The Institute of Environmental Toxicology, Joso-shi, Ibaraki, Japan.
Mutat Res. 2012 Feb 18;742(1-2):26-30. doi: 10.1016/j.mrgentox.2011.11.008. Epub 2011 Nov 28.
Conducting the single-cell gel electrophoresis (Comet) assay in the urinary bladders of rodents is technically problematic because the bladder is small and thin, which makes it difficult to collect its mucosal cells by scraping. We performed the Comet assay using a simple mincing method in which tissues are minced with scissors. We then compared data obtained with this method with data obtained using the scraping method. Sprague-Dawley rats of both sexes were orally given twice the known carcinogens N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), or o-anisidine (OA). Three hours after the second administration, the bladder of each rat was divided into two parts and each part was processed by either the mincing or the scraping method. Both mincing and scraping methods detected DNA damage in MNU-, EMS-, but not OA-treated rats, and thus the mincing method had a sufficient capability to detect DNA damaging agents. The morphological analysis of the prepared cell suspensions revealed that more than 80% of the cells collected by the mincing method were from the epithelium. Because the mincing method requires only one-half of a bladder, the other half remains intact and can be used for histopathological examination. We conclude that the mincing method is easier and more appropriate for the Comet assay in urinary bladder tissue than the scraping method.
在啮齿动物的膀胱中进行单细胞凝胶电泳（彗星）分析在技术上存在问题，因为膀胱体积小且壁薄，通过刮取很难收集其黏膜细胞。我们使用简单的切碎方法进行彗星分析，即用剪刀切碎组织。然后，我们将这种方法获得的数据与刮取法获得的数据进行了比较。雄性和雌性 Sprague-Dawley 大鼠经口给予已知致癌剂 N-甲基-N-亚硝脲（MNU）、乙磺酸乙酯（EMS）或邻茴香胺（OA）两次。第二次给药后 3 小时，将每只大鼠的膀胱分为两部分，每部分分别通过切碎或刮取方法处理。切碎和刮取方法均可检测到 MNU、EMS 处理的大鼠的 DNA 损伤，但 OA 处理的大鼠未检测到 DNA 损伤，因此切碎方法具有足够的能力来检测 DNA 损伤剂。制备的细胞悬浮液的形态分析表明，切碎法收集的细胞中超过 80%来自上皮细胞。由于切碎法仅需要一半的膀胱，另一半保持完整，可以用于组织病理学检查。我们得出结论，与刮取法相比，切碎法在膀胱组织的彗星分析中更简单、更合适。
