Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring.

2.50
Hdl Handle:
http://hdl.handle.net/10146/36492
Title:
Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring.
Authors:
Forchhammer, Lykke; Brauner, Elvira Vaclavik; Folkmann, Janne Kjaersgaard; Danielsen, Pernille Hogh; Nielsen, Claus; Jensen, Annie; Loft, Steffen; Friis, Gitte; Moller, Peter
Abstract:
The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced investigators scored pre-made slides of nuclei differently, but each investigator scored constantly over time. Scoring of 200 nuclei per treatment was associated with the lowest residual variation. Alkaline unwinding for 20 or 40 min and electrophoresis for 20 or 30 min yielded different dose-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators and this variation could not be reduced by use of investigator-specific calibration curves. The mean numbers of lesions per 10(6) bp in MNBCs from seven humans were 0.23 [95% confidence interval (CI): 0.14-0.33] and 0.31 (95% CI: 0.20-0.55) for strand breaks (SBs) and oxidized guanines, respectively. In conclusion, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay.
Citation:
Mutagenesis 2008, 23 (3):223-231
Journal:
Mutagenesis
Issue Date:
May-2008
URI:
http://hdl.handle.net/10146/36492
DOI:
10.1093/mutage/gen006
PubMed ID:
18326868
Additional Links:
http://mutage.oxfordjournals.org/cgi/content/full/23/3/223
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).
Appears in Collections:
Articles

Full metadata record

DC FieldValue Language
dc.contributor.authorForchhammer, Lykke-
dc.contributor.authorBrauner, Elvira Vaclavik-
dc.contributor.authorFolkmann, Janne Kjaersgaard-
dc.contributor.authorDanielsen, Pernille Hogh-
dc.contributor.authorNielsen, Claus-
dc.contributor.authorJensen, Annie-
dc.contributor.authorLoft, Steffen-
dc.contributor.authorFriis, Gitte-
dc.contributor.authorMoller, Peter-
dc.date.accessioned2008-08-27T08:41:22Z-
dc.date.available2008-08-27T08:41:22Z-
dc.date.issued2008-05-
dc.identifier.citationMutagenesis 2008, 23 (3):223-231en
dc.identifier.issn1464-3804-
dc.identifier.pmid18326868-
dc.identifier.doi10.1093/mutage/gen006-
dc.identifier.urihttp://hdl.handle.net/10146/36492-
dc.description.abstractThe comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced investigators scored pre-made slides of nuclei differently, but each investigator scored constantly over time. Scoring of 200 nuclei per treatment was associated with the lowest residual variation. Alkaline unwinding for 20 or 40 min and electrophoresis for 20 or 30 min yielded different dose-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators and this variation could not be reduced by use of investigator-specific calibration curves. The mean numbers of lesions per 10(6) bp in MNBCs from seven humans were 0.23 [95% confidence interval (CI): 0.14-0.33] and 0.31 (95% CI: 0.20-0.55) for strand breaks (SBs) and oxidized guanines, respectively. In conclusion, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay.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).en
dc.language.isoenen
dc.relation.urlhttp://mutage.oxfordjournals.org/cgi/content/full/23/3/223en
dc.subjectCell Nucleusen
dc.subjectComet assayen
dc.subjectDNA damageen
dc.subjectDNAen
dc.subjectData Interpretationen
dc.subjectLeukocytes, Mononuclearen
dc.subjectObserver Variationen
dc.subjectOxidative stressen
dc.subject.meshCell Nucleus-
dc.subject.meshComet Assay-
dc.subject.meshDNA-
dc.subject.meshDNA Damage-
dc.subject.meshData Interpretation, Statistical-
dc.subject.meshHumans-
dc.subject.meshLeukocytes, Mononuclear-
dc.subject.meshObserver Variation-
dc.subject.meshOxidative Stress-
dc.titleVariation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring.en
dc.typeArticleen
dc.identifier.journalMutagenesisen

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