Gontijo A M, Elias F N, Salvadori D M, de Oliveira M L, Correa L A, Goldberg J, Trindade J C, de Camargo J L
Departamentos de Patologia, Faculdade de Medicina, Universidade Estadual Paulista, 18618-000, Botucatu, São Paulo, Brazil.
Cancer Epidemiol Biomarkers Prev. 2001 Sep;10(9):987-93.
A protocol for DNA damage assessment by the single-cell gel (SCG)/comet assay in human urinary bladder washing cells was established. Modifications of the standard alkaline protocol included an increase to 2% of sodium sarcosinate in the lysis solution, a reduction in the glass-slide area for comet analysis, and a cutoff value for comet head diameter of at least 30 microm, to exclude contaminating leukocytes. Distinguishing cell populations is crucial, because significant differential migration was demonstrated for transitional and nontransitional cells, phenomena that may confound the results. When applying the modified protocol to urinary bladder cells from smokers without urinary bladder neoplasia, it was possible to detect a significant (P = 0.03) increase in DNA damage as depicted by the tail moment (6.39 +/- 3.23; mean +/- 95% confidence interval; n = 18) when compared with nonsmokers (1.94 +/- 1.41; n = 12). No significant differences were observed between ex-smokers and current smokers regarding comet parameters. Inflammation was not a confounding factor, but DNA migration increased significantly with age in nonsmokers (r = 0.68; P = 0.014). Thus, age matching should be a concern when transitional cells are analyzed in the SCG assay. As it is well known, DNA damage may trigger genomic instability, a crucial step in carcinogenesis. Therefore, the present data directly support the classification of individuals with smoking history as patients at high risk for urinary bladder cancer.
建立了一种用于评估人膀胱冲洗细胞中DNA损伤的单细胞凝胶（SCG）/彗星试验方案。对标准碱性方案的修改包括：裂解液中肌氨酸钠的浓度增加到2%；彗星分析的载玻片面积减小；彗星头部直径的截断值至少为30微米，以排除污染的白细胞。区分细胞群体至关重要，因为已证实移行细胞和非移行细胞存在显著的差异迁移现象，这可能会混淆结果。将修改后的方案应用于无膀胱肿瘤的吸烟者的膀胱细胞时，与不吸烟者（1.94±1.41；n = 12）相比，尾矩（6.39±3.23；平均值±95%置信区间；n = 18）所显示的DNA损伤显著增加（P = 0.03）。在彗星参数方面，戒烟者和现吸烟者之间未观察到显著差异。炎症不是一个混杂因素，但在不吸烟者中，DNA迁移随年龄显著增加（r = 0.68；P = 0.014）。因此，在SCG试验中分析移行细胞时，年龄匹配应予以关注。众所周知，DNA损伤可能引发基因组不稳定，这是致癌过程中的关键一步。因此，本数据直接支持将有吸烟史的个体归类为膀胱癌高危患者。
