title: DNA-repair measurements by use of the modified comet assay: an inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG).
abstract: The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories were also less consistent in ordering the repair activity of the other two cell lines, probably because the DNA-repair activity by extracts from these cell lines were very similar (on average approximately 60-65% of the cell line with the highest repair capacity). A significant correlation was observed between the repair activity found in the provided and the self-made cell extracts (r=0.71, P<0.001), which indicates that the predominant source for inter-laboratory variation is derived from the incubation of the extract with substrate cells embedded in the gel. Overall, we conclude that the incubation step of cell extracts with the substrate cells can be identified as a major source of inter-laboratory variation in the modified comet assay for base-excision repair.
authors.affiliation: Department of Toxicology, Maastricht University, Maastricht, The Netherlands.
authors.forename: Roger W L
authors.initials: RW
authors.lastname: Godschalk
authors.forename: Clara
authors.initials: C
authors.lastname: Ersson
authors.forename: Patrizia
authors.initials: P
authors.lastname: Riso
authors.forename: Marisa
authors.initials: M
authors.lastname: Porrini
authors.forename: Sabine A S
authors.initials: SA
authors.lastname: Langie
authors.forename: Frederik-Jan
authors.initials: FJ
authors.lastname: van Schooten
authors.forename: Amaya
authors.initials: A
authors.lastname: Azqueta
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Collins
authors.forename: George D D
authors.initials: GD
authors.lastname: Jones
authors.forename: Rachel W L
authors.initials: RW
authors.lastname: Kwok
authors.forename: David H
authors.initials: DH
authors.lastname: Phillips
authors.forename: Osman
authors.initials: O
authors.lastname: Sozeri
authors.forename: Alessandra
authors.initials: A
authors.lastname: Allione
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Matullo
authors.forename: Lennart
authors.initials: L
authors.lastname: Möller
authors.forename: Lykke
authors.initials: L
authors.lastname: Forchhammer
authors.forename: Steffen
authors.initials: S
authors.lastname: Loft
authors.forename: Peter
authors.initials: P
authors.lastname: Møller
mesh_terms: D002460:Cell Line / Q000528:radiation effects; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004784:Environmental Monitoring; D006801:Humans; D009000:Monocytes / Q000166:cytology / Q000528:radiation effects*
keywords: 8-Oxo-7,8-dihydroguanine; Base-excision repair; Biomonitoring; Oxidatively damaged DNA; Validation