Technical University of Denmark, National Food Institute, Søborg, Denmark.
Mutat Res. 2012 Dec 12;749(1-2):70-5. doi: 10.1016/j.mrgentox.2012.07.003. Epub 2012 Sep 5.
The impact on the sensitivity of the in vitro comet assay by increasing the number of cells scored has only been addressed in a few studies. The present study investigated whether the sensitivity of the assay could be improved by scoring more than 100 cells. Two cell lines and three different chemicals were used: Caco-2 cells were exposed to ethylmethane sulfonate and hydrogen peroxide in three concentrations, and HepG2 cells were exposed to ethylmethane sulfonate, hydrogen peroxide and benzo[a]pyrene in up to four concentrations, in four to five independent experiments. The scoring was carried out by means of a fully automated scoring system and the results were analyzed by evaluating the % tail DNA of 100-700 randomly selected cells for each slide consisting of two gels. By increasing the number of cells scored, the coefficients of variance decreased, leading to an improved sensitivity of the assay. A two-way ANOVA analysis of variance showed that the contribution from the two variables "the number of cells scored" and "concentration" on the total variation in the coefficients of variance dataset was statistically significant (p<0.05). The increase in sensitivity was demonstrated by the possibility to detect an increase in % tail DNA with statistical significance at lower concentrations. The results indicated that for low levels of DNA damage, below 9% tail DNA, scoring of 600 cells increased the sensitivity compared with scoring of 100 cells. For relatively low levels of DNA damage, about 9-16% tail DNA, scoring of 300 cells increased the sensitivity. Thus, the recommendation for the optimum number of cells scored would be 600 and 300 for low and relatively low levels of DNA damage, respectively. The findings from this study could be particularly important for bio-monitoring studies where small differences in DNA-damage levels could be relevant.
增加细胞评分数量对体外彗星试验灵敏度的影响仅在少数研究中得到探讨。本研究旨在探讨评分超过 100 个细胞是否可以提高试验的灵敏度。本研究使用了两种细胞系和三种不同的化学物质：将 Caco-2 细胞暴露于乙基甲磺酸酯和过氧化氢的三种浓度中，将 HepG2 细胞暴露于乙基甲磺酸酯、过氧化氢和苯并[a]芘的四种浓度中，进行了四到五次独立实验。通过使用全自动评分系统进行评分，对每个载玻片上的 100-700 个随机选择的细胞的%尾部 DNA 进行分析，每个载玻片由两个凝胶组成。通过增加评分的细胞数量，变异系数降低，从而提高了试验的灵敏度。方差双向 ANOVA 分析显示，“评分的细胞数量”和“浓度”这两个变量对变异系数数据集总变异的贡献具有统计学意义（p<0.05）。通过在较低浓度下可以检测到具有统计学意义的%尾部 DNA 增加，证明了灵敏度的提高。结果表明，对于低于 9%尾部 DNA 的低水平 DNA 损伤，与评分 100 个细胞相比，评分 600 个细胞可提高灵敏度。对于相对较低水平的 DNA 损伤，约 9-16%尾部 DNA，评分 300 个细胞可提高灵敏度。因此，建议对于低水平和相对较低水平的 DNA 损伤，评分的最佳细胞数量分别为 600 个和 300 个。本研究的结果对于生物监测研究可能特别重要，因为 DNA 损伤水平的微小差异可能是相关的。
