Characterizing Valproic Acid-Induced DNA Double Strand Break Repair
Cutler, Geoffrey Lloyd
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The teratogenic effects of valproic acid (VPA) are well known, though its teratogenic mechanism remains unknown. VPA induces oxidative stress, which may lead to double strand breaks (DSBs) in DNA. Though the cell may repair this damage via homologous recombination (HR) and non-homologous end joining (NHEJ), repair is not always error-free; genomic instability may arise from gene deletions, amplifications, rearrangements, and loss of heterozygosity. Such alterations may underpin VPAʼs teratogenicity. The present study evaluated VPAʼs ability to induce NHEJ and HR and characterized the changes in expression of two proteins key to HR (RAD51) and NHEJ (XRCC4). Using pKZ1 transgenic mice (C57BL/6 genetic background), we sought to measure NHEJ events via X-gal staining. Although consistent staining was observed in adult male brain (positive control), no staining was observed in embryos 12 or 24 hours after in utero exposure to a teratogenic dose of VPA (500 mg/kg, maternal subcutaneous dose) on gestational day 9 (GD9). To determine whether the lack of staining observed in embryos was due to low/absent expression of key DSB-repair proteins, we measured mRNA/protein expression of RAD51 and XRCC4 in C57BL/6, GD9-exposed embryos and maternal brain. One hour after treatment, XRCC4 was increased at the protein level in brain and embryo. RAD51 was not increased in embryos and not detected in adult brain. These data suggest that embryos do possess the protein mediators of NHEJ and HR and that VPA-induced changes in expression of XRCC4 may influence the type of repair pursued, potentially affecting DSB repair fidelity (accuracy). Determination of fidelity of VPA-induced HR was attempted with the Chinese hamster ovary cell line (CHO33) using DNA sequencing; low template concentration and purity precluded successful sequencing of DNA from recombinant colonies and the assessment of fidelity. Overall, these data demonstrate that the lack of X-gal staining observed in pKZ1 embryos is not due to an underexpression of at least one key protein in the NHEJ pathway. Furthermore, a VPA-induced change in the the type of repair pathway pursued by the embryo may have teratological implications.