Coller H A, Khrapko K, Torres A, Frampton M W, Utell M J, Thilly W G
Division of Toxicology and Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge 02139, USA.
Cancer Res. 1998 Mar 15;58(6):1268-77.
Seventeen separate mitochondrial hot spot mutations in a 100-bp target sequence (mitochondrial bp 10,030-10,130) were detected and measured in bronchial epithelial cell samples isolated from smokers and nonsmokers. Among the individuals sampled were three pairs of monozygotic twins in which one twin had never smoked and had a nonsmoking spouse, and the other had a smoking history of >10 pack-years. Individual point mutations present at frequencies as low as 10(-6) were detected. Partially denaturing electrophoresis was used to separate mutant from nonmutant sequences on the basis of their melting temperatures, and the target sequence was subsequently amplified via high-fidelity PCR with Pfu DNA polymerase. Tests were performed to determine whether mismatch intermediates or DNA adducts present in the cellular DNA were converted to mutants during PCR. Hot spot mutations were clearly observed in bronchial epithelial cells, and the same hot spots were observed consistently in different samples. Significant numerical variability in the mutant fractions for individual mutants was observed among samples and are ascribed to unequal mitochondrial segregation in stem and transition cells. The mutational spectra in smokers' samples did not differ significantly from the mutational spectra in nonsmokers' samples for this 100 bp of mitochondrial DNA. No smoking-specific hot spots were detected. The overall mutant fractions in smokers' samples were not elevated compared to those of nonsmokers. As much variability was observed between two samples from the same individual's lung as between a sample from a smoker and a sample from a nonsmoker. These findings demonstrate that inhaled tobacco smoke does not induce prominent point mutations in this 100-bp target mitochondrial sequence in smokers' bronchial epithelial cells. Endogenous factors (e.g., DNA replication errors or DNA damage by endogenous reactive chemicals) are suggested to be more likely to represent the most important contributors to mitochondrial mutagenesis.
在从吸烟者和非吸烟者分离出的支气管上皮细胞样本中，检测并测量了一个100bp靶序列（线粒体碱基对10,030 - 10,130）中的17个独立的线粒体热点突变。采样的个体中有三对同卵双胞胎，其中一对双胞胎从不吸烟且配偶不吸烟，另一对有超过10包年的吸烟史。检测到频率低至10(-6)的个别点突变。部分变性电泳用于根据突变序列和非突变序列的解链温度分离它们，随后使用Pfu DNA聚合酶通过高保真PCR扩增靶序列。进行测试以确定细胞DNA中存在的错配中间体或DNA加合物在PCR过程中是否转化为突变体。在支气管上皮细胞中清楚地观察到热点突变，并且在不同样本中一致地观察到相同的热点。在样本中观察到个别突变体的突变分数存在显著的数值变异性，这归因于干细胞和过渡细胞中线粒体的不均等分离。对于这100bp的线粒体DNA，吸烟者样本中的突变谱与非吸烟者样本中的突变谱没有显著差异。未检测到吸烟特异性热点。与非吸烟者相比，吸烟者样本中的总体突变分数没有升高。同一个人肺部的两个样本之间观察到的变异性与吸烟者样本和非吸烟者样本之间的变异性一样大。这些发现表明，吸入的烟草烟雾不会在吸烟者支气管上皮细胞的这个100bp靶线粒体序列中诱导明显的点突变。内源性因素（例如，DNA复制错误或内源性反应性化学物质引起的DNA损伤）被认为更有可能是线粒体诱变的最重要因素。
