DBD-FISH on Neutral Comets: Simultaneous Analysis of DNA Single- and Double-Strand Breaks in Individual Cells

• Published in: Experimental cell research 2001-10, Vol.270 (1), p.102-109
• Main Authors: Fernández, J.L., Vázquez-Gundı́n, F., Rivero, M.T., Genescá, A., Gosálvez, J., Goyanes, V.
• Format: Article
• Language: English
• Subjects: Comet Assay; DNA - drug effects; DNA - radiation effects; DNA Damage; DNA double-strand breaks; DNA single-strand breaks; DNA, Single-Stranded - drug effects; DNA, Single-Stranded - radiation effects; DNA-breakage detection-FISH (DBD-FISH); Humans; hydrogen peroxide; Hydrogen Peroxide - pharmacology; In Situ Hybridization, Fluorescence - methods; ionizing radiation; Leukocytes - drug effects; Leukocytes - radiation effects; Nucleic Acid Conformation - drug effects; Nucleic Acid Conformation - radiation effects
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1006/excr.2001.5328

Humanblood leukocytes exposed to X-rays were immersed in an agarose microgel on a slide, extensively deproteinized, and electrophoresed under neutral conditions. Following this single-cell gel electrophoresis assay, characteristics of DNA migration (i.e., area of the comet) are related to the DNA double-strand breaks (dsbs) yield. After electrophoresis, comets were briefly incubated in an alkaline unwinding solution, transforming DNA breaks and alkali-labile sites into restricted single-stranded DNA (ssDNA) motifs. These motifs behave as target sites for hybridization with a whole genome probe, following the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure. As DNA breakage increases with dose, more ssDNA is produced 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 fluorescence intensity of the DBD-FISH signal from the comet is related to the ssb level, whereas the surface area of the same comet signal is indicative of the dsb yield. Thus, both DNA break types may be simultaneously analyzed in the same cell. This was confirmed in a repair assay performing the DBD-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-exposed human leukocytes.