Alkaline nuclear dispersion assays for the determination 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. The separation process can be obtained either electrically (comet assay, linear migration of DNA fragments) or chemically (alkaline dispersion assays, radial diffusion of DNA fragments). Once separated and stained, intact and fragmented DNA can be observed with 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 simpler, are 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 (FHA) is currently the simplest and quickest nuclear dispersion assay; recent modifications of FHA have further improved the assay and pave the way to a full exploitation of its analytical potential. In this chapter the development, procedures, applications, and limits of these dispersion assays, with a particular focus on FHA, will be illustrated.