Abstract

Although the comet assay is now a popular method to assess DNA damage induced by environmental genotoxins at a single cell level, it has not been excluded the, criticism that some or all of the comet images may be the results of apoptosis- and necrosis-mediated DNA fragmentation. In this study, therefore, we query whether necrosis in mouse stomach and liver can lead to false positive results of in vivo comet assay and whether the validity the diffusion assay detecting cells with DNA fragmentation due to apoptosis or necrosis to exclude false positive results in in vivo comet assay of. Carbon tetrachloride (CCl4), allyl alcohol (AA), and D-galactoseamine (D-Gal) that are non-genotoxic but induce necrosis in the liver and high concentration sodium chloride solution (NaCl, 250mg/mL w/v) and dimethylamine (DMA) that are
non-genotoxic but induce necrosis in the stomach mucosa were given to mice. Also, diethylnitrsoamine (DEN) and ethylmethane sulfonate (EMS) that are genotoxic were given to mice at non-cytotoxic level.In the liver and stomach mucosa, not only the alkaline comet assay but also the neutral comet and diffusion assays to detect DNA fragmentation, TUNNEL assay, and histopathological observation were conducted. The stomach mucosa of mice received NaCl and DMA and the liver of mice received AA, CCl4, and D-Gal showed necrosis, the incidence of TUNNEL-positive cells, and the increases in Halo-image in the diffusion assay but not increased tail length in the alkaline comet assay. Therefore, the possibility is not considered that the generation of DNA fragments due to cell death is responsible for an increase in tail length in the
alkaline comet assay but that the increase in Halo-image well agrees with cell death observed by histopathological observation and TUNNEL staining. Therefore, if a concurrently diffusion assay performed in parallel with the alkaline comet assay is positive, the possibility could not be excluded that the positive result of the comet assay is a false positive due to cell death.