Journal of The Electrochemical Society, 160 (7) G3139-G3143 (2013) G3139 0013-4651/2013/160(7)/G3139/5/$31.00 © The Electrochemical Society JES FOCUS ISSUE ON ORGANIC AND BIOLOGICAL ELECTROCHEMISTRY Quantitative Measurement of Electrochemically Induced DNA Damage Using Capillary Electrophoresis Donald H. Atha, Niyatie Ammanamanchi, Mofiyin Obadina, and Vytas Reipa *, z National Institute of Standards and Technology, Biosystems and Biomaterials Division, Materials Measurement Laboratory, Gaithersburg, Maryland 20899, USA 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 capillary electrophoresis (CE) to control and measure the effect of oxidative stress on DNA. Calf thymus DNA and a fluorescently labeled DNA sizing ladder were subjected to fixed oxidizing potentials using a reticulated vitreous carbon electrode (RVC) and their fragmentation was measured with the use of CE. The resulting electropherograms showed that the oxidative treatment resulted in DNA fragmentation. Poly adenosine (Poly A) 40mer and poly guanosine (Poly G) 40-mer oligonucleotides were exposed to a controlled oxidative environment at constant potential values E = 0.5 V, 1.0 V, 1.5 V, and 2 V (vs Ag/AgCl) for 1 hour in 0.1 mol/L potassium phosphate buffer pH 7.3. The treated DNA fragments were analyzed by CE. The areas of the CE peaks were measured and the percentage of DNA fragmentation was calculated. Only minor fragmentation was observed when oligonucleotides were exposed to E = 0.5 V, with strand scission starting at electrode potentials E > 1.0 V. The E = 2.0 V treatment resulted in approximately 50% fragmentation of Poly A, compared to approximately 15% for the Poly G. These results, using DNA as a test model, demonstrate that controlled-potential electrolysis can be used to produce desired levels of oxidative damage to biomaterials without the need to use oxidative chemicals, which are difficult to control and relate to thermodynamic models. © 2013 The Electrochemical Society. [DOI: 10.1149/2.021307jes] All rights reserved. Manuscript submitted March 1, 2013; revised manuscript received April 23, 2013. Published May 3, 2013. This paper is part of the JES Focus Issue on Organic and Biological Electrochemistry. Nearly all living organisms store their genetic information in DNA. Any mutation in DNA can lead to disruption in cell processes. A com- mon DNA damage causal factor is oxidative stress that reflects an imbalance between the systemic manifestation of reactive oxygen species (ROS) and a cell’s ability to readily detoxify it. External or in- ternal disturbances in the normal redox state of cells such as inflamma- tion, ionizing radiation, elevated iron content, and some nanoparticles can produce potent oxidizing species that potentially damage cellular components, including proteins, lipids, and DNA. 1 For DNA, oxida- tive stress leads to detectable structural changes such as base lesions and strand breaks. 2,3 For experimental studies, structural changes in DNA similar to the effects observed in genomic DNA can be gener- ated by reactive oxygen species (ROS) produced by extraneous factors either directly or through intermediates. 4 ROS are either free radicals, reactive anions containing oxygen atoms, or molecules containing oxygen atoms that can produce free radicals. The relative strength of the reactive oxidative species that are implicated in causing the damage in biomolecules can be character- ized by their formal potential. 5 Therefore, establishing the relationship between the strength of the oxidative action and biomolecule fate is essential to understanding the mechanistic framework of oxidative damage. Environments that mimick oxidative stress typically are gen- erated by chemicals, radiation, UV light, or purging with oxygen. 6 Experiments using radical-mediated nucleic acid damage are complicated by short radical lifetimes and quantifying their concentration. 7 Chemically, the test subject (proteins, peptides, DNA, oligonucleotides, etc.) is exposed to the oxidizing agents, such as per- oxide solution, menadione or elevated iron concentration that would catalyze the production of hydroxyl radicals. Then, biochemical con- sequences are analyzed. However, oxidant strength is difficult to con- trol and quantify in such experiments due to radical instability, result- ing in data presented in relative and/or qualitative terms. Our goal is to explore preparative DNA electrochemical oxidation in order to generate a well controlled oxidative environment using high surface area reticulated vitreous carbon (RVC) electrodes. This experimental design enables us to equilibrate bulk solution DNA with a microporous RVC electrode, biased at potentials in the range from 0.5 V to 2.0 V vs Ag/AgCl, the range of the most common oxidants. ∗ Electrochemical Society Active Member. z E-mail: vytautas.reipa@nist.gov By equilibrating solution DNA with a positively biased high surface area electrode, we can establish electron exchange between them ei- ther directly or through products of water electrolysis that serve as mediators. As a first stage, we compared the fragmentation of calf thymus DNA produced by our electrochemical treatment to fragmen- tation by gamma irradiation. We then compared fragmentation in a well characterized DNA sizing ladder and homogeneous preparations of Poly A and Poly G 40-mer oligonucleotides. Methods The electrochemical reactor.— The stable oxidative environment was created by exposing the DNA solution to the desired working elec- trode potential at potentiostatic conditions, maintained by the EG&G Model 273 Potentiostat. A high-surface-area reticulated vitreous car- bon (RVC) electrode (100 pores per inch, Electrosynthesis, Inc.) cut to fit a standard 12 mm spectrophotometer cell served as a working electrode for the bulk electrolysis. The estimated total active electrode area S = 50–60 cm 2 . A 50 × 50 mm Pt gauze (100 mesh, Alfa Aesar Inc.), folded and enclosed in a dialysis tubing (2000 MW cutoff,) was used as a counter electrode, while a flexible Ag/AgCl (3 mol/L KCl, E 0 = 0.210 V vs normal hydrogen electrode, NHE) electrode (MI- 402, Microelectrodes Inc.), served as a reference electrode. Unless noted otherwise, the potential values throughout this study are quoted relative to the Ag/AgCl, 3 mol/L KCl reference electrode. The cell contained 2 mL of DNA solution in 0.1 mol/L potassium phosphate buffer at pH 7.3 that completely filled the working electrode pores due to capillary action. Following a 1 h exposure at a fixed electrode potential the cell was dismantled, test solution removed, and the working electrode cleaned by sonication for 10 min in a detergent solution followed by a vigorous rinsing with distilled/deionized water. Experiments were performed at 20 ◦ C while gently purging with N 2 to remove the dissolved oxygen and facilitate solution mixing. DNA sample preparation and treatment.— Calf thymus DNA (Sigma) at 0.1 mg/mL was dissolved in 0.1 mol/L potassium phos- phate buffer, pH 7.3 and used for electrochemical treatment as well as a control for CE measurements with Beckman P/ACE instrument. For comparison, γ-irradiated (60 Gy) calf thymus DNA (0.1 mg/mL), prepared as previously described, 8 was dissolved in 0.1 mol/L potassium phosphate buffer, pH 7.3. Genescan 6-carboxy-X- rhodamine (ROX) 500 standard DNA sizing ladder (Applied ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 129.6.179.231 Downloaded on 2016-08-31 to IP