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.