Controlled potential electro-oxidation of genomic DNA

<div><p>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.</p></div

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.

<p>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.</p

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

<p>HOMO values are from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190907#pone.0190907.ref065" target="_blank">65</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190907#pone.0190907.ref068" target="_blank">68</a>] 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).</p

Electrochemical cell used for DNA oxidation.

<p>BDD WE is shown in blue, Si CE- black.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p

Identification and Quantification of Human DNA Repair Protein NEIL1 by Liquid Chromatography/Isotope-Dilution Tandem Mass Spectrometry

Accumulated evidence points to DNA repair capacity as an important factor in cancer and other diseases. DNA repair proteins are promising drug targets and are emerging as prognostic and therapeutic biomarkers. Thus, the knowledge of the overexpression or underexpression levels of DNA repair proteins in tissues will be of fundamental importance. In this work, mass spectrometric assays were developed for the measurement in tissues of the human DNA repair protein NEIL1 (hNEIL1), which is involved in base excision and nucleotide excision repair pathways of oxidatively induced DNA damage. Liquid chromatography/isotope-dilution tandem mass spectrometry (LC–MS/MS), in combination with a purified and fully characterized recombinant ¹⁵N-labeled analogue of hNEIL1 (¹⁵N-hNEIL1) as an internal standard, was utilized to develop an accurate method for the quantification of hNEIL1. Both hNEIL1 and ¹⁵N-hNEIL1 were hydrolyzed with trypsin, and 18 tryptic peptides from each protein were identified by LC–MS/MS on the basis of their full-scan mass spectra. These peptides matched the theoretical peptides expected from trypsin hydrolysis of hNEIL1 and provided a statistically significant protein score that would unequivocally identify hNEIL1. The product ion spectra of the tryptic peptides from both proteins were recorded, and the characteristic product ions were defined. Selected-reaction monitoring was used to analyze mixtures of hNEIL1 and ¹⁵N-hNEIL1 on the basis of product ions. Additional confirmation of positive identification was demonstrated via separation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in-gel tryptic digestion followed by LC–MS/MS analysis. These results suggest that the developed assays would be highly suitable for the <i>in vivo</i> positive identification and accurate quantification of hNEIL1 in tissues

DNA Damaging Potential of Photoactivated P25 Titanium Dioxide Nanoparticles

Titanium dioxide nanoparticles (TiO₂ NPs) are found in numerous commercial and personal care products. Thus, it is necessary to understand and characterize their potential environmental health and safety risks. It is well-known that photoactivated TiO₂ NPs in aerated aqueous solutions can generate highly reactive hydroxyl radicals (^•OH), which can damage DNA. Surprisingly, recent <i>in vitro</i> studies utilizing the comet assay have shown that nonphotoactivated TiO₂ NPs kept in the dark can also induce DNA damage. In this work, we utilize stable isotope-dilution gas chroma­tography/tandem mass spectrometry to quantitatively characterize the levels and types of oxidatively generated base lesions in genomic DNA exposed to NIST Standard Reference Material TiO₂ NPs (Degussa P25) under precisely controlled illumination conditions. We show that DNA samples incubated in the dark for 24 h with TiO₂ NPs (0.5–50 μg/mL) do not lead to the formation of base lesions. However, when the same DNA is exposed to either visible light from 400 to 800 nm (energy dose of ∼14.5 kJ/m²) for 24 h or UVA light at 370 nm for 30 min (energy dose of ∼10 kJ/m²), there is a significant formation of lesions at the 50 μg/mL dose for the visible light exposure and a significant formation of lesions at the 5 and 50 μg/mL doses for the UVA light exposure. These findings suggest that commercial P25 TiO₂ NPs do not have an inherent capacity to oxidatively damage DNA bases in the absence of sufficient photoactivation; however, TiO₂ NPs exposed to electromagnetic radiation within the visible portion of the light spectrum can induce the formation of DNA lesions. On the basis of these findings, comet assay processing of cells exposed to TiO₂ should be performed in the dark to minimize potential artifacts from laboratory light