Woods Julie A, Dunne Colum, Collins J Kevin, Shanahan Fergus, O'Brien Nora M
Department of Food Science, Food Technology, and Nutrition, University College, Cork, Republic of Ireland.
Nutr Cancer. 2002;42(1):62-9. doi: 10.1207/S15327914NC421_9.
The alkaline single-cell gel electrophoresis (comet) assay was used to investigate the genotoxicity of fecal water (FW) isolated from 47 Irish subjects using Caco-2 colonocytes as target cells. Two methods of comet assay analysis were compared to determine the extent of DNA damage and to categorize the samples as having no, low-to-moderate, or high genotoxicity. FW was isolated from stool samples by centrifugation and tested for its ability to induce DNA damage in Caco-2 cells. DNA damage was assessed using the comet assay by measuring the extent of DNA migration from the nucleus (microns, tail length) or by classifying the nuclei into five different categories depending on their morphology. Data collected from the two methods were used to categorize the FW samples on the basis of their genotoxic activity. Both methods showed good agreement. There was an approximately 50:50 split, with half the samples having some level of genotoxic activity and half having no genotoxicity. About one-third of the samples were considered to be highly genotoxic. There was a trend for low pH of the FW to be associated with increased DNA damage, but this was not significant. The results presented in this report show a relatively high incidence of genotoxic FW in samples derived from a free-living Irish population. Our data demonstrate the suitability of classifying nuclei on the basis of their morphology as a means of determining DNA damage. This procedure is very rapid and, therefore, advantageous in analyzing a large number of slides in the absence of an image analysis system.
采用碱性单细胞凝胶电泳（彗星试验），以Caco - 2结肠癌细胞作为靶细胞，研究了从47名爱尔兰受试者分离出的粪便水（FW）的遗传毒性。比较了两种彗星试验分析方法，以确定DNA损伤程度，并将样本分类为无、低至中度或高遗传毒性。通过离心从粪便样本中分离出FW，并检测其在Caco - 2细胞中诱导DNA损伤的能力。使用彗星试验通过测量DNA从细胞核迁移的程度（微米，尾长）或根据细胞核形态将其分为五个不同类别来评估DNA损伤。从这两种方法收集的数据用于根据FW样本的遗传毒性活性对其进行分类。两种方法显示出良好的一致性。样本大致呈50:50分布，一半样本具有一定程度的遗传毒性活性，另一半没有遗传毒性。约三分之一的样本被认为具有高遗传毒性。FW的低pH值有与DNA损伤增加相关的趋势，但不显著。本报告中的结果表明，在来自自由生活的爱尔兰人群的样本中，具有遗传毒性的FW发生率相对较高。我们的数据证明了根据细胞核形态对其进行分类作为确定DNA损伤方法的适用性。该程序非常快速，因此在没有图像分析系统的情况下分析大量载玻片时具有优势。
