A P-32 POSTLABELING METHOD FOR THE DETECTION OF ADDUCTS IN THE DNA OFHUMAN FIBROBLASTS EXPOSED TO SULFUR MUSTARD

Since the toxicities of sulfur mustard are attributed to DNA alkylatio n, levels of DNA modification in exposed cells should correlate with t he intensity of exposure. We have found that P-32-postlabeling can be used successfully to detect the major adduct, 7-hydroxyethylthioethyld eoxyguanosine 5'-phosphate (HETEpdG), that is formed in DNA by sulfur mustard. This method has been used to establish a correlation between exposure and adduct formation in human fibroblasts grown in cell cultu re and exposed to sulfur mustard concentrations between 2.5 and 15 mu M. DNA was recovered from these cells using a salt precipitation metho d to remove proteins and was found to have an HETEpdG content which in creased linearly with SM concentration. This relationship shows that o ne HETEpdG per 10(6) nucleotides is produced at a SM concentration of 2.3 mu M. Growth of fibroblast cells, assayed by trypan-blue exclusion , is somewhat inhibited by 2 mu M SM, indicating that P-32-postlabelin g has the requisite sensitivity to detect adducts at levels of SM that are minimally toxic.