Abstract: Sulfur mustard (HD) is a vesicating agent that alkylates cellular DNA and produces DNA strand breaks. The comet assay (single-cell gel electrophoresis) was used to detect HD-induced DNA single strand breaks in human peripheral blood lymphocytes (PBL). Specific steps taken to perform the comet assay in our laboratory are outlined. Briefly, PBL were mixed with agarose, mounted on a microscope slide, and lysed. The cellular DNA was treated with an alkaline buffer for denaturation, electrophoresed, and stained with ethidium bromide for visualization. Under a fluorescence microscope, cells with undamaged DNA appeared as intact comet heads without tails. Damaged DNA with single-strand breaks migrated in the direction of the anode, and this produced the appearance of a comet. The amount of DNA that migrates is proportional to the number of DNA single-strand breaks, as determined in past studies. Using an image analysis system, relative amounts of DNA damage were obtained by measuring the length of the comet tail and the intensity of fluorescence in the tail. The comet assay has the capacity to measure the ability of a compound to increase the rate of DNA repair in HD-damaged cells. Thus, it may potentially be used for rapid in vitro screening of antivesicant compounds.
| Limitations: |
 APPROVED FOR PUBLIC RELEASE |
| Description: |
Rept. for 26 Jun 97-16 Dec 98 |
| Pages: |
27 |
| Report Date: |
JUL 1999 |
| Report Number: |
A895663 |
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