Cross-Linking Interferes with Assessing Sulfur Mustard-Induced DNA Damage in Human Peripheral Blood Lymphocytes Using the Comet Assay

Abstract

Sulfur mustard (SM) is a blistering agent that produces DNA strand breaks. To detect SM-induced DNA single strand breaks in human peripheral blood lymphocytes (PBL), cells were exposed to various concentrations of SM (10 to 1000 μM), and the comet assay (single-cell gel electrophoresis) was performed. We observed a SM concentration- and time-dependent increase in detectable DNA damage. To test whether SM-induced DNA cross-linking inhibits DNA migration in the comet assay, PBL were exposed to a) SM alone (10 to 1000 μM), b) H2O2 (0.001%), which produces DNA single strand breaks with no cross-links, or c) SM followed at 2, 4, or 6 h by H2O2. With H2O2 alone, a large amount of strand breakage was detected. With H2O2 plus SM, detectable H2O2-induced strand breaks decreased as SM concentration increased up to 30 μM; at 30 μM and above, the response with H2O2 plus SM was similar to that with SM alone. Interference with the detection of H2O2-induced DNA strand breaks appears to be SM concentration-dependent up to 30 μM, and independent of SM concentration at ≥30 μM. This is presumably due to SM-induced cross-linking. It follows that cross-linking in DNA of SM-exposed PBL also interferes with DNA migration and detection of DNA strand breaks when cells are exposed to SM alone.