CUPRAC colorimetric and electroanalytical methods determining antioxidant activity based on prevention of oxidative DNA damage

Abstract

An unbalanced excess of oxygen/nitrogen species (ROS/RNS) can give oxidative hazard to DNA and other biomacromolecules under oxidative stress conditions. While the ‘comet’ assay for measuring DNA damage is neither specific nor practical, monitoring oxidative changes on individual DNA bases and other oxidation products needs highly specialized equipment and operators. Thus, we developed a modified CUPRAC (cupric ion reducing antioxidant capacity) colorimetric method to determine the average total damage on DNA produced by Fenton oxidation, taking advantage of the fact that the degradation products of DNA but not the original macromolecule is CUPRAC−responsive. The DNA−protective effects of water-soluble antioxidants were used to devise a novel antioxidant activity assay, considered to be physiologically more realistic than those using artificial probes. Our method, based on the measurement of DNA oxidative products with CUPRAC colorimetry proved to be 2 orders-of-magnitude more sensitive than the widely used TBARS (thiobarbituric acid-reactive substances) colorimetric assay used as reference. Additionally, the DNA damage was electrochemically investigated using pencil graphite electrodes (PGEs) as DNA sensor platform in combination with differential pulse voltammetry (DPV). The interaction of the radical species with DNA in the absence/presence of antioxidants was detected according to the changes in guanine oxidation signal.