DBD-FISH on neutral comets: Simultaneous analysis of DNA single- and double-strand breaks in individual cells

Human blood leukocytes exposed to X-rays were immersed in an agarose microg el on a slide, extensively deproteinized, and electrophoresed under neutral conditions. Following this single-cell gel electrophoresis assay, characte ristics of DNA migration (i.e., area of the comet) are related to the DNA d ouble-strand breaks (dsbs) yield. After electrophoresis, comets were briefl y incubated in an alkaline unwinding solution, transforming DNA breaks and alkali-labile sites into restricted single-stranded DNA (ssDNA) motifs. The se motifs behave as target sites for hybridization with a whole genome prob e, following the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure. As DNA breakage increases with dose, more ssDNA is pr oduced in the comet by the alkali and more DNA probe hybridizes, resulting in an increase in the mean fluorescence intensity. Since radiation-induced DNA single-strand breaks (ssbs) are far more frequent than dsbs, the mean f luorescence intensity of the DBD-FISH signal from the comet is related to t he ssb level, whereas the surface area of the same comet signal is indicati ve of the dsb yield. Thus, both DNA break types may be simultaneously analy zed in the same cell. This was confirmed in a repair assay performing the D BD-FISH on neutral comets from a human cell line defective in the repair of dsbs. Otherwise, treatment with hydrogen peroxide, a main inducer of ssbs, increased the mean fluorescence intensity, but not the surface, of X-ray-e xposed human leukocytes. (C) 2001 Academic Press.