The Comet Assay In Vivo in Humans

Abstract

In the last decades, the comet assay has been used as a molecular biomarker to detect DNA damage in several human biomonitoring studies. Nevertheless, many of these studies are often inconclusive or offer poor quality data due to study design constraints. This chapter describes not only the experimental protocol to carry the alkaline version of the comet assay in human cells but also considers many of the obstacles that the researcher faces while carrying a human biomonitoring study, such as sample size, sample type, sample collection, statistics, and result communication. Understanding assay limitations, both experimental and biological, is essential to improve both data quality and data relevance obtained and to guarantee that comet assay continues to provide enhanced reliability as a biomarker in human biomonitoring studies.Key words Comet assay , Human biomonitoring , Exposure , Lymphocytes , Whole blood Comet Assay for Human BiomonitoringComet assay (also designated of single cell gel electrophoresis) is a rapid, simple, visual, and sensitive method to detect DNA breakage in eukaryotic cells [ 1 ].Briefl y, cells are embedded in agarose on a microscope slide, lysed with detergent to remove cell and nuclear membranes, and treated with a high salt solution. Nucleoids are formed, containing non-nucleosomal but still supercoiled DNA. Any breaks present in the DNA cause the supercoiling to relax locally and loops of DNA are then free to extend toward the anode during electrophoresis. At the end of this procedure, damaged cells will resemble comets (hence the name comet assay ) while non-damaged cells maintain their round appearance. Comets are viewed by fl uorescence microscopy after staining with a suitable fl uorescent DNA-binding dye [ 2 ].The pH of unwinding solution is responsible for differences in method sensitivity, as the conversion of alkali-labile sites to strand breaks is a process dependent on the pH and time of alkaline treatment. Under alkaline (pH > 13) conditions, the assay potentially