23] Comet assay to detect nitric oxide-dependent DNA damage in mammalian cells

Abstract

Publisher Summary This chapter discusses the use of comet assay to detect nitric oxide-dependent DNA damage in mammalian cells. After hydrogen peroxide, nitric oxide (NO) is the most prevalent mutagen to which human DNA is exposed. NO may interact with cellular amines to form N-nitroso compounds. Its oxidation products nitrite and the higher nitrogen oxides also have biological activity. NO produce DNA strand breakage in mammalian cells by the DNA precipitation assay, in situ nick translation, and the comet assay. The comet assay is a sensitive method for the detection of DNA strand breaks in mammalian cell. The comet assay detects release of DNA from a highly supercoiled DNA-protein complex. In comparison with other sensitive methods, the comet assay is relatively robust and economical in its use of material. It has the specific advantage that as a single-cell assay, it can detect nonuniform response within a cell population, and characterize the behavior of different cell types within a mixed population. Only a small proportion of DNA-damaging agents, including ionizing radiation, bleomycin, and hydrogen peroxide, induces direct breakage of the DNA phosphodiester backbone.