is to demonstrate, through morphological and molecular evidences, that the hESCs-based in vitro test system described responds to valproic acid exposure with similar changes as those reported in vivo in humans, and therefore represents a promising alternative test for detecting teratogenic effects during early neural development.…”