Valproic acid teratogenicity: a toxicogenomics approach

Abstract

Embryonic development is a highly coordinated set of processes that depend on hierarchies of signaling and gene regulatory networks, and the disruption of such networks may underlie many cases of chemically induced birth defects. The antiepileptic drug valproic acid (VPA) is a potent inducer of neural tube defects (NTDs) in human and mouse embryos. As with many other developmental toxicants however, the mechanism of VPA teratogenicity is unknown. Using microarray analysis, we compared the global gene expression responses to VPA in mouse embryos during the critical stages of teratogen action in vivo with those in cultured P19 embryocarcinoma cells in vitro. Among the identified VPA-responsive genes, some have been associated previously with NTDs or VPA effects [vinculin, metallothioneins 1 and 2 (Mt1, Mt2), keratin 1-18 (Krt1-18)], whereas others provide novel putative VPA targets, some of which are associated with processes relevant to neural tube formation and closure [transgelin 2 (Tagln2), thyroid hormone receptor interacting protein 6, galectin-1 (Lgals1), inhibitor of DNA binding 1 (Idb1), fatty acid synthase (Fasn), annexins A5 and A11 (Anxa5, Anxa11)], or with VPA effects or known molecular actions of VPA (Lgals1, Mt1, Mt2, Id1, Fasn, Anxa5, Anxa11, Krt1-18). A subset of genes with a transcriptional response to VPA that is similar in embryos and the cell model can be evaluated as potential biomarkers for VPA-induced teratogenicity that could be exploited directly in P19 cell-based in vitro assays. As several of the identified genes may be activated or repressed through a pathway of histone deacetylase (HDAC) inhibition and specificity protein 1 activation, our data support a role of HDAC as an important molecular target of VPA action in vivo.

Figure 1. NTDs and apoptosis in VPA-exposed mouse embryos. Whole 10-dpc embryos, stained with the TUNEL technique, viewed from the right (A,B) and front (C,D). Abbreviations: ba, first branchial arch; fb, forebrain; mb, midbrain; nfs, neural folds. Control (A,C) and VPA-treated embryos (B,D) were removed (48 hr posttreatment) from the uteri of NMRI dams after ip administration of sodium valproate (600 mg/kg body weight) on 8.0 dpc. Note that VPA-exposed embryos exhibit unfused neural folds, resulting in apparent signs of failed anterior neural tube closure (black arrowheads in B). Apoptotic cells (dark) are seen along the line of neural fold fusion in control embryos (white arrowheads in C) but not in VPA-exposed embryos (D), where instead a transversal band of apoptotic cells can be seen in the neuroepithelium of the forebrain (D). Angles of views in C and D are indicated by white arrows in A and B, respectively.

Figure 2. Microarray analysis of transcriptional response to VPA in (A) mouse embryos and (B) P19 mouse embryocarcinoma cells. The log posterior odds for each clone to be differentially expressed are plotted against the log₂ fold change of expression for all cDNA clones in the NIA array 1 (see “Materials and Methods”), based on analysis including four replicate microarray slides. The horizontal line marks the threshold (log odds > 1) for selection of a clone as differentially expressed. Upregulated clones are labeled red, and downregulated clones are labeled blue. The clones under the threshold line labeled red or blue were selected (log odds > 1) by leaving out either one of the four replicate slides from the analysis (see “Materials and Methods”). Arrows indicate clones representing the six genes Kpnb1, Krt1-18, Lgals1, Mt2, Upp, and Vcl selected for reanalysis by qPCR (Figure 3). (A) Transcriptional response in 8.25-dpc embryos (6 hr posttreatment) from pregnant NMRI mice after ip administration of sodium valproate (600 mg/kg body weight) on 8.0 dpc. (B) Transcriptional response in P19 cells cultured in the presence of 1 mM sodium valproate for 24 hr.

Figure 3. Comparison of log₂-transformed expression ratios in embryos and P19 cells as determined by microarray analysis and qPCR. : *Significant difference between VPA-treated and control, p < 0.05.

Figure 4. Hierarchical two-way clustering of all 220 genes expected to be transcriptionally responsive to VPA (log odds > 1) in either embryos (Supplemental Material, Tables 1 and 2) or cells (Supplemental Material, Tables 3 and 4), using the mean log₂ fold change of expression (represented by a blue–red color scale; bottom) on both sets of four replicate microarray slides (embryos: EM1–EM4 and cells: CM1–CM4; see “Materials and Methods”). The thick vertical lines to the right of the heat map mark the 29 clones with log odds > 1 that respond similarly to VPA in both embryos and cells (black), and the additional 41 clones with log₂ fold changes > 0.3 or < −0.3 in both embryos and cells (gray). At the right, four discernible clusters (C1–C4) are marked with thin vertical lines.

Figure 5. Transcriptional response in embryos 1.5 and 6 hr posttreatment after ip administration of sodium valproate (600 mg/kg body weight) on 8.0 dpc, as determined by qPCR. : *Significant difference between VPA-treated and control, p < 0.05.