Characterization of Valproic Acid-Initiated Homologous Recombination
Valproic acid , homologous recombination , DNA damage , DNA repair
Oxidative stress and histone deacetylase (HDAC) inhibition has 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 this present study we evaluated the role of HDAC inhibition in VPA-initiated HR. HDAC inhibition may indirectly alter repair activity as a result of increased expression of genes involved in HR or indirectly by causing DNA damage which initiates repair. The first objective was to investigate the ability of VPA to cause HDAC inhibition in the Chinese hamster ovary (CHO) 33 cell line. Using immunblotting, an increase in acetylated histone H3 and H4 protein levels was observed throughout 24 hr exposure to 5 mM VPA. Secondly, to investigate whether VPA affects the activity of DNA DSB repair, CHO 33 cells were transfected with either the endonuclease I-SceI 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-induced HDAC inhibition increases susceptibility to DNA damage, immunocytochemistry revealed an increase in the number of γ-H2AX foci throughout 24 hr exposure to 5 mM VPA. To determine if oxidative stress may play 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 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. 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 other roles in DNA maintenance and cell cycle arrest in initiating DNA DSBs and HR repair.