Mark J. Eliason and Dr. Kim O’Neill, Microbiology
The single-cell gel electrophoresis assay or “Comet Assay” is a novel test which can be used to empirically ascertain the degree of damage sustained by the DNA of individual cells when exposed to cytotoxic chemicals. In this experiment a known cell line was grown in varying concentrations of grape seed extract (GSE), a reputed antioxidant, and then exposed to hydrogen peroxide, an oxidant, known to cause breaks in the DNA. When the comet assay was subsequently run the effect that the varying concentrations of grape seed extract had in protecting the cells from damage could be quantified.
To determine the concentrations of grape seed extract (GSE) that the cells would be exposed to, growth curves were created by growing the cells in different concentrations of GSE over several days and recording the growth (the number of cells/ml) and viability (the percentage of living cells divided by the total number of the cells ) at 12 hour intervals for 48 hours. Comparing the viability and growth of the different concentrations with a control grown without GSE established that the highest concentration of GSE these cells could be exposed to was 10)g/ml. Based on this data, 2.5)g/ml, 5)g/ml and 10)g/ml were selected as the three concentrations to be used in this experiment.
One problem encountered as the growth curves were established was the maintenance of high viability for the experiment. The Raji cell line used in the experiment needed to have a viability of at least 84% for the data collected to be accepted as reliable. After several unsuccessful attempts to raise the viability, it was determined that the use of an alternate cell line might be able to sidestep this problem. HL-60R’s were opted for as it was quite easy to maintain their viability at around 96-97%.
The cells were prepared the day before the experiment by removing them from the nutritional media they grew in and adding the appropriate amount of GSE to create the desired concentration. The cells were then put back in the incubator for 12 hours to allow time for the uptake of the GSE into the cells. The media was washed from the cells, and they were exposed to hydrogen peroxide for 10 minutes. The hydrogen peroxide was washed from the cells, and they were imbedded in an agarose gel which was then affixed to a glass slide. By exposure to a lysis solution, the cell membranes and other cellular debris were removed from the cell leaving only the DNA. The DNA was treated with propidium iodide, a compound which inserts itself into the DNA strand and emits light when placed in contact with fluorescent light. When the slides with the prepared DNA were then examined under a fluorescent microscope, light given off by the treated DNA would allow a computer hooked up to the microscope to project that image onto a monitor. To examine the degree of damage the DNA sustained an electric current (10 volts for 20 minutes) was run through the gel. As the fragmented strands of DNA have a negative charge they would migrate toward the positively charged anode, straightening out in the process and form a characteristic shape which can be examined with a computer to assess the damage done to the DNA.
The greater the DNA has been oxidized the smaller the fragments of DNA will be. Smaller molecules can move with greater ease through the gel, and therefore will appear to have moved farther from the “head” of the DNA. A comet-like shape generally appears. As the smaller pieces move the farthest, forming the tip of the “tail,” with the rest of the comet formed by the intermediate sizes. From each slide, the first fifty “comets” that appeared were selected for analysis. The computer measured the length of each comet’s tail and the relative amount of DNA comprising both the head and tail and used this data to assign a relative value to each of the comets.
As this data was compiled, a discrepancy was noticed. When the assay was repeated and the tail values were compared, the results did not show a reliable reproduction. The HL-60R cells were later understood to have a mutation which enabled them to rapidly repair oxidative damage, thus interfering with the analysis of the protection afforded the cells by the GSE. The experiments were repeated using the Raji cells as had originally been planned. (The difficulty in maintaining acceptable viability had been resolved.) Raji were used and after several weeks of assays the data obtained from the slides was compiled.
With p value set at .05 the data showed that there was a statistically significant difference in the length of the tail of the negative controls (cells that were not grown in GSE) and the cells grown in 2.5)g/ml of GSE. Based on the length of the tails the 5)g/ml and the 10)g/ml concentrations also showed significant decrease in tail moments. There was no statistically significant difference in the length of the tails of the 2.5)g/ml and 5)g/ml concentrations which was contrary to the assumption that a linear relationship would be seen. In light of the discrepancy between the results of the different cell lines it would be prudent to repeat the experiment using several other cell lines before any definite conclusions are drawn. However, the data obtained from the experiment seems to support the hypothesis that GSE does in fact afford cells an appreciable protection from at least some kinds of oxidative damage.