Hoffmann Heike, Speit Günter
Universitätsklinikum Ulm, Abteilung Humangenetik, D-89070 Ulm, Germany.
Mutat Res. 2005 Mar 7;581(1-2):105-14. doi: 10.1016/j.mrgentox.2004.11.015. Epub 2005 Jan 12.
The comet assay (single-cell gel electrophoresis, SCG) is being increasingly used in human biomonitoring for the detection of genotoxic exposures. Cigarette smoking is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds. Therefore, smoking should represent a relevant mutagenic exposure and lead to genotoxic effects in exposed cells. However, our previous investigations as well as several other published studies on human biomonitoring failed to show an effect of smoking on DNA migration in the comet assay, while some other studies did indicate such an effect. Although many factors can contribute to the generation of discrepant results in such studies, clear effects should be obtained after high exposure. We therefore performed a comparative study with healthy male heavy smokers (>20 cigarettes per day) and non-smokers (n=12 in each group). We measured the baseline comet assay effects in fresh whole blood samples and isolated lymphocytes. In addition, the amount of 'formamidopyrimidine DNA-glycosylase (FPG)-sensitive sites' was determined by a combination of the standard comet assay with the bacterial FPG protein. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline DNA damage was comparatively analysed. Duplicate slides from each sample were processed and analysed separately. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. Finally, to compare the comet assay results with another genetic endpoint, all blood samples were investigated in parallel by the micronucleus test (MNT). Baseline and gamma radiation-induced micronucleus frequencies were determined. None of these approaches revealed a significant difference between heavy smokers and non-smokers with regard to a genotoxic effect in peripheral blood cells.
彗星试验（单细胞凝胶电泳，SCG）在人类生物监测中越来越多地用于检测遗传毒性暴露。吸烟是多种潜在诱变和致癌化合物的一个有充分文献记载的来源。因此，吸烟应代表一种相关的诱变暴露，并导致暴露细胞产生遗传毒性效应。然而，我们之前的调查以及其他一些关于人类生物监测的已发表研究未能显示吸烟对彗星试验中DNA迁移的影响，而其他一些研究确实表明有这种影响。尽管许多因素可能导致此类研究结果出现差异，但在高暴露后应能获得明显的效应。因此，我们对健康男性重度吸烟者（每天>20支香烟）和非吸烟者进行了一项比较研究（每组n = 12）。我们测量了新鲜全血样本和分离淋巴细胞中的彗星试验基线效应。此外，通过标准彗星试验与细菌FPG蛋白相结合的方法测定了“甲酰胺嘧啶DNA糖基化酶（FPG）敏感位点”的数量。此外，还比较分析了一种修复抑制剂（阿非迪霉素，APC）对基线DNA损伤的影响。对每个样本的重复玻片分别进行处理和分析。在所有实验中，均纳入了一个参考标准（未处理的V79细胞）以校正试验变异性。最后，为了将彗星试验结果与另一个遗传终点进行比较，通过微核试验（MNT）对所有血样进行了平行研究。测定了基线和γ辐射诱导的微核频率。这些方法均未显示重度吸烟者和非吸烟者在外周血细胞遗传毒性效应方面存在显著差异。
