Valproic Acid Induces the Hyperacetylation of P53, Expression of P53 Target Genes, and Markers of the Intrinsic Apoptotic Pathway in Midorganogenesis Murine Limbs

Abstract

In utero exposure to valproic acid (VPA), an anticonvulsant and histone deacetylase inhibitor (HDACi), increases the risk of congenital malformations. Although the mechanisms leading to the teratogenicity of VPA remain unsolved, several HDAC inhibitors increase cell death in cancer cell lines and embryonic tissues. Moreover, P53, the master regulator of apoptosis, is an established HDAC target. The purpose of this study was to investigate the effects of VPA on P53 signaling and markers of apoptosis during midorganogenesis in vitro limb development. Timed-pregnant CD1 mice (gestation day 12) were euthanized; embryonic forelimbs were excised and cultured in vitro for 3, 6, 12, or 24 hr in the presence or absence of VPA or valpromide (VPD), a non-HDACi analog of VPA. Quantitative RT-PCR and Western blots were used to assess the expression of candidate genes and proteins involved in P53 signaling and apoptosis. P53 hyperacetylation and a decrease (Survivin/Birc5 and Bcl2) or an increase (p21/Cdkn1a) in the expression of p53 target genes was observed only in VPA-exposed limbs. VPA exposure also triggered an increase in markers of apoptosis and DNA damage; the concentrations of cleaved caspase 9 and caspase 3, cleaved-poly (ADP-ribose) polymerase, and γ-H2AX were increased in VPA-exposed limbs. VPD treatment caused a small but significant increase in cleaved caspase 3. Thus, in vitro exposure to an HDACi such as VPA leads to P53 hyperacetylation, enhances the expression of P53 target genes, and triggers an increase in apoptosis that may contribute to teratogenicity