Department of Applied Science, Hong Kong Institute of Vocational Education (Shatin), Sha Tin, New Territories, Hong Kong.
Hum Exp Toxicol. 2012 Nov;31(11):1144-50. doi: 10.1177/0960327112446816. Epub 2012 May 31.
The comet assay is a well-established, simple and sensitive method to measure DNA damage in single cell and is commonly used in human trials to investigate the effects of pollution, occupational hazards and potential genoprotective agents. Peripheral blood lymphocytes are most commonly used in human biomonitoring studies, but lymphocytes collected from the mouth offer a potentially attractive, noninvasive alternative. The aim of the current study was to develop a buccal cell lymphocyte comet assay procedure. Cells were collected from mouthwash of three healthy volunteers and tested individually. The comet assay was performed under different pH and times of alkaline treatment, electrophoresis run times and hydrogen peroxide concentrations. Optimal conditions for buccal lymphocytes in comet assay were found to be pH >13 for unwinding and electrophoresis buffers, 10-min alkaline unwinding treatment and 20-min electrophoresis run time. We successfully utilized our optimized assay conditions to demonstrate the genoprotective activity of quercetin. This newly established procedure offers an alternative noninvasive sampling method for the investigation of DNA protection and/or damaging effect.
彗星试验是一种成熟、简单和灵敏的方法，可用于测量单细胞中的 DNA 损伤，常用于人体试验中，以研究污染、职业危害和潜在的遗传保护剂的影响。外周血淋巴细胞最常用于人体生物监测研究，但从口腔采集的淋巴细胞提供了一种有吸引力的非侵入性替代方法。本研究旨在开发一种口腔细胞淋巴细胞彗星试验程序。从 3 名健康志愿者的漱口液中收集细胞并单独进行测试。在不同的 pH 值和碱性处理时间、电泳运行时间和过氧化氢浓度下进行彗星试验。发现彗星试验中口腔淋巴细胞的最佳条件为：解旋和电泳缓冲液的 pH 值>13、碱性解旋处理 10 分钟和电泳运行时间 20 分钟。我们成功地利用优化的试验条件证明了槲皮素的遗传保护活性。新建立的程序为研究 DNA 保护和/或损伤效应提供了一种替代的非侵入性采样方法。
