A simple technique for quantitation of low levels of DNA damage in individual cells

Abstract

Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H2O2 (9.1 to 291 microM) at 4 degrees C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H2O2. An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells.

Biomarkers responses of the earthworm Eisenia fetida to acetochlor exposure in OECD soil

“…The comet assay (alkali method) was measured according to the method described by Singh et al (1988). Cell suspensions (25 ll) (about 10 5 cells) were mixed with 75 ll of 0.5% low-melting (37°C) agarose and spreaded on a normal melting agarose-precoated slide.…”

“…Comet assay was proved to be a sensitive and useful tool for measuring DNA damage and repair of different chemical pollutants in a variety of organisms, from plants to mammals (Singh et al, 1988;Cotelle and Ferard, 1999;Reinecke and Reinecke, 2004). Its application to earthworm is very valuable for monitoring and detection of genotoxicity of different chemical pollutants in terrestrial ecosystem (Verschaeve and Gilles, 1995;Salagovic et al, 1996;Cotelle and Ferard, 1999).…”

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“…apurinic) sites in cells exposed to genotoxic carcinogens [15]. In this study, we did not use a modified comet assay with a purified DNA repair enzyme, formamidopyrimidine DNA-glycosylase (FPG), even though this method is more sensitive than the assay for detecting FPG-sensitive sites than our classic comet assay [31,32].…”

“…The alkaline version of the Comet assay was performed according to the method of Singh et al (1988) with minor modifications [15]. Briefly, fully-frosted microscope slides were pre-coated with 1 ml of 0.75% normal melting point agarose (NMA; Promega, Madison, WI) and stored at 4°C.…”

“…In general, this method of analysis can be applied to stained cells adherent to slides, eliminating the cell loss that inevitably occurs due to repeated centrifugations during sample preparation for flow cytometry. And also, the alkaline version of the single cell gel electrophoresis (SCGE) or Comet assay represents a sensitive technique for the detection of single-stranded DNA breaks [15]. We used the Comet assay to measure the extent of single-stranded DNA breaks in epithelial cells treated with H 2 O 2 , and compared this with the number of base modifications at the single cell level.…”