Alterations in P53 Gene Expression in Experimental Colon Carcinoma of Wistar Rat

Experimental Groups
The animals were randomly divided into two groups: 1) the first group (N=10) was subcutaneously (s.c.) injected with normal saline as a control group, and 2) the second group (N=10) received twice-weekly s.c. injections of DMH dissolved in EDTA (40 mg/kg) for 8 weeks to induce colon carcinoma. All procedures were conducted in accordance with the ethical guidelines of the Islamic Azad University, Sanandaj Branch.

Dissection of Rats and Colon Sampling
Eight weeks after tumor induction, sampling was conducted from the distal colon in animals of both groups for histologic evaluation and immunohistochemical analysis regarding P53 gene alterations. For this purpose, rats were anesthetized using ether, then ethically euthanized via intracardial injection of potassium chlorate. A fine incision was made in the lumbar area of the rats, and colon samples were transferred to a 10% formalin solution. A day later, the formalin solution was changed, and all specimens were stored for 2 weeks in darkness under monitored temperature and humidity.

Histologic Sections Stained with Hematoxylin and Eosin (H&E)
For the preparation of histologic sections, all specimens were initially dehydrated using a Tissue Processor device as follows: specimens were dehydrated by passing through ethanol concentrations of 50%, 70%, 80%, and 96%, as well as two absolute ethanol containers, respectively, for 2h per container. Samples were cleared using xylene impregnation twice (2h per container). Finally, impregnation was performed three times in pure paraffin (melting temperature: 58°C), for 2 hours per step. Then, specimens were placed into special blocks, filled with melted paraffin, cooled down, and ultimately subjected to a rotational microtome device to prepare histologic cuts (thickness: 4 µm). The resulting slices were placed in a 50°C water bath, then transferred onto a glass slide and subjected to a 37°C incubator for drying.

H&E staining was applied to each prepared tissue specimen following this procedure: excess paraffin was removed from each specimen using xylene impregnation twice (10 min per container). The hydration of the samples was accomplished through absolute, 95%, and 70% ethanol (1 min per container), washed with distilled water, and placed in hematoxylin stain (20 min), then rinsed. Subsequently, a 20-second impregnation was done in a combination of hydrochloric acid (1 cc) and 70% ethanol (99 cc), then washed again. Next, five-time soaking was done in lithium carbonate to achieve a light blue color, washed for 15 min, transferred to eosin for 5 min, then dehydrated using 70% and absolute ethanol (2 min per container). To clear the slices, they were also passed through two xylene wells (2 min per well). For long-term storage of prepared sections, they were covered on a glass slide with a cover glass and histologic adhesive.

Immunohistochemical Analysis
A new section (3 µm thick) was prepared from paraffin- embedded blocks of those slides positive for colon cancer in histologic evaluation. Immunohistochemical staining for P53 gene alterations was performed using the Benchmark XT automatic slide preparation system (Ventana Company, Arizona, USA), as follows: slices were initially placed in a water bath (56-60°C, 5 min), then transferred onto positively charged Poly-L-lysine coated glass slides and soaked in three xylene wells (5 min each) to remove paraffin. The sample hydration was done by placing them in absolute, 95%, 75%, and 50% ethanol (10 min each) and twice washing with distilled water. The antigen retrieval step was done using heat induction, so that slides were incubated in Tris-EDTA at 98 °C for 20 min, then cooled down to ambient temperature. Triple washing was done with distilled water for 5 min, and slides were incubated in 3% hydrogen peroxide for 10 min at room temperature, to terminate the endogenous peroxidase activity that may cause false-positive results. Twice washing (5 min) was again performed and slides were incubated in tris-buffered saline (TBS) for 5 min. A hydrophobic barrier pen was utilized to delineate the sample section and a UV inhibitor blocker agent was applied to each slide for an hour at room temperature. This blocker agent was further eliminated by TBS washing, specific anti-P53 antibodies (Ventana Company, Arizona, USA) were applied to the slides for 30 min, washed with TBS, and secondary antibodies were used for 20 min. The slides were triple washed with TBS (5 min each), diaminobenzidine (DAB) substrate and H2O2 were added to form a brown color, the washing step was repeated, then a UV copper solution was added (5 min) for better coloring. After TBS washing, hematoxylin stain was applied for 3 min, rinsed with tap water (3 min) and hydrated using 50%, 75%, 95% and absolute ethanol (10 min each), placed three times in xylene, then finally sealed with proper adhesive and alterations in the P53 gene were evaluated using the standard protocol of the World Association of Pathologists.[]

Statistical Analysis
All data were analyzed using SPSS version 22 for Windows. The statistical analysis was performed using the T-test with a 5% level of significance.