Controlled Potential Electrooxidation of Genomic DNA
Vytautas Reipa, Donald H. Atha, Sanem Hosbas Coskun, Christopher M. Sims, Bryant C. Nelson
Exposure of mammalian cells to oxidative stress can result in DNA damage that adversely affects many cell processes. We used bulk electrolysis in an electrochemical system and gas chromatographic mass spectrometric analysis (GC/MS/MS) to control and measure, respectively, the effect of electrochemically produced reactive oxygen species on calf thymus DNA (ct-DNA). DNA in 0.01 mol/L potassium phosphate buffer (pH 7.3) was electro-oxidized for 1 h at four fixed oxidizing potentials (E = 0.5V, 1.0V, 1.5V and 2V (vs Ag/AgCl)) using a high surface area boron-doped diamond (BDD) electrode and the resulting DNA damage in the form of oxidatively-modified DNA lesions was measured using GC/MS/MS. We have shown that there are two distinct base lesion formation modes in the explored electrode potential range, corresponding to 0.5 V 1.5 V. The lower potential range exhibited lesion levels up to 50 % above the negative control (a sample left at the floating open circuit potential). However, quite remarkably a statistically significant gradual decrease in all measured lesions was detected when DNA was exposed at increasingly higher fixed BDD potentials from E = 0.5 V to E = 1.5 V. A rapid increase in all base lesion yields was measured when ct-DNA was exposed at E = 2V, the potential at which hydroxyl radicals were efficiently produced by the BDD electrode. The present results demonstrate that controlled potential electrooxidation of double-stranded DNA can be used to purposely increase the levels of oxidatively modified DNA lesions in discrete samples. It is envisioned that these DNA samples may potentially serve as analytical control or quality assurance reference materials for the determination of oxidatively induced DNA damage.