Albino Rat Experiment

Forty male albino rats (120–150 g) were purchased from AL-Zyade Experimental Animals Production Center, Giza, Egypt. Rats were housed in polypropylene cages with mesh wire covering at 22-25°C with 60-65% relative humidity and under a 12-hr light/dark cycle. Clean food and water were provided ad libitium. After 2 weeks of acclimatization, the rats were randomly divided into four groups, each group had 10 animals as the following; group 1: control group fed with free basal diet, group 2: fed with basal diet containing aflatoxin, group 3: fed with basal diet containing bee pollen, and group 4: fed with basal diet containing aflatoxin and bee pollen with the same previously mentioned concentrations. All experimental procedures were approved by

Serum samples were collected by allowing blood samples collected from the inner canthus of the eye to clot on ice for 30 minutes (min) and then centrifuged at 3000 rpm for 15 min. Immediately after blood collection, all rats were euthanized by cervical dislocation. Liver tissues were grossly examined and divided into three parts, the first part was preserved at -20°C for tissue biochemical investigations, the second part was preserved at -20 °C in phosphate buffer saline (PBS) for Comet assay, and the third part was fixed in 10% neutral buffered formalin (NBF) for

(2004) with some modifications. Briefly, 10 µL of liver homogenate were mixed with 90 µL of low melting point agarose (0.7% in PBS) at 37 ºC and loaded on a fully frosted slide coated with 110 µl of normal melting point agarose (1% in PBS). Then this slide was coverslipped and the agarose layer was left 10 min at 4 °C to solidify. The cover slip was removed and the previous step was repeated for 5 min at 4 ºC. The slides were placed for 2 hrs at 4 °C in a lysis buffer containing (2.5 mol/L NaCl, 100 mmol/L Na2EDTA, 10 mmol/L Tris, [pH 10] and a freshly prepared 1% Triton X-100 and 10% Dimethyl sulfoxide were added to the buffer just before use). Next, slides were covered and incubated for 20 min at 4 °C with electrophoresis alkaline buffer containing (300 mmol/L NaOH, 1 mmol/L Na2EDTA [pH > 13]) in the electrophoresis chamber. This step allowed DNA unwinding and the expression of alkali-labile DNA damage sites. Electrophoresis was performed for 30 min at 25 V and 300 mA. DNA from undamaged cells did not migrate and appeared circular while, DNA from damaged cells migrated to the anode and appeared as a comet. After finishing electrophoresis, the slides were washed 3 times for 5 min each with neutralization buffer 0.4 mol/L Tris [pH 7.5] and then slides were stained with 50 µL of ethidium bromide (2 mg/mL), coverslipped and examined using Optika