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

2.50
Hdl Handle:
http://hdl.handle.net/10146/113465
Title:
An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay.
Authors:
Johansson, Clara; Moller, Peter; Forchhammer, Lykke; Loft, Steffen; Godschalk, Roger W. L.; Langie, Sabine A. S.; Lumeij, Stijn; Jones, George D. D.; Kwok, Rachel W. L.; Azqueta, Amaya; Phillips, David H.; Sozeri, Osman; Routledge, Michael N.; Charlton, Alexander J.; Riso, Patrizia; Porrini, Marisa; Allione, Alessandra; Matullo, Giuseppe; Palus, Jadwiga; Stepnik, Maciej; Collins, Andrew R.; Moller, Lennart
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.
Citation:
Mutagenesis 2010, 25 (2):125-132
Journal:
Mutagenesis
Issue Date:
Mar-2010
URI:
http://hdl.handle.net/10146/113465
DOI:
10.1093/mutage/gep055
PubMed ID:
19948595
Additional Links:
http://mutage.oxfordjournals.org/content/25/2/125.long; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825342/?tool=pubmed
Type:
Article
Language:
en
ISSN:
1464-3804
Sponsors:
Environmental Cancer Risk, Nutrition and Individual Susceptibility, a network of excellence operating within the European Union 6th Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No 513943); the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS); the European Union 6th Framework Program strategic targeted research project ‘COMICS’ (Contract No 037575); Swedish Research Council (Vetenskapsra°det).
Appears in Collections:
Articles

Full metadata record

DC FieldValue Language
dc.contributor.authorJohansson, Claraen
dc.contributor.authorMoller, Peteren
dc.contributor.authorForchhammer, Lykkeen
dc.contributor.authorLoft, Steffenen
dc.contributor.authorGodschalk, Roger W. L.en
dc.contributor.authorLangie, Sabine A. S.en
dc.contributor.authorLumeij, Stijnen
dc.contributor.authorJones, George D. D.en
dc.contributor.authorKwok, Rachel W. L.en
dc.contributor.authorAzqueta, Amayaen
dc.contributor.authorPhillips, David H.en
dc.contributor.authorSozeri, Osmanen
dc.contributor.authorRoutledge, Michael N.en
dc.contributor.authorCharlton, Alexander J.en
dc.contributor.authorRiso, Patriziaen
dc.contributor.authorPorrini, Marisaen
dc.contributor.authorAllione, Alessandraen
dc.contributor.authorMatullo, Giuseppeen
dc.contributor.authorPalus, Jadwigaen
dc.contributor.authorStepnik, Maciejen
dc.contributor.authorCollins, Andrew R.en
dc.contributor.authorMoller, Lennarten
dc.date.accessioned2010-10-19T12:00:55Z-
dc.date.available2010-10-19T12:00:55Z-
dc.date.issued2010-03-
dc.identifier.citationMutagenesis 2010, 25 (2):125-132en
dc.identifier.issn1464-3804-
dc.identifier.pmid19948595-
dc.identifier.doi10.1093/mutage/gep055-
dc.identifier.urihttp://hdl.handle.net/10146/113465-
dc.description.abstractThe 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.en
dc.description.sponsorshipEnvironmental Cancer Risk, Nutrition and Individual Susceptibility, a network of excellence operating within the European Union 6th Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No 513943); the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS); the European Union 6th Framework Program strategic targeted research project ‘COMICS’ (Contract No 037575); Swedish Research Council (Vetenskapsra°det).en
dc.language.isoenen
dc.relation.urlhttp://mutage.oxfordjournals.org/content/25/2/125.longen
dc.relation.urlhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825342/?tool=pubmeden
dc.subjectOxidative Stressen
dc.subjectDNA Damageen
dc.subjectCells, Cultureden
dc.subjectComet Assayen
dc.subjectHumansen
dc.subjectLaboratoriesen
dc.subjectReference Standardsen
dc.subjectValidation Studies as Topicen
dc.subject.meshAutomatic Data Processing-
dc.subject.meshCells, Cultured-
dc.subject.meshComet Assay-
dc.subject.meshDNA Damage-
dc.subject.meshDNA-Formamidopyrimidine Glycosylase-
dc.subject.meshGamma Rays-
dc.subject.meshGuanine-
dc.subject.meshHumans-
dc.subject.meshLaboratories-
dc.subject.meshMonocytes-
dc.subject.meshObserver Variation-
dc.subject.meshOxidative Stress-
dc.subject.meshReference Standards-
dc.subject.meshValidation Studies as Topic-
dc.titleAn ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay.en
dc.typeArticleen
dc.identifier.journalMutagenesisen

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