Quantitative Measurement of Electrochemically Induced DNA Damage Using Capillary Electrophoresis

Abstract

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.Nearly all living organisms store their genetic information in DNA. Any mutation in DNA can lead to disruption in cell processes. A common 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 internal disturbances in the normal redox state of cells such as inflammation, 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, oxidative 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 generated 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 characterized 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 generated by chemicals, radiation, UV li...

Controlled potential electro-oxidation of genomic DNA

“…A BDD working electrode (WE) was utilized to establish a stable oxidizing environment, an environment whereby DNA samples with quantifiable levels of oxidatively modified lesions could be produced. Previously, with capillary electrophoresis, we have quantified nucleic acid fragmentation under a constant oxidizing potential treatment using a reticulated carbon WE [ 36 ]. Here we equilibrate solution-state DNA with a high surface area BDD WE, biased at constant potentials in the range from 0.5 V to 2.0 V. When polarized at 2V the BDD electrode becomes an efficient • OH radical producer while limiting oxygen evolution due to its high overpotential [ 37 , 38 ].…”

Cellular Reference Materials for DNA Damage Using Electrochemical Oxidation

“…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]. ese studies show that the production of significant DNA damage under physiological conditions in this electrode potential range is consistent with our current studies of mammalian cells.…”

Developing an elegant and integrated electrochemical-theoretical approach for detection of DNA damage induced by 4-nonylphenol

“…Various instrumental techniques such as electrophoresis [16, 17, 18], high-performance liquid chromatography (HPLC) [19, 20, 21], photoelectrochemical methods [22, 23, 24] and fluorescence methods [25, 26, 27] have been reported for detection of DNA damage. Despite their sensitivity, these methods have some disadvantages such as complexity, long assay time, high cost and labor-intensive.…”