Calibration of the comet assay for the measurement of DNA damage in mammalian cells |
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Authors: | Vanessa Pitozzi Stefania Pallotta Manuela Balzi Marta Bucciolini Aldo Becciolini Piero Dolara |
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Institution: | 1. Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy;2. Medical Physics Unit, University of Florence, Florence, Italy;3. Laboratory of Radiation Biology of the Department of Clinical Physiopathology, University of Florence, Florence, Italy |
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Abstract: | 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. γ-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 γ- 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/109 Da, respectively, (0.50 and 0.52 breaks/106 bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods. |
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Keywords: | Oxidative DNA damage radiation damage comet assay human lymphocytes |
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