Technical recommendations to perform the alkaline standard and enzyme-modified comet assay in human biomonitoring studies

Publication date: Available online 1 May 2019Source: Mutation Research/Genetic Toxicology and Environmental MutagenesisAuthor(s): Amaya Azqueta, Damian Muruzabal, Elisa Boutet-Robinet, Mirta Milic, Maria Dusinska, Gunnar Brunborg, Peter Møller, Andrew R. CollinsAbstractThe comet assay (single cell gel electrophoresis) is widely used as a biomonitoring tool to assess DNA damage - strand breaks, as well as oxidised bases; it can also be adapted to measure DNA repair. It is based on the ability of breaks in the DNA to relax supercoiling, allowing DNA loops to extend from the nuclear core (nucleoid) under an electric field to form a comet-like tail. Most commonly, it is applied to white blood cells. The range of detection is between a few hundred breaks per cell and a few thousand, encompassing levels of damage that can be repaired and tolerated by human cells. Its applications include monitoring various diseases, studying the influence of nutrition on DNA stability, and investigating effects of environmental and occupational mutagens. Here we address the issue of inter-laboratory variation in comet assay results. This variation is largely due to differences in methods. Imposing a standard protocol is not practical, but users should be aware of the crucial parameters that affect performance of the assay. These include the concentration of agarose in which the cells are embedded; the duration of cell lysis, and of enzyme incubation when oxidised bases are being measured; the dura...
Source: Mutation Research Genetic Toxicology and Environmental Mutagenesis - Category: Genetics & Stem Cells Source Type: research