Matijasevic Z, Precopio M L, Snyder J E, Ludlum D B
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655-0126, USA.
Carcinogenesis. 2001 Apr;22(4):661-4. doi: 10.1093/carcin/22.4.661.
DNA damage is thought to be the initial event that causes sulfur mustard (SM) 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 either SM or its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES). Damaged plasmids were transfected into wild-type and nucleotide excision repair (NER) deficient Chinese hamster ovary cells; these cells were also transfected with a second reporter plasmid containing RENILLA: luciferase as an internal control on the efficiency of transfection. Transfected cells were incubated at 37 degrees C for 27 h and then both firefly and RENILLA: luciferase intensities were measured on the same samples with the dual luciferase reporter assay. Bioluminescence in lysates from cells transfected with damaged plasmid, expressed as a percentage of the bioluminescence from cells transfected with undamaged plasmid, is increased by host cell repair activity. The results show that NER-competent cells have a higher reactivation capacity than NER-deficient cells for plasmids damaged by either SM or CEES. Significantly, NER-competent cells are also more resistant to the toxic effects of SM and CEES, 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 mechanisms protect cells from mustard toxicity and under what conditions these mechanisms are most effective.
DNA损伤被认为是导致硫芥（SM）毒性的初始事件，而细胞修复这种损伤的能力被认为提供了一定程度的天然保护。为了研究修复过程，我们用SM或其单功能类似物2-氯乙基乙基硫醚（CEES）损伤了含有萤火虫荧光素酶基因的质粒。将受损质粒转染到野生型和核苷酸切除修复（NER）缺陷的中国仓鼠卵巢细胞中；这些细胞还被转染了第二个报告质粒，该质粒含有海肾荧光素酶作为转染效率的内部对照。将转染后的细胞在37℃下孵育27小时，然后用双荧光素酶报告基因检测法在相同样品上测量萤火虫荧光素酶和海肾荧光素酶的强度。宿主细胞的修复活性会使转染受损质粒的细胞裂解物中的生物发光增加，以未受损质粒转染细胞的生物发光百分比表示。结果表明，对于被SM或CEES损伤的质粒，具有NER能力的细胞比NER缺陷细胞具有更高的再激活能力。值得注意的是，具有NER能力的细胞对SM和CEES的毒性作用也更具抗性，这表明NER不仅擅长修复由这两种试剂引起的DNA损伤，还能降低它们的毒性。这种宿主细胞修复检测现在可用于确定还有哪些细胞机制能保护细胞免受芥子气毒性的影响，以及这些机制在何种条件下最有效。
