Cloutier J F, Drouin R, Castonguay A
Laboratory of Cancer Etiology and Chemoprevention, Faculty of Pharmacy, Laval University, Quebec City, Québec G1K 7P4, Canada.
Chem Res Toxicol. 1999 Sep;12(9):840-9. doi: 10.1021/tx990025f.
The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) present in tobacco smoke is a major carcinogen involved in tobacco-induced lung cancer. Its complex bioactivation along two pathways, which leads to methylation and pyridyloxobutylation of DNA, makes the study of NNK-induced DNA damage difficult. We selected two nitroso compounds, N-methyl-N-nitrosourea (MNU) and N-nitroso(acetoxymethyl)methylamine (NDMAOAc), with which to map NNK-induced DNA methylation frequency at every nucleotide position. We address the issue of how sequence context and complex chromatin structures, present in living cells, regulate the formation of modified purines through methylation generated by MNU and NDMAOAc. For comparison purposes, purified DNA was treated with dimethyl sulfate (DMS). We used ligation-mediated polymerase chain reaction to map and conduct a high-resolution footprinting analysis of the DNA damage along the p53 gene (exons 5-8), the ras gene family (exons 1 and 2 of H-, K-, and N-ras genes), and the c-jun promoter in living cells. The distribution of piperidine-sensitive DNA damage induced in cellular DNA and purified DNA by MNU or NDMAOAc was identical. MNU and NDMAOAc methylate more frequently the central guanines in a run of guanines, suggesting a regioselective mechanism for DNA methylation. In contrast, DMS methylates more frequently guanines at the 5'-end of a guanine run; this frequency decreased from the 5'- to the 3'-end. While the presence of adenines in a guanine run does not affect the distribution pattern, the presence of pyrimidines does change said pattern. Our data lead us to suggest that NNK would also methylate DNA sequences in a way similar to that of MNU or NDMAOAc. Footprinted areas of DNA methylated with MNU or NDMAOAc correspond to a consensus sequence for transcription factors AP-1, NF-Jun, CCAAT box, SP-1, and RSRF, as observed in c-jun promoters. Our results are in line with the fact that NNK metabolites, generated through the alpha-hydroxylation pathways, could potentially be mutagenic, since these activated metabolites can methylate guanines. In p53 and ras genes, the frequency of methylation of guanines parallels the frequency of mutations of those same guanines in lung cancer.
烟草烟雾中存在的亚硝胺4-(甲基亚硝氨基)-1-(3-吡啶基)-1-丁酮(NNK)是导致烟草诱导肺癌的主要致癌物。其沿着两条途径的复杂生物活化过程会导致DNA甲基化和吡啶氧基丁基化，这使得研究NNK诱导的DNA损伤变得困难。我们选择了两种亚硝基化合物，N-甲基-N-亚硝基脲(MNU)和N-亚硝基(乙酰氧基甲基)甲胺(NDMAOAc)，用于绘制NNK在每个核苷酸位置诱导的DNA甲基化频率。我们探讨了活细胞中存在的序列背景和复杂染色质结构如何通过MNU和NDMAOAc产生的甲基化来调节修饰嘌呤的形成。为了进行比较，用硫酸二甲酯(DMS)处理纯化的DNA。我们使用连接介导的聚合酶链反应来绘制并对活细胞中p53基因(外显子5-8)、ras基因家族(H-、K-和N-ras基因的外显子1和2)以及c-jun启动子的DNA损伤进行高分辨率足迹分析。MNU或NDMAOAc在细胞DNA和纯化DNA中诱导的对哌啶敏感的DNA损伤分布是相同的。MNU和NDMAOAc更频繁地使鸟嘌呤序列中的中央鸟嘌呤甲基化，这表明DNA甲基化存在区域选择性机制。相比之下，DMS更频繁地使鸟嘌呤序列5'端的鸟嘌呤甲基化；这种频率从5'端到3'端降低。虽然鸟嘌呤序列中腺嘌呤的存在不影响分布模式，但嘧啶的存在确实会改变该模式。我们的数据使我们推测，NNK也会以类似于MNU或NDMAOAc的方式使DNA序列甲基化。如在c-jun启动子中观察到的，用MNU或NDMAOAc甲基化的DNA足迹区域对应于转录因子AP-1、NF-Jun、CCAAT框、SP-1和RSRF的共有序列。我们的结果与以下事实一致，即通过α-羟基化途径产生的NNK代谢物可能具有致突变性，因为这些活化的代谢物可以使鸟嘌呤甲基化。在p53和ras基因中，鸟嘌呤的甲基化频率与肺癌中相同鸟嘌呤位点的突变频率平行。
