SEVEN BIOMARKERS OF ROS ACTIVITY IN DNA

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DNA modifications, especially the 8-hydroxyguanine modification, have been widely used as indicators of reactive oxygen species (ROS) activity. The question we propose to address is the choice of most appropriate DNA modifications to be used as indicators of ROS activity. Analysis of DNA oligomers exposed to ROS in a fully oxygenated environment indicate that the most appropriate modifications are 8- hydroxyguanine, the formamido remnant of pyrimidine bases and tandem base lesions in which both modifications are present on adjacent bases. As oxygen becomes less available the appropriate DNA modifications are thymine glycol, 5-hydroxymethyluracil, 6-hydroxy-5,6-dihydrothymine and dihydrothymine. The comparative usefulness of these seven DNA modifications as indicators of ROS activity will be evaluated in DMSO-HL 60 cells. These cells can be stimulated by phorbol myristate acetate (PMA) to release ROS. The amounts of the seven modifications in DNA extracted from PMA stimulated cells will be compared with background levels measured in non-stimulated cells. The method of analysis will be by an adaptation of 32P-postlabeling wherein the various DNA modifications are measured in the form of modified dinucleoside monophosphates in nuclease P1 digests of the extracted DNA. The measurement of the seven DNA modifications at the dimer level has several advantages and is feasible because the DNA modifications of interest increase the resistance of the phosphoester bond 3' to the modified dinucleoside to hydrolysis by nuclease P1. A feature of the assay is the use of dimer carriers to unequivocally locate the lesion of interest in the final step of the assay. Another feature is the introduction of internal controls to make the assay quantitative. Our view is that meaningful and reliable biomarkers of ROS activity are key to establishing possible connections between factors in the environment that may generate ROS and specific human diseases.