Comet assay as a reliable tool to enhance knowledge about antioxidant potential in natural matrices

Evaluating the antioxidant activity in natural matrices has been among our primary research challenges (1 ). However, most of the established methodologies still have significant limitations and interferences, especially in examining whether antioxidant activity is actually translated from in vi1ro to in vivo systems. Furthermore, several antioxidant in vitro assays still pose difficulties when comparing results between different procedures and researchers; in fact, there is not a unique method that can provide unequivocal results {2). In alternative, we have also used cell culture, which might represent a closer approach to in vivo systems, but this methodology presents also operational problems: cells adapt to the imposed oxidative stress {changing their properties). some cells suffer mutation, while others segregate antioxidant compounds {like pyruvate) and antioxidants are unstable in cell culture media and may generate one or more pro-oxidants or react with components of cell culture media. In addition, the fluctuating 02 levels in cell culture media have very different antioxidant compositions from in vivo extracellular fluids (3). In vivo assays could represent a way to overcome these difficulties, but, despite some developments, electron spin resonance methods to detect free radicals in humans, immuno spin-trapping and hydroxylation of aromatic compounds as a method to detect hydroxyl radicals , have proven to be difficult {3). Moreover, the number of reports identifying new potential antioxidant compounds grows rapidly, demanding their fast and reliable evaluation. In contrast, from the available literature, it is possible to conclude that there have been major advances to accurately measure end products of oxidative damage to proteins, lipids, and DNA. Comet assay, in particular, might represent a suitable solution due to its sensitiveness for detecting low levels of DNA damage, small number of cells and low amount of the test substance, low cost, ease of application and flexibility. In addition, a high potential research field is focused on the link between antioxidants and DNA repair, this being an indirect mechanism to confront oxidative stress {4). Therefore, Comet assay comprises a valuable complementary tool for our research on antioxidant activity.