title: An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay.
abstract: The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/10(6) bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/10(6) bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose-response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories.
authors: Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 57 Huddinge, Sweden.
authors: Clara
authors: C
authors: Johansson
authors: Peter
authors: P
authors: Møller
authors: Lykke
authors: L
authors: Forchhammer
authors: Steffen
authors: S
authors: Loft
authors: Roger W L
authors: RW
authors: Godschalk
authors: Sabine A S
authors: SA
authors: Langie
authors: Stijn
authors: S
authors: Lumeij
authors: George D D
authors: GD
authors: Jones
authors: Rachel W L
authors: RW
authors: Kwok
authors: Amaya
authors: A
authors: Azqueta
authors: David H
authors: DH
authors: Phillips
authors: Osman
authors: O
authors: Sozeri
authors: Michael N
authors: MN
authors: Routledge
authors: Alexander J
authors: AJ
authors: Charlton
authors: Patrizia
authors: P
authors: Riso
authors: Marisa
authors: M
authors: Porrini
authors: Alessandra
authors: A
authors: Allione
authors: Giuseppe
authors: G
authors: Matullo
authors: Jadwiga
authors: J
authors: Palus
authors: Maciej
authors: M
authors: Stepnik
authors: Andrew R
authors: AR
authors: Collins
authors: Lennart
authors: L
authors: Möller
mesh_terms: D002478:Cells, Cultured; D020552:Comet Assay; D004249:DNA Damage / Q000528:radiation effects*; D043382:DNA-Formamidopyrimidine Glycosylase / Q000378:metabolism*; D001330:Electronic Data Processing; D005720:Gamma Rays; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007753:Laboratories / Q000592:standards* / Q000706:statistics & numerical data*; D009000:Monocytes / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects; D015588:Observer Variation; D018384:Oxidative Stress / Q000528:radiation effects*; D012015:Reference Standards; D054928:Validation Studies as Topic
chemical_list: C024829:8-hydroxyguanine; D006147:Guanine; D043382:DNA-Formamidopyrimidine Glycosylase