An ECVAG† trial on assessment of oxidative damage to DNA measured by the comet assay

Clara Johansson 3 10 Peter Mller 1 10 Lykke Forchhammer 1 10 Steffen Loft 1 10 Roger W. L. Godschalk 0 10 Sabine A. S. Langie 0 10 Stijn Lumeij 0 10 George D. D. Jones 7 10 Rachel W. L. Kwok 7 10 Amaya Azqueta 6 10 David H. Phillips 5 10 Osman Sozeri 5 10 Michael N. Routledge 4 10 Alexander J. Charlton 4 10 Patrizia Riso 9 10 Marisa Porrini 9 10 Alessandra Allione 8 10 Giuseppe Matullo 8 10 Jadwiga Palus 2 10 Maciej Stepnik 2 10 Andrew R. Collins 6 10 Lennart Mo ller 3 10 0 Department of Health Risk Analysis and Toxicology, Maastricht University , Maastricht, The Netherlands 1 Section of Environmental Health, Department of Public Health, University of Copenhagen , Copenhagen K, Denmark 2 Toxicology and Carcinogenesis Department, Nofer Institute of Occupational Medicine , qodz, Poland 3 Department of Biosciences and Nutrition, Karolinska Institutet , Huddinge, Sweden 4 The Molecular Epidemiology Unit, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds , Leeds, UK 5 Section of Molecular Carcinogenesis, Institute of Cancer Research , Sutton, UK 6 Department of Nutrition, Faculty of Medicine, University of Oslo , Oslo, Norway 7 Department of Cancer Studies and Molecular Medicine, University of Leicester , Leicester, UK 8 Department of Genetics, Biology and Biochemistry, Institute for Scientific Interchange Foundation , Turin, Italy 9 Department of Food Science and Microbiology, Division of Human Nutrition, University of Milan , Milan, Italy 10 Inter-laboratory variation in oxidative damage to DNA - 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/106 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/106 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. Alkaline single cell gel electrophoresis (the comet assay) is a method used to measure single strand breaks (SSB) and alkali-labile sites (ALS). One reason for the increasing interest in using the method is the low number of cells required to measure DNA lesions. A range of different types of DNA lesions can be measured by adding lesion-specific enzymes (1). A common modification of the assay is to measure the level of 8-oxoguanine as well as other altered purines by incorporating a digestion with the bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG) (1). In addition, the comet assay can be modified to measure DNA incision activity reflecting base excision repair (2) and nucleotide excision repair (3). Several guidelines for the comet assay have been published (47), but there are still considerable differences in protocols used by different research groups. These differences affect inter-laboratory comparisons of results and there is no general agreement about the normal background level of DNA lesions measured by the comet assay. Important steps in the comet assay procedure that may affect the variability are: (i) cell treatment/ dilution in agarose, (ii) duration of enzyme treatment, (iii) duration and pH of alkaline treatment, (iv) electrophoresis conditions and (v) slide scoring. In addition, the fact that different laboratories use different end points (i.e. %DNA in tail, tail moment, tail length and arbitrary units as well as various descriptions of the distribution of images) when reporting their results further complicates the comparison of data between different laboratories. Mller et al. (8) have previously shown that there is substantial variation when different investigators score the same slides by visual classification of comets. Forchhammer et al. (9) recently reported that inter-individual differences in visual scoring could be reduced to a large extent by using investigator- and protocol-specific calibration curves. The aim of this study was to assess variation in estimates of oxidatively damaged DNA, in terms of FPG-sensitive sites, measured with the comet assay by 10 different European Comet assay Validation Group (ECVAG) partners using their own protocols when analyzing coded samples. Materials and methods Study design In order to investigate the inter-laboratory variation in the analysis of oxidation damage to DNA, 10 laboratories measured the level of DNA damage in four c-ray irradiated calibration samples and three coded samples of human cells using their own protocols. The three coded samples contained cells with different amounts of 8-oxoguanine in their DNA. Cryopreserved calibration curve samples, coded samples and aliquots of FPG from the same batch were distributed on dry ice to the participating laboratories. Laboratories were instructed to analyse the calibration samples together with the coded samples, in order to create laboratory-specific calibration curves. Each laboratory completed a questionnaire on their comet assay protocol (Table I). Reagents and enzymes Trypsinethylenediaminetetraacetic acid (EDTA), Dulbeccos Minimal Essential Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, foetal bovine serum (FBS), penicillinstreptomycin and sodium pyruvate were purchased from Invitrogen Corporation, Karlsruhe, Germany. FPG was supplied by one of the participating laboratories (Department of Nutrition, University of Oslo, Norway). Preparation of ca (...truncated)