Abstract

The need for a complementary short-term mutagenicity bioassay with robust endpoints to the Ames assay has become increasingly crucial to in order to avoid false negative results. The alternative short-term test  (STT) used in conjunction with the Ames increases the validity and decreases the number of false positive outcomes. As a result, Caco-2 cells (Human intestinal epithelial cell model) and RAW264.7 cells (mouse microphage-like cell line) were treated for 24 h with graded doses of hydrogen peroxide (0, 5, 10, 20, and 40 µM) (oxidative stress-inducing mutagen). Single- and double-strand DNA damage was quantified using single-cell gel electrophoresis (Comet assay). The head intensity, tail intensity, tail migration, and tail moment of the damaged DNA were analysed using an epifluorescence microscope with a gated camera and installed comet IV image analysis software. In Caco-2 and RAW264.7 cells, a significant drop in head intensity and a corresponding dose-dependent increase in tail intensity, tail migration, and tail moment are seen when were quantified w compared to the solvent control. The single cell gel electrophoresis (Comet assay) is a very sensitive, robust, and statistically reliable method for determining DNA damage utilising many parameters. As such, the comet assay is advised as a complement to existing short-term bioassays for mutagenicity, such as the Ames assay.

Key words: Mutagenicity, hydrogen peroxide, RAW264.7 cells, Caco-2 cells, single cell gel electrophoresis (Comet assay)