DNA Damage (Comet assay)
Three milliliters of heparinized venous blood samples were taken from each patient just before (group 1), immediately after (Group 2), and one week after the scan (group 3) from all patients. The groups were involved from the same patients, and they were analyzed three times. MNL isolation from the heparinized blood for the comet assay was performed by density gradient separation (Histopaque 1077; Sigma-Aldrich, Inc., St. Louis, MO, USA). For this purpose, 1mL of blood was carefully layered over 1mL of Histopaque and centrifuged for 35 min. at 500 x_g and 25°C. The interface band containing mononuclear leukocyte was washed with phosphate- buffered saline (PBS) and then collected by 15 min. centrifugation at 400 x g. The resulting pellets were resuspended in PBS to obtain 20,000 cells in 10 mL. MNL DNA damage was evaluated by modifying the alkaline single cell gel electrophoresis (comet assay) method whose principle is based on the different migration of DNA molecules with different molecular weights and different electrical charges at the alkaline pH. After electrophoresis, when DNA molecules are stained with DNA- specific fluorescent dyes and examined under a fluorescent microscope, stained DNA can be evaluated by eye or kinetic reading programme [17]. In this study, the images of 100 randomly chosen nuclei (50 nuclei from each of two duplicated slides) were analyzed visually for each subject. Each image was classified according to the intensity of the fluorescence in the comet tail and was given a value of 0, 1, 2, 3 or 4 (from undamaged, class 0, to maximally damaged, class 4) and total score of a slide was between 0 and 400 arbitrary units. Samples from each volunteer were displayed on duplicate slides. The imaged DNAs were at least 50 DNA on each slide, a total of DNA images were recorded and scored on the computer.