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.

Fig 1. Electrochemical cell used for DNA oxidation.

BDD WE is shown in blue, Si CE- black.

Fig 2. Oxidatively modified DNA base lesions measured in this study.

(A) 4,6 diamino-5-formamidopyrimidine (FapyAde), (B) 2,6-diamino-4 hydroxy-5-formamidopyrimidine (FapyGua), (C) 8-hydroxyguanine (8-OH-Gua), (D) 5-hydroxy-5-methylhydantoin (5-OH-5-MeHyd), and (E) 8 -hydroxyadenine (8-OH-Ade).

Fig 3. Chronoamperometry curves of the BDD electrode for two fixed potentials (upper panel) and a cyclic voltammetry trace (scan rate v = 10 mV/s, lower panel) in 0.01 mol/L potassium phosphate buffer (pH = 7) + 250 μg/mL DNA.

Fig 4. Level of 8-OH-Gua lesions, measured following 1 h DNA electrolysis at four fixed potentials.

Asterisks indicate significantly increased lesion level results compared to the control samples using one-way Analysis of Variance (ANOVA) followed by Dunnett’s multiple comparison test. The analytical positive control sample was not included in the ANOVA analysis. Four asterisks indicate p < 0.0001. Control and fixed potential sample data represent the mean of 5 independent measurements. The analytical positive control data represents the mean of 2 independent measurements. Uncertainties are standard deviations.

Fig 5. Level of FapyGua lesions, measured following 1 h DNA electrolysis at four fixed potentials.

Asterisks indicate significantly increased lesion level results compared to the control samples using one-way Analysis of Variance (ANOVA) followed by Dunnett’s multiple comparison test. The analytical positive control sample was not included in the ANOVA analysis. One asterisk indicates p < 0.05. Control and fixed potential sample data represent the mean of 5 independent measurements. The analytical positive control data represents the mean of 2 independent measurements. Uncertainties are standard deviations.

Fig 6. Level of 8-OH-Ade lesions, measured following 1 h DNA electrolysis at four fixed potentials.

Asterisks indicate significantly increased lesion level results compared to the control samples using one-way Analysis of Variance (ANOVA) followed by Dunnett’s multiple comparison test. The analytical positive control sample was not included in the ANOVA analysis. One, three or four asterisks indicate p < 0.05, p < 0.001 or p < 0.0001, respectively. Control and fixed potential sample data represent the mean of 4 to 5 independent measurements. The analytical positive control data represents the mean of 2 independent measurements. Uncertainties are standard deviations.

Fig 7. Level of FapyAde lesions, measured following 1 h DNA electrolysis at four fixed potentials.

Asterisks indicate significantly increased lesion level results compared to the control samples using one-way Analysis of Variance (ANOVA) followed by Dunnett’s multiple comparison test. The analytical positive control sample was not included in the ANOVA analysis. One or four asterisks indicate p < 0.05 or p < 0.0001, respectively. Control and fixed potential sample data represent the mean of 3 to 5 independent measurements. The analytical positive control data represents the mean of 2 independent measurements. Uncertainties are standard deations.

Fig 8. Level of 5-OH-5-MeHyd lesions, measured following 1 h DNA electrolysis at four fixed potentials.

Asterisks indicate significantly increased lesion level results compared to the control samples using one-way Analysis of Variance (ANOVA) followed by Dunnett’s multiple comparison test. The analytical positive control sample was not included in the ANOVA analysis. One, three or four asterisks indicate p < 0.05, p < 0.001 or p < 0.0001, respectively. Control and fixed potential sample data represent the mean of 4 to 5 independent measurements. The analytical positive control data represents the mean of 2 independent measurements. Uncertainties are standard deviations.

Fig 9. Redox ambivalence of the guanine oxidation intermediate radical cation.

Fig 10. Schematic representation of the nucleic acid base highest occupied molecular orbital (HOMO) in aqueous solutions relative to the electrochemical potential scale.

HOMO values are from [–68] with bar widths representing a spread in literature values. BDD shows a continuum of the anode electronic density, red arrow—Fermi level variation with applied potential (see text).