Lachapelle M, Marion M, Krzystyniak K, Fournier M, Denizeau F
Department of Biological Sciences, University of Quebec at Montreal, Quebec, Canada.
J Toxicol Environ Health. 1994 Dec;43(4):441-51. doi: 10.1080/15287399409531933.
The localization of DNA and RNA adducts was studied at the ultrastructural level using antibodies directed against O6-metG and the protein A-gold technique. Primary rat hepatocyte cultures were exposed for 2-24 h to 5 mM N-nitrosodimethylamine (NDMA) or 0.1 mM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In both cases, the O6-metG immunoreactive sites were concentrated in the nucleus and in the rough endoplasmic reticulum (RER) rich cytoplasmic regions. The highest gold labeling density measured was observed at 2 h of NDMA or MNNG treatment. However, after a 24-h exposure, very little labeling was observed in both the nuclear and the cytoplasmic compartments. The rate of disappearance of immunoreactive sites was faster in the cytoplasm than in the nucleus, Untreated control preparations showed no specific immunogold labeling. Furthermore, when cells were exposed first to NDMA and MNNG for a few hours and then to culture medium containing no genotoxin, and subsequently were reexposed to NDMA or MNNG for a few hours, very little labeling of both the nuclear and cytoplasmic compartments was observed. Control preparations without a second genotoxin exposure showed a normal labeling pattern. Control preparations without genotoxin showed no gold labeling. Our results provide evidence for the existence of a cytoplasmic O6-metG repair mechanism that behaves like its nuclear counterpart.
利用针对O6-甲基鸟嘌呤（O6-metG）的抗体和蛋白A-金技术，在超微结构水平上研究了DNA和RNA加合物的定位。将原代大鼠肝细胞培养物暴露于5 mM N-亚硝基二甲胺（NDMA）或0.1 mM N-甲基-N'-硝基-N-亚硝基胍（MNNG）中2至24小时。在这两种情况下，O6-metG免疫反应位点都集中在细胞核和富含粗面内质网（RER）的细胞质区域。在NDMA或MNNG处理2小时时观察到最高的金标记密度。然而，暴露24小时后，在细胞核和细胞质区室中均观察到很少的标记。免疫反应位点在细胞质中的消失速度比在细胞核中更快，未处理的对照制剂未显示特异性免疫金标记。此外，当细胞先暴露于NDMA和MNNG数小时，然后暴露于不含基因毒素的培养基中，随后再暴露于NDMA或MNNG数小时时，在细胞核和细胞质区室中均观察到很少的标记。未进行第二次基因毒素暴露的对照制剂显示出正常的标记模式。未含基因毒素的对照制剂未显示金标记。我们的结果为细胞质中存在一种类似于细胞核对应机制的O6-metG修复机制提供了证据。
