Vock E H, Vamvakas S, Gahlmann R, Lutz W K
Department of Toxicology, University of Würzburg, Germany.
Toxicol Sci. 1998 Nov;46(1):83-9. doi: 10.1006/toxs.1998.2553.
The question was addressed whether methylenediphenyl-4,4'-diisocyanate (MDI), a bifunctional electrophile, can induce DNA double-strand breaks (DSB) by repair of interstrand DNA crosslinks or whether DSB are the result of cell death. Cultured human lung epithelial cells (A549) were treated with MDI, methylene-4,4'-dianiline (MDA; a potential hydrolysis product of MDI), the nitrogen mustard melphalan, and the detergent Triton X-100. All chemicals were dissolved in ethylene glycol dimethyl ether which was added to a cell monolayer covered with phosphate-buffered saline. After 2 h, the treatment solution was exchanged against medium, and 8, 24, and 72 h after treatment initiation, the induction of DNA double-strand breaks was assessed by pulsed-field gel electrophoresis. At the same time, the viability was determined with the MTT test (intracellular reduction of the tetrazolium dye MTT). At the 8-h time point, 1 and 10 microM melphalan induced DSB without concomitant effect on cell viability. With all other chemicals, the dose-response curves for DNA fragmentation and viability were mirror images. Approximate 50% lethal concentrations were 200, 3000, and 100 microM for MDI, MDA, and Triton X-100, respectively. For these chemicals, the observed DSB were the consequence of extragenomic damage in the course of cell death rather than of an interaction with DNA. The mechanistic difference of melphalan was supported by analysis of nuclear morphology. Apoptotic bodies were observed only after melphalan treatment, whereas MDI and Triton X-100 produced only irregular clumping of chromatin (72-h time point). DNA fragment length analysis showed a time-independent pattern, with sizes between 1 and 4 Mbp for melphalan, while MDI, and Triton X-100 induced smaller DNA fragments in a time-dependent manner. It is concluded that DSB observed in cells treated with MDI are unlikely the result of DNA crosslink formation.
问题在于，双功能亲电试剂亚甲基二苯基-4,4'-二异氰酸酯（MDI）是否能通过修复链间DNA交联来诱导DNA双链断裂（DSB），或者DSB是否是细胞死亡的结果。用MDI、亚甲基-4,4'-二苯胺（MDA；MDI的一种潜在水解产物）、氮芥美法仑和去污剂曲拉通X-100处理培养的人肺上皮细胞（A549）。所有化学物质都溶解在乙二醇二甲醚中，然后加入覆盖有磷酸盐缓冲盐水的细胞单层中。2小时后，将处理溶液换成培养基，并在处理开始后的8、24和72小时，通过脉冲场凝胶电泳评估DNA双链断裂的诱导情况。同时，用MTT试验（四唑盐染料MTT的细胞内还原）测定细胞活力。在8小时时间点，1和10微摩尔美法仑诱导了DSB，同时对细胞活力没有伴随影响。对于所有其他化学物质，DNA片段化和活力的剂量反应曲线是镜像关系。MDI、MDA和曲拉通X-100的近似50%致死浓度分别为200、3000和100微摩尔。对于这些化学物质，观察到的DSB是细胞死亡过程中基因组外损伤的结果，而不是与DNA相互作用的结果。核形态分析支持了美法仑的机制差异。仅在美法仑处理后观察到凋亡小体，而MDI和曲拉通X-100仅产生不规则的染色质聚集（72小时时间点）。DNA片段长度分析显示出一种与时间无关的模式，美法仑处理后的片段大小在1至4兆碱基之间，而MDI和曲拉通X-100以时间依赖性方式诱导产生较小的DNA片段。得出的结论是，在用MDI处理的细胞中观察到的DSB不太可能是DNA交联形成的结果。
