Repair of sulfur mustard-induced DNA damage in mammalian cells measured bya host cell reactivation assay

DNA damage is thought to be the initial event that causes sulfur mustard (S M) toxicity, while the ability of cells to repair this damage is thought to provide a degree of natural protection. To investigate the repair process, we have damaged plasmids containing the firefly luciferase gene with eithe r SM or its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES), Dama ged plasmids were transfected into wild-type and nucleotide excision repair (NER) deficient Chinese hamster ovary cells; these cells were also transfe cted with a second reporter plasmid containing Renilla luciferase as an int ernal control on the efficiency of transfection. Transfected cells were inc ubated at 37 degreesC for 27 h and then both firefly and Renilla luciferase intensities were measured on the same samples with the dual luciferase rep orter assay. Bioluminescence in lysates from cells transfected with damaged plasmid, expressed as a percentage of the bioluminescence from cells trans fected with undamaged plasmid, is increased by host cell repair activity. T he results show that NER-competent cells have a higher reactivation capacit y than NER-deficient cells for plasmids damaged by either SM or GEES. Signi ficantly, NER-competent cells are also more resistant to the toxic effects of SM and GEES, indicating that NER is not only proficient in repairing DNA damage caused by either agent but also in decreasing their toxicity. This host cell repair assay can now be used to determine what other cellular mec hanisms protect cells from mustard toxicity and under what conditions these mechanisms are most effective.