Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

This protocol is significant because the use of frozen tissue simplifies logistics when evaluating multiple tissues, permits sample transfer to a remote laboratory for analysis, and enables deferring a decision to analyze a tissue for months. The main advantage of this technique is that the use of frozen cube tissue does not require personnel trained in the comet assay at necropsy, and minimizes opportunity for technical errors and mechanical DNA damage introduced during sample manipulation. This technique has application in pre-clinical drug development, the safety assessment of chemicals entering the environment, and food additives.

Frozen tissues can be used in the comet assay to evaluate genotoxic potential, DNA repair, protective effects against DNA damage induced by chemotherapy or radiation, and for environmental bio-monitoring. Ideally, experiments should be designed to allow processing of each tissue sample to the freezing step before organs harvested from the next animal become available for processing. Frozen tissues can be sued in the comet assay to evaluate genotoxic potential, DNA repair, protective effects against DNA damage induced by chemotherapy or radiation, and for environmental bio-monitoring.

Technical trainer Carole Bobbitt, and investigative toxicology lab manager Eileen Gilas will be serving as prosectors today. Research associate Lincoln Martin, and histology technician Amelie Ladesseur will be demonstrating sample handling. To begin this procedure, remove any attached connecting tissue, organs, and debris from an organ of interest.

Immediately swish the organ vigorously in a medium sized weigh boat containing approximately seven milliliters of cold, freshly prepared mincing solution to remove residual blood and debris. Transfer the organ to another clean weigh boat containing sufficient cold mincing solution to keep the tissue submerged and maintain it on ice until further processing. For the liver, cut a five millimeter longitudinal section of the left lobe and gently swish in about seven milliliters of cold mincing solution.

Then place the strip of tissue into a clean medium sized weigh boat containing approximately seven milliliters of cold mincing solution and maintain on ice until ready to process further. Remove a section of the liver and place it into an embedding cassette. Perform fixing, trimming, and paraffin embedding according to the laboratory standard procedures for possible future histopathology evaluation.

For the duodenum, cut a 10 millimeter portion of the duodenum proximal to the stomach. Insert a 21 to 25 gauge needle in one end of the duodenum and flush it with one milliliter of cold mincing solution to remove any debris and bacteria. Flip the duodenum over, flush it again with another one milliliter of mincing solution and discard the needle.

Slice the duodenum open and rinse it in about seven milliliters of mincing solution. Then rinse the duodenum again and maintain it in mincing solution on ice until ready to process further. Cut and fix a portion of the remaining duodenum for possible future histopathology evaluation as previously described for the liver.

For the brain, it may be useful to first divide the brain into two hemispheres, with one hemisphere being saved for possible histopathology. Dissect the brain regions of interest, then gently rinse them, and maintain on ice in mincing solution until ready to process further, as described previously for the liver and duodenum. For the stomach, remove and discard the forestomach.

Cut open the glandular stomach, and wash it free from food by swishing it in a medium sized weigh boat containing about seven milliliters of cold mincing buffer. Remove a five millimeter strip of glandular stomach proximal to the duodenum for fixation for possible histopathology evaluation as described previously. Place the remaining stomach into a medium sized weigh boat containing approximately seven milliliters of cold mincing buffer and incubate on ice for 15 to 30 minutes.

After this, transfer the stomach to a clean piece of paraffin and use a scalpel blade or plastic cell scraper to gently scrape the surface epithelium at least two times to remove debris. Pick up the gastric mucosa with forceps and use a pipette to rinse it with one milliliter of cold mincing buffer. Transfer the rinsed stomach tissue to a clean surface or dish and add cold mincing solution.

Use the back of a scalpel blade or a plastic cell scraper to carefully scrape the stomach epithelium four to five times to release the cells. Then, use a pipette to collect the mincing solution containing the released cells, and transfer it to a microcentrifuge tube on ice. For mincing tissue samples, cut a section of liver, brain, or duodenum and transfer it to a labeled microcentrifuge tube containing one milliliter of cold mincing solution.

Use mincing scissors to rapidly mince the sample until it is finally dispersed, while ensuring that the sample stays cold. The sample should look slightly cloudy. However, it is common for some pieces to remain that will settle to the bottom of the tube.

