Calibration of the comet assay for the measurement of DNA damage in mammalian cells.
We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. Gamma-rays from 60Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with gamma- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10(9) Da, respectively, (0.50 and 0.52 breaks/10(6) bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.
Pitozzi
Vanessa
V
Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy.
Pallotta
Stefania
S
Balzi
Manuela
M
Bucciolini
Marta
M
Becciolini
Aldo
A
Dolara
Piero
P
Giovannelli
Lisa
L
D000328:Adult; D000818:Animals; D002138:Calibration; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D003037:Cobalt Radioisotopes / Q000737:chemistry; D020552:Comet Assay / Q000379:methods* / Q000592:standards*; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D005260:Female; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D008297:Male; D051379:Mice; D008875:Middle Aged
D003037:Cobalt Radioisotopes