DNA-repair measurements by use of the modified comet assay:An inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG)

The measurement of DNA-repair activity by extracts from cells or tissues of the single-cell gel electrophoresis (comet) assay has a high become widely used in biomonitoring studies. We assessed the inter- variation in reported values of DNA-repair activity on substrate cells been incubated with Ro19-8022 plus light to generate oxidatively damaged Eight laboratories assessed the DNA-repair activity of three cell lines epithelial and two fibroblast cell lines), starting with cell pellets or cell extracts provided by the coordinating laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level incisions between the laboratory with the lowest (0.002incisions/106bp) highest (0.988incisions/106bp) incision activity. Nevertheless, six out laboratories reported the same cell line as having the highest level of DNA-repair activity. The two laboratories that reported discordant another cell line having the highest level of DNA-repair activity) were that reported to have little experience with the modified comet assay to DNA repair. The laboratories were also less consistent in ordering the activity of the other two cell lines, probably because the DNA-repair extracts from these cell lines were very similar (on average of the cell line with the highest repair capacity). A significant observed between the repair activity found in the provided and the self- extracts (r=0.71, P<0.001), which indicates that the predominant source inter-laboratory variation is derived from the incubation of the extract substrate cells embedded in the gel. Overall, we conclude that the step of cell extracts with the substrate cells can be identified as a source of inter-laboratory variation in the modified comet assay for base-excision repair