Abstract

Analysis of Oxidation Products in Isolated and Cellular DNA by LC-ESI-MS/MS

Guru S Madugundu, Research Scholar, University of Sherbrooke

Reactive oxygen species (ROS) are produced endogenously, during normal aerobic metabolism, and exogenously, upon exposure to ionizing radiation, UV light and environmental toxins. ROS are involved in numerous biological functions. As a consequence of ROS production, the reaction of ROS with biomolecules inevitably leads to oxidative damage. ROS mediated damage to DNA is particularly deleterious because, if the damage is not efficiently removed by DNA repair, it can cause cell death or contribute to mutations. For example, ROS mediated oxidation of cytosine (C) causes CG to TA transition mutations in the mammalian genome. Interestingly, oxidation of the minor base, 5-methylcytosine (5mC), is likely responsible for prevalent mutational hotspots at CG dinucleotide sequences in cancer cells. The purpose of our present work is to develop suitable methods to quantify oxidant induced lesions in isolated and cellular DNA. To this end, we have developed several methods to measure DNA damage based on liquid chromatography coupled to electrospray tandem mass spectrometry and isotopic dilution as a gold standard for the analysis of lesions in isolated and cellular DNA. These analyses include the major oxidation products of cytosine (C), thymine (T), and 5-methylcytosine (5mC) as well as 8-oxo-7, 8-dihydroguanine (8oxoG) as a reference lesion.