2.5.2. Evaluation of the Micronuclei (MN) Appearance
The bone marrow cells were prepared following the method of Schmid (20) with certain adjustments described by Bhilwade et al. (2004) (21). Each animal’s femur was taken, and the excess tissues and muscles were removed. The femoral marrow was flushed out using fetal bovine serum (FBS) into a centrifuge tube and centrifuged at 1500 rpm for 10 min to obtain cells pellet, which was thoroughly mixed, then smeared on coded and cleaned frosted slides, air-dried and fixed with absolute methanol. The slides were stained in May–Gruenwald for 5min, then with 10% Giemsa for 10 min, followed by thorough washing with DW. The slides were dried and examined under a light microscope. At least 1000 cells/animal were screened for scoring the frequency of micronucleated polychromatic erythrocytes (MNPCEs) (22).
2.6. Single-cell gel electrophoresis (SCGE) /Comet assay
The comet assay was performed following the method described by Dhawanet al. (23) based on the original procedure developed by Singh et al. (24). Concisely, the BM was flushed out with the chilled Hanks’ Balanced Salt Solution (HBSS) buffer into a microcentrifuge tube; then 5μl was mixed with 75μl of 0.5% low melting agarose solution prepared in 0.9% normal saline and transferred onto frosted slides, which were kept in lysis buffer (20 mM EDTA, 10% DMSO and 0.1% Triton X-100) for 2 hours at 4 °C. Then the slides were removed from the lysis buffer and placed on a horizontal electrophoresis gel box; then the slides were kept in freshly prepared alkaline buffer (Electrophoresis Buffer) with pH>13 for 20 min to unwind the DNA strands. Electrophoresis was carried out for 30 min at 24 volts (~0.74 V/cm), 300 milliamperes. The slides were gently washed in a neutralising buffer for 5min, which was repeated twice to remove the alkaline buffer and then dried. The slides were stained with 80μL 1X Ethidium Bromide, and a minimum of 50 cells/slide was captured using a 40x objective on a fluorescent microscope. The comet images were analysed using “Open Comet” digital imaging software. The percent (%) DNA in Tail, which is the fraction of DNA in the comet tail divided by the total amount of DNA associated with a cell multiplied by 100, was measured to assess the extent of oxidative DNA damage.