A comparison of cell-collecting methods for the Comet assay in urinary bladders of rats

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.

► MNU (N-methyl-N-nitrosourea) and EMS (ethyl methanesulfonate) gave positive results.
► OA (o-anisidine) gave negative results.
► The detecting capabilities of the scraping and mincing methods appeared comparable.
► More than 80% of the cells collected by the mincing method were from the epithelium.
► The mincing method can be used for the Comet assay in urinary bladders.