Controlled potential electro-oxidation of genomic DNA

Abstract

Exposure of mammalian cells to oxidative stress can result in DNA damage that adversely affects many cell processes. Lack of dependable DNA damage reference materials and standardized measurement methods, despite many case-control studies hampers the wider recognition of the link between oxidatively degraded DNA and disease risk. 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 was electro-oxidized for 1 h at four fixed oxidizing potentials (E = 0.5 V, 1.0 V, 1.5 V and 2 V (vs Ag/AgCl)) using a high surface area boron-doped diamond (BDD) working electrode (WE) 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 < E < 1.5 V and E > 1.5 V. Amounts of all four purine lesions were close to a negative control levels up to E = 1.5 V with evidence suggesting higher levels at the lowest potential of this range (E = 0.5 V). A rapid increase in all base lesion yields was measured when ct-DNA was exposed at E = 2 V, the potential at which hydroxyl radicals were efficiently produced by the BDD electrode. The present results demonstrate that controlled potential preparative 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.

Cellular Reference Materials for DNA Damage Using Electrochemical Oxidation

“…Since the asynchronous culture of cells was treated over a period of 12 h, they would be equally affected by treatment during their following their exposure to a range of the oxidizing potentials. We also have demonstrated that soluble genomic DNA is electro-oxidized on boron doped diamond electrodes under potentiostatic conditions [16]. Our GC/MS/MS measurements of purified calf thymus DNA showed that base lesions sensitive to data asymmetry.…”

“…However, several factors inherent to chemical use are difficult to control and hamper the data comparability. e concentration of hydrogen peroxide is particularly difficult to quantify, primarily due to its instability in storage and in cellular media, (8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxy-5-methylhydantoin) were produced during electrochemical treatment at 퐸 = 2.0 V for 1 h [16]. Also, in an earlier study, using capillary electrophoresis, we found extensive strand breakage in calf thymus DNA when exposed for 1 h at 퐸 = 3.0 V and in Poly A and Poly G nucleotides exposed 1 h at 퐸 = 1.0 V [21].…”

“…In that study, we assayed viability in the cultured mammalian cells in a redox potential gradient and found that the electrochemical oxidation mimics oxidative stress and could be used to test the effect of antioxidants. In a separate study, a controlled potential preparative electro-oxidation of soluble calf thymus DNA produced DNA lesions,that were quantified by gas chromatographic mass spectrometry (GC/MS/MS) [16]. In the current report, we examine the use of electrochemical oxidation to produce a controlled amount of DNA strand breaks in cultured mammalian cells.…”

Genotoxic Effects of Etoposide, Bleomycin, and Ethyl Methanesulfonate on Cultured CHO Cells: Analysis by GC-MS/MS and Comet Assay

“…Oxidatively induced damage to DNA bases can be generated by ROS attack (predominantly hydroxyl radical ( ⋅ OH)) on purine and pyrimidine nucleotides in the nucleotide pool and on intact duplex DNA. Because guanine possesses the lowest oxidation potential [34], oxygen radical attack often results in the incorporation of 8-OH-dGTP into the nucleotide pool along with the simultaneous induction and cellular accumulation of the highly mutagenic lesion, 8-OH-Gua [9,29]. 8-OH-Gua promotes G → T transversion mutations that can also lead to strand breakage in the process [10].…”

“…Measurement of Modified DNA Bases by GC-MS/MS. Quadruplicate samples containing 50 μg each of genomic DNA extracted from etoposide-, bleomycin-, and EMStreated cells were enzymatically hydrolyzed, derivatized by trimethylsilylation, and analyzed using DNA extraction and isotope dilution GC-MS/MS methodology exhaustively described in previous studies [26][27][28][29]. Samples of 40 Gy gamma irradiated DNA from calf thymus were used as positive controls (e.g., identification of modified DNA bases).…”