2.3. DNA Comet Assay

The alkaline single cell gel electrophoresis analysis (comet assay) has previously been used to study the potential of chemical-induced DNA damage in individual cells and genotoxicity in mice [40, 41].

In this study, male C57Bl/6 mice were randomly divided into four groups of 5 animals each and administered single doses of the test drugs orally as follows:

The high-dose group: 9000 mg/kg UNCP

The therapeutic dose group: 900 mg/kg UNCP

The negative control group: solvent, 1% starch

The positive control group: methyl methanesulfonate (40 mg/kg)

After treatment, male C57Bl/6 mice were euthanised by dislocation of the cervical vertebrae 3 hours after the single oral administration of test agents. The rectum, epithelium, liver, bone marrow, and kidney were isolated as quickly as possible to prevent invalid results from prolonged manipulations.

The epiphyses of the femurs were cut off and bone marrow cells washed from the diaphysis using 2 ml of phosphate-buffered saline (PBS) precooled to 4°C containing 20 mM EDTA-Na2 and 10% DMSO (pH 7.5). The liver, kidney, and rectum were homogenised in 3 ml of the same buffer. The tubes were held for 5 minutes at room temperature to precipitate large fragments, after which 1.5 ml of the top layer was transferred to a new tube. Cell suspensions in volumes of 60 μl were introduced into a test tube with 240 μl of 0.9% low-melting-point agarose solution (<42°C) in PBS heated to 42°C (microthermostat, “TERMIT”) and resuspended. Then, 60 μl of the agarose solution with the cells was applied to precoated 1% versatile agarose slides that were covered with a coverslip and placed on ice. All subsequent operations were carried out in a dark room with yellow light. After hardening of the agarose (about 10 minutes), the coverslips were carefully removed, and micropreparations were placed in a glass cuvette (Schifferdecker type). This preparation was then poured into a 4°C lysis buffer (10 nM Tris-HCl (pH 10), 2.5 M NaCl, 100 mM EDTA-Na2, 1% Triton X-100, and 10% DMSO) and incubated for at least 1 hour. After lysis, the micropreparations were transferred to the electrophoresis chamber (Sub Cell GT, “Bio-Rad”). The chamber was filled with an electrophoresis buffer (300 mM NaOH, 1 mM EDTA-Na2, pH > 13). The prepared micropreparations were then incubated for 20 minutes to produce alkaline labile sites and alkaline DNA denaturation. Then, electrophoresis was performed for 20 minutes at a field strength of 1 V/cm and a current strength of ∼300 mA. The micropreparations were then transferred into a glass cuvette and fixed in a 70% solution of ethyl alcohol (for 15 min). After fixing, the micropreparations were dried and stained with SYBR Green I fluorescent dye (Sigma-Aldrich, USA) (1 : 10,000 in TE buffer with 50% glycerol) in the dark for 20 minutes and microscopy was performed. The analysis was carried out on a fluorescence microscope (Micromed 3 Lum, Russia) combined with a high-resolution digital camera (x200). The comet images were analysed using CASP software 8.0 (CASP, Wroclaw, Poland). The choice of this software was due to its free download availability on the Internet. As a measure of DNA damage, the percentage of tail DNA (tail DNA%) was used [38]. One hundred cells were analysed from each micropreparation. As a negative control, 2% starch (solvent) was used. Methyl methanesulfonate (methyl mesylate) 40 mg/kg, an alkylating agent and a carcinogen, which is also a reproductive toxicant served as a positive control. The criterion for toxicity was a statistically significant increase in the number of DNA comets, i.e., means of the treated groups and control groups [38]. Data were processed statistically with the help of the Wilcoxon–Mann–Whitney test. P values < 0.05 were considered significant.