To use the tissue while it is fresh, maintain the tubes containing the samples on ice. To freeze the tissue samples, secure the tube's lid immediately following the mincing or collection and drop the tube into a Dewar flask containing liquid nitrogen. At the conclusion of tissue harvest, use a ladle to retrieve the sample tubes, and place them on dry ice to sort.

Transfer the tubes to a freezer box on dry ice and store the box in a freezer at minus 80 degrees Celsius. To prepare frozen cubes of tissue, cut several small pieces of tissue and individually drop them directly into a medium sized weigh boat or other suitable vessel containing liquid nitrogen for a few seconds. After the pieces have frozen completely, transfer the frozen tissue cubes individually to labeled microcentrifuge tubes, or cryo-tubes, on dry ice.

At the conclusion of tissue harvest, transfer the tubes to a freezer box on dry ice and store the box in a freezer at minus 800 degrees Celsius. When using freshly minced or scraped tissue maintained on wet ice, prepare comet slides within an hour of harvest. When using frozen minced or scraped tissue, place tubes of frozen tissue into a water bath at room temperature until the samples are completely thawed while ensuring that they are kept cold.

Once the samples are thawed, immediately transfer the tubes onto ice. For cubed tissue, retrieve the tubes containing the cubes from the freezer and maintain on dry ice until slide preparation. Aliquot one milliliter of merchant's medium or a mincing solution into a labeled 1.7 milliliter microcentrifuge tube for each sample and maintain on ice.

Working rapidly to avoid thawing, place a cube of tissue into the open end of a room temperature tissue mincing device. Quickly insert a size matched syringe plunger into the device to push the tissue into the mesh end, which should then be placed into the microcentrifuge tube containing the cold mincing solution. Immerse the mesh end of the device for approximately five to 10 seconds in the cold mincing solution to allow the tissue to thaw completely.

Completely depress the plunger until cells are seen extruding through the mesh pores. Then pull the plunger up approximately 2.5 centimeters and press down again completely. Repeat several times, until the mincing solution becomes turbid.

To prepare slides, mix a cell suspension gently, and transfer 50 microliters to a tube containing 450 microliters of 0.5%low melting point agarose at 37 degrees Celsius. Prepare and score the slides as outlined in the text protocol. In the first study, liver was harvested from two cohorts of male Sprague Dawley vehicle control rats staggered by one week.

Slides were prepared from freshly minced tissue, frozen minced tissue, and frozen cubed tissue. Since the OECD recommended upper limit for percent tail DNA in fresh rat liver is 6%these results demonstrate that any of the freezing methods can work well for evaluating tissue. Overall, the percent hedgehogs paralleled the percent tail DNA, although frozen tissues exhibited greater variability.

In the second study, female B6C3F1 mice were administered vehicle or the mutagen ethyl methanesulfonate. The percent tail DNA results indicate that liver tissue frozen as cubes and subsequently processed using the tissue mincing device yielded results very similar to those obtained for freshly minced tissue. A statistically significant increase in EMS induced DNA damage was measured in tissue samples processed by all three methods.

In the third study, liver samples collected from female B6C3F1 mice in a 90 day chemical toxicity study were analyzed. DNA damage in frozen minced tissue was high across all the dose groups, with substantial animal to animal variability. The percent tail DNA results correlated with percent hedgehogs, indicating that hedgehogs contributed substantially to the high level of DNA damage that was observed in these samples.

In contrast, both DNA damaged and percent hedgehogs were very low in the flash frozen cubed tissue that had been prepared in parallel with the minced tissue samples. Thus, the highly damaged cells manifested as hedgehogs in the minced tissue samples were clearly not the result of a biological process, but were likely introduced by mechanical disruption, and, or tissue warming during the mincing procedure prior to being flash frozen. Therefore, the data for minced tissue were considered to be unreliable.

By comparison, high quality data were obtained from the frozen cubed tissue, even after prolonged storage. It is critical to keep harvested tissues cold and moist, and to work quickly to freeze the samples. Also, frozen cube tissue should be thawed immediately prior to the homogenization step.

Frozen tissue samples are suitable for other downstream applications, including for example, modifications of the comet assay to detect oxidative damage or cross-linking, and evaluation of changes in gene expression. Caution should be exercised while handling scalpel blades to cut or scrape tissues, and when handling liquid nitrogen during the freezing process.