Initial studies on quantitative DNA induced oxidation by gel electrophoresis (GE)-ICP-MS

Abstract

One of the most important consequences of oxidative stress is the induction of oxidation in DNA molecules. Permanent modification of genetic material resulting from “oxidative damage” incidents represents the first step involved in mutagenesis, carcinogenesis, and aging. Therefore, there is an urgent need for monitoring DNA oxidative damage in a quantitative way. For this purpose, the present work evaluates the use of gel electrophoresis (GE) on-line coupled to inductively coupled plasma-mass spectrometry (ICP-MS) to address induced oxidative events in plasmid DNA pBlueScript SK (2961 bp). Oxidative stress is induced in the samples by addition of Fe²⁺ and H₂O₂ through the Fenton reaction. After optimization of the set-up for large DNA fragments, the GE separation of the different oxidation products (as induced by the Fenton reaction) were followed by ³¹P⁺ monitoring with ICP-MS and compared with conventional slab gels. The main advantage with the proposed set-up is that the quantification of each resulting oxidation product could be performed using just an inorganic phosphate as internal standard.