The overall goal of the in vivo alkaline comet assay and the enzyme-modified alkaline comet assay, is to concurrently measure direct and oxidative DNA damage at the single-cell level. This method was first proposed as a regulatory tool for safety analysis for FDA regulated products. It can help answer key questions in fields ranging from regulatory safety evaluation, to molecular epidemiology, to basic cancer research.
This technique is sensitive and versatile, and also it is relatively inexpensive. It is capable of detecting DNA damage in virtually any animal tissue. Generally, individuals knew this method will struggle to quickly prepare single cell suspensions from various tissues, and it can be a challenge.
Visual demonstration of this method is critical because it requires a certain amount of art, as well as science. After euthanizing rats with carbon dioxide gas, according to the text protocol, palpate to confirm that the rat has no heartbeat, and pinch the toes to confirm that the rat does not withdraw its limb. Use ethanol to disinfect the skin, then use surgical scissors to open the abdominal cavity, before removing the intact liver.
With scissors, separate the left lateral lobe from the rest of the organ, and place it on a cutting board. Next, using a disposable single-edged razor blade, cut two adjacent 3 to 4 millimeter thick transverse sections. Place one transverse section on filter paper, then, into 10%neutral buffered formalin, or NBF.
Place the second liver section into five milliliters of ice-cold mincing buffer in a six centimeter petri dish, and rinse three times to remove the residual blood. Then, transfer the liver section to a new two milliliter centrifuge tube, containing one milliliter of ice-cold mincing buffer. With fine scissors, mince the section to release the cells, then with a 40 micrometer cell strainer, strain the cell suspension to remove any tissue lumps.
Place the single cell suspension on ice, and with a hemocytometer, verify that the concentration is approximately two times 10 to the sixth cells per milliliter. To prepare comet slides, mix one volume of the single cell suspension with 10 volumes of molten 37 degree Celsius 0.5%low-melting agarose solution. Immediately pipette 100 microliters onto a previously prepared agarose-coated slide, and use a cover slip to cover the agarose cell mixture.
Lay the slides flat, and cool at four degrees Celsius for 30 minutes. After the gel has solidified, gently remove the cover slips and immerse the slides in pre-chilled lysis buffer. Incubate in the dark at four degrees Celsius for one hour to overnight.
Following the incubation, remove six of the eight slides prepared for each tissue sample from the lysis buffer, and use room temperature enzyme buffer to rinse them three times for five minutes each. Then, use tissues to blot excess solution. Next, use enzyme buffer to dilute hOGG1 and Endo III enzyme stocks, to one to one thousand, and place on ice.
Place the slides into petri plates lined with moisten paper towels, then apply 200 microliters of the following solutions to the slides in each treatment group. Enzyme buffer alone to two slides, hOGG1 solution to two slides, and Endo III solution to two slides, and use Parafilm to cover the slides. Then place the slides at 37 degrees Celsius, incubating the Endo III treated and reference slides for 45 minutes, and the hOGG1 treated slides for 30 minutes.
Following the incubation, drain the slides and for the reference slides, use cold neutralization buffer to remove residual detergent and salts. Then use distilled water to rinse the enzyme treated slides. Randomly place the slides in a horizontal gel electrophoresis tank, avoiding any spaces.
Then add freshly made electrophoresis buffer to the tank, while avoiding bubbles, until it just covers the slides. Set the power supply to 0.7 volts per centimeter, and incubate the slides in the alkaline buffer for 20 minutes, to allow unwinding of the DNA and expression of the Alkali-liable damage. Record the slide placement, the temperature of the buffer, and the current.
After the incubation for unwinding, place the electrophoresis tank at four degrees Celsius. Then press the Run button on the power supply, and if needed, adjust the current to 300 milliamps by raising or lowering the buffer level, keeping it just above the slides. Run the samples for 20 minutes, before again recording the slide placement, the temperature of the buffer, and the current.
Following electrophoresis, gently lift the slides from the tank, and immerse a neutralization buffer to neutralize the excess Alkali. Then drain the slides and repeat the neutralization two more times. With cold 100%ethanol, fix the slides for five minutes.
Allow the slides to air dry and store in a dry environment. To stain the DNA, place the slides on a cardboard or metal tray, and add 200 microliters of nucleic acid fluorescent stain working solution to each slide, before covering with a cover slip. Protect the slides from light and incubate for at least 30 minutes.
Then to read the slides, gently blot away the excess staining solution, taking care not to disturb the cover glass. Next, place the comet slide on the stage of a fluorescence microscope equipped with a video camera, and randomly choose a field containing cells. After opening the comet assay software, using the mouse, select a cell, then left-click the center of the comet head of the cell.
The software will calculate parameters for the cell, and add to a data file. Once all the cells have been scored in the current field, move to another field and repeat the scoring of any suitable comet cell that appears without bias. Score 10 random fields and at least 75 comets per slide.
Finally, save the data into a spreadsheet for later analysis. As previously reported, CPA is a synthetic hormonal drug that induces rat liver tumors in a sex-specific manner, with five-fold higher doses needed to induce liver tumors in male rats, compared to females. As shown here, the in vivo alkaline comet assay, and the enzyme-modified comet assay, demonstrated that a five-fold higher dose of CPA was needed, to induce a significant increase in DNA damage in male livers, compared to females.
Hydroxysteroid sulfotransferase, or HST, is expressed at 15-fold higher levels in adult female rats, compared to adult males. HST metabolism is a rate-limiting step in the activation of CPA to DNA-binding metabolites. As seen here, CPA-induced oxidative DNA damage was generally greater in male compared to female rat livers, and therefore, less likely to be rate-limiting in tumor formation in male rats.
As reported in these tables, histopathology evaluation of livers from CPA-treated rats, showed no evidence of agent-induced apoptosis or necrosis, confirming previous conclusions that the positive comet assay results were not a secondary effect of cytotoxicity. While doing this procedure, it is important to collect single cell suspension, and prepare comet assay slides as quickly as possible. After its development, this technique paved the way for researchers in the field of genetic toxicology to explore DNA damage, and repair quantitatively in virtually any animal tissue and in human samples, as well.
After watching this video, we hope you can have a better understanding of how to use the in vivo alkaline comet assay together with the enzyme-modified comet assay, to concurrently measure posts of the rat and oxidative DNA damage, at the single cell level in animal tissues. Please remember that working with DNA damage reagent can be extremely hazardous. Please always wear the personal protective equipment when you perform these procedures.
