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Sensitivity of the standard and Fpg-modified comet assay for the estimation of DNA damage in peripheral blood lymphocytes after exposure to gamma rays

The comet assay is a rapid and sensitive technique for measuring DNA damage. This assay detects single and double stranded breaks, sites of incomplete repair, alkali labile sites, DNA-DNA and DNA-protein cross-links. In addition, particular enzymes such as formamidopyrimidine glycosilase (Fpg) can be used for detection of oxidative damage at the level of DNA molecule by cleavage of 8-oxodG, FaPyGua, FaPyAde and other ring-opened purines caused by reactive oxygen species (ROS). The aim of this study was to test the sensitivity of both standard alkaline and Fpg-modified comet assay and to detect the type of DNA damage caused by gamma rays. In that manner, human lymphocytes were exposed to gamma radiation doses of 0.1 Gy and 4 Gy in vitro. With the standard assay increase in DNA damage was noticed for both exposure doses but it was significant only at higher dose of 4 Gy whereas at lower dose there were no statistically significant increase in neither of the standard comet assay parameters. Fpg-modified protocol showed significant increase in all the parameters measured for both exposure doses indicating that the modified version is capable of detecting wider scale of DNA damage induced by gamma radiation. In addition, with modified protocol it is possible to detect ROS mediated DNA damage, thus significant increase in modified comet parameters in comparison to the standard ones suggests that gamma radiation did induce oxidative damage in DNA molecule. Correlation between different protocols of the comet assay suggests that Fpg-modified version is more sensitive to gamma radiation by virtue of measuring oxidative DNA damage in addition to the basal DNA strand breaks. Results obtained lead to the conclusion that gamma rays affects DNA molecule by ROS that are the most frequent product of gamma radiation. Additionally, human lymphocytes proved to be sensitive to ionizing radiation depending on the radiation dose and are suitable biomarkers for this type of research.

Human peripheral blood lymphocytes; ROS; Gamma radiation; Alkaline comet assay; Fpg- modified comet assay

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