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Mutagenesis Advance Access published online on March 7, 2008
Mutagenesis, doi:10.1093/mutage/gen006
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© The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

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

Lykke Forchhammer, Elvira Vaclavik Bräuner, Janne Kjærsgaard Folkmann, Pernille Høgh Danielsen, Claus Nielsen, Annie Jensen, Steffen Loft, Gitte Friis and Peter Møller*

Department of Environmental Health, