The fast-halo assay for the assessment of DNA damage at the single-cell level

Over the past three decades the development of methods for visualizing at the cell level the extent of DNA breakage significantly contributed to genotoxicity testing: their availability greatly improved the knowledge in the field of genetic toxicology. These procedures are based on the separation and visualization of DNA fragments resulting from cleavage of nuclear DNA from intact DNA. The separation can be obtained either electrically (comet assay, linear migration of DNA fragments) or chemically (fast halo assay, radial diffusion of DNA fragments). Once separated and stained, intact and fragmented DNA can be observed at the fluorescence or light microscope. Appropriate computer-assisted image analysis allows quantitative determination of the extent of DNA breakage. These procedures have been proven to be sensitive, flexible and reliable and, as compared to former methods, they are more simple, less time and money consuming and have the unique capability of detecting DNA damage at the single cell level. This last feature has the additional advantage of allowing the identification of cellular subpopulations characterized by different sensitivity to the damaging agent. The fast halo assay is currently the simplest and quickest; recent modifications of FHA further implemented the assay and pave the way to a full exploitation of its analytical potential. In this chapter the development, the procedures, the applications and the limits of FHA will be illustrated.