Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals.
The alkaline version of the single cell gel electrophoresis assay, popularly known as the Comet assay, is widely used to evaluate the genotoxic potential of chemicals and environmental contaminants, and for environmental monitoring purposes. In recent years, this assay has increasingly been recognized as a potentially valuable tool for regulatory studies. The assay commonly utilises commercially available software programmes to evaluate the extent of DNA damage at the single-cell level. These programmes provide a large number of measurement outcomes (i.e., tail length, %Tail DNA, various measures of tail moment, etc.) to evaluate the extent of DNA migration and DNA damage. At the moment, however, there is no general agreement with respect to the most relevant measurements or parameters to use. This study was carried out to establish which measurement(s) in the Comet assay are most significantly correlated with DNA damage, and should thus be adopted for routine use. Pooled peripheral blood samples from 3 healthy human individuals were irradiated with a range of doses of (137)Cs gamma-radiation (0, 1, 2, 4 and 8 Gy). Following irradiation, the Comet assay was performed according to a standard protocol, and different parameters were recorded by use of Komet 5.0 software (Kinetic Imaging Ltd., Liverpool, UK). Following a correlation analysis, the Olive Tail Moment (OTM), the Tail Extent Moment and the percentage of DNA in the tail (%Tail DNA) gave good correlations that were not significantly different from each other. Further retrospective analysis from other in vitro and in vivo Comet assay experiments with chemical agents also suggested that OTM and %Tail DNA gave good correlation with the dose of genotoxic agents used. Since OTM and %Tail DNA are the most commonly used parameters in many manuscripts, these two could continue to be applied for routine use. However, since OTM is measured in arbitrary units and different image-analysis systems give different values, the %Tail DNA could be considered more meaningful and easy to conceptualise. Other parameters might not be considered of significant use in genotoxicological studies.
Kumaravel
T S
TS
Covance Laboratories Limited, Otley Road, Harrogate HG3 1PY, UK. tm.kumaravel@covance.com
Jha
Awadhesh N
AN
D020552:Comet Assay / Q000592:standards*; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D005720:Gamma Rays; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000187:drug effects / Q000528:radiation effects; D009153:Mutagens / Q000633:toxicity*; D059010:Single-Cell Analysis; D012984:Software
D009153:Mutagens