Comet Assay

Assessment of DNA damage was performed using a standard protocol for CA (37, 38). A mixture of 5 μl of whole blood with 70 μl of 0.6% low melting point agarose was prepared at about 37°C and rapidly pipetted onto a fully frosted microscope slide coated with a layer of 80 μl of normal-melting-temperature agarose (0.6%). Coverslip was removed after the solidification process, and the slides were immersed in cold lysing solution (2.5 M of NaCl, 100 mM of Na₂EDTA·2H₂O, 10 mM of Tris-HCl, and sodium sarcosinate, adjusted to pH 10.0 with solid NaOH; and the volume was made up of 1 L of dH₂O) for 1 h in the refrigerator. The slides were then incubated in freshly prepared electrophoresis buffer (60 ml of NaOH and 10 ml of EDTA were made up of 2 L of cold dH₂O) at 10°C and a depth of 0.25 cm for 20 min. Electrophoresis of DNA was carried out at 1–10°C for 20 min using 25 V with the current adjusted to 300 mA, followed by buffer neutralisation with 0.4 M of Tris (pH 7.5) for two times. Slides were drained, and 30 μl of EtBr (0.2 mg/ml) was added. Slides were placed in a humidified air-tight container in a refrigerator to prevent drying of the gel and analysed immediately after the excess liquid was blotted. Coded slides were examined at ×200 magnification using a fluorescence microscope (AxioCam MRC, Carl Zeiss, Germany) whereby 500 randomly selected non-overlapping cells on each slide were analysed microscopically by categorising cells as undamaged cells without a tail (Grade 0 = CA0), cells with a tiny tail (Grade 1 or mild damage = CA1), cells with a dim tail (Grade 2 or moderate damage = CA2), cells with a clear tail (Grade 3 or severe damage = CA3), and only tail (Grade 4 or maximally damage = CA4). A total damage score for each slide was calculated by multiplying the number of cells assigned to each grade of damage by the numeric value of the grade and summing overall grades, giving a maximum possible score of 2,000, corresponding to 500 cells at grade 4 (CA total) (39).