Detection of DNA Single Strand Breaks Induced by Chemical Mutagens Using the Acellular Comet Assay

Abstract

In the acellular comet assay, slides with gels prepared from untreated cells are exposed after lysis to the test agents and then processed according to the standard comet assay protocol. The sensitivity of acellular comet assay was compared with that of standard assay using human lymphoblastoid WTK1 cells. Selected model mutagens were N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), bleomycin (BLM), and UVC. In the acellular assay, lysed slides were exposed to model chemical mutagens for 2 h or irradiated to UVC, and then electrophoresed immediately after chemical mutagen-treatment or 2 h after UVC-irradiation. The slides for standard assay were prepared immediately after 2 h-exposure to each chemical mutagen or 2 h after UVC-irradiation. In both assays, slides were electrophoresed at pH>13 or pH 12 for 20 min after 20 min unwinding. BLM was positive at pH>13 and pH 12 in both assays. UVC was positive in the standard assay but not in the acellular assay. In spite of positive responses of alkylating agents in the acellular assay at pH>13 and pH 12, they were positive at pH>13 but not at pH 12 in the standard assay. The positive responses in the acellular assay were greater than those in the standard assay. Our present results suggest that acellular comet assay can detect DNA single strand breaks (SSBs) as initial lesions but not alkali-labile sites generated from DNA lesion such as alkylated bases and that the sensitivity to detect SSBs as initial lesions is lower in the standard than in the acellular assay.