In vitro detection of indirect-acting genotoxins in the comet assay using Hep G2 cells

The induction of DNA damage by four known promutagens (cyclophosphamide (CP), benzo(a)pyrene (BP), dimethylbenz(a)anthracene and 2-acetylaminofluorene (2AAF) was investigated on Hep G2 using the alkaline single cell electroporesis (SCGE) test, most often referred as the "comet assay". After a 3-day incubation, lysed cells embedded in agarose were electrophoresed under alkaline conditions, dyed with a SYBRgold fluorogen and analysed by the Komet software. Among the comet parameters provided by the image analysis program, statistical analysis did not identify any in particular that could best represent the DNA damages. All promutagens, when compared with the control, caused a statistically significant increase in DNA migration as determined by different parameters such as Olive tail moment, tail extent moment, tail/head or tail length. The data demonstrated the ability and the sensitivity of the comet assay when performed on Hep G2 in the detection of DNA damage induced by promutagens, and its suitability in mutagenicity testing in in vitro short-term assays.