2.4. Cytokinesis-block micronucleus (CBMN) assay

The CBMN assay was carried out using the standard technique proposed by earlier investigations (21), with minor modifications (22). Hence, 0.5ml of peripheral blood samples were added to 4.5ml of a cell culture medium (RPMI-1640) containing sodium bicarbonate supplemented with 10% fetal calf serum (FCS), antibiotics, and 1% L-glutamine. One hundred microliters of Phytohaemagglutinin (PHA, SIGMA) was added to stimulate lymphocytes. Forty-four hours after PHA stimulation, Cytochalasin-B (Cyt-B, Sigma) at a final concentration of 6μg/ml was added, and binucleated lymphocytes were harvested after 72 h. The cells were then collected by centrifugation at 2,000 RPM for 10 min (BOECHO U-320 R), and supernatant was decanted. Two to three microliters of fresh hypotonic solution 0.075 M KCl was added to the solution remaining at the bottom of tubes and centrifuged at 1,200 RPM for 7 min. Again, the supernatant was poured off, and 5ml of fixature including methanol:glacial acetic acid (6/1) were quickly mixed with solution at the bottom of the tubes. After 20 min, the tubes were centrifuged at 1,200 RPM for 7 min. the induction of MN was evaluated using a double-blind score of 1000 binucleated cells (BNC) with a light microscope set to 40x magnification. Only BN cells were included in the microscopic analysis. All slides were coded before analysis.