A RAPID METHOD FOR QUANTITATING THE EXTENT OF DNA DAMAGE INDUCED BY SULFUR MUSTARD IN HUMAN LYMPHOCYTES FOR DRUG SCREENING

We previously characterized the effects of sulfur mustard on deoxyribonucleic acid (DNA) patterns in exposed human lymphocytes and demonstrated how poly (ADP-ribose) polymerase inhibitors (PARPI) alter these effects. These studies were conducted utilizing labor-intensive and time-consuming DNA isolation andgelelectrophoresis procedures.Toconduct mechanistic studiesandto screen antivesicant therapeutic regimens for their capacity to block or alter the DNAdamaging effects of sulfur mustard, a faster and less labor-intensive method was developed. Preparations of human lymphocytes, isolated from the blood of normal volunteers, were exposed to sulfur mustard (1 × 10 -8 M to 1 × 10 -3 M) andincubatedat 37 o C for 0-24h.The effects of sulfur mustard on the DNA of the lymphocytes were determinedusing flow cytometry by measuring theincrease in the fluorescence of the DNA peak caused by the uptake of propidium iodide (PI) by the fragmented DNA. The increase in the fluorescence of the DNA depended on both the concentration of sulfur mustard to which the cells were exposed and the lengthof time following exposure to sulfur mustard. Anincrease inthe binding of PI to the sulfur mustard-exposed lymphocyte DNA is detected as early as 1hpostexposure.Theincrease inPIfluorescencerose sharplyduring thefirst 4 h and then appeared to increase linearly between 4 and 24 h after sulfur mustard exposure. This method results in a more rapid and less labor-intensive determination of DNA damage, enabling kinetic and mechanistic determination of DNA damage.