Characterization of valproic acid-initiated homologous recombination

Oxidative stress and histone deacetylase (HDAC) inhibition have been implicated as potential mechanisms in valproic acid (VPA) teratogenicity. Reactive oxygen species (ROS) can target DNA to cause oxidative DNA damage and DNA double strand breaks (DSBs) which can be repaired through homologous recombination (HR). HR is not an error free process and can result in detrimental genetic changes. In the present study, we evaluated the potential role of HDAC inhibition in VPA-initiated HR. HDAC inhibition may indirectly alter repair activity or cause DNA DSBs which initiates repair.;Secondly, to investigate whether VPA affects the activity of DNA DSB repair, CHO 33 cells were transfected with either the endonuclease I- Sce1 plasmid to induce a site specific DSB, or the empty plasmid, pGem. However, no increase in the difference in HR between VPA and media exposed I-Sce1 transfected cells compared to cells transfected with pGem was observed, which suggests that VPA does not affect DNA repair activity.;Thirdly, to determine if VPA increases susceptibility to DNA DSBs, immunocytochemistry revealed an increase in the number of gamma-H2AX foci after 10 to 24 hr exposure to VPA. To determine if oxidative stress plays a role in mediating VPA-induced DNA DSBs, another recombination study was carried out in which cells were pretreated with 400 U/ml of PEG-catalase, an antioxidant, prior to VPA treatment. The observed protective effect of PEG-catalase against VPA-induced HR and the generation of intracellular ROS by VPA suggest ROS may also play a role in VPA-initiated HR. ROS can cause oxidative DNA damage which can lead to DNA DSBs. However, in our DNA oxidation study, no increase in the oxidized nucleosides, 8-hydroxy-2'-deoxyguanosine and 5-hydroxycytosine was observed after VPA treatment. These studies suggest that HDAC inhibition and ROS signalling may play roles in DNA maintenance and cell cycle arrest in initiating DNA DSBs and HR repair.;The first objective was to investigate the ability of VPA to cause HDAC inhibition in the Chinese hamster ovary (CHO) 33 cell line. As a consequence of HDAC inhibition, an increase in acetylated histone H3 and H4 protein levels were observed after 10 to 24 hr exposure to 5 mM VPA.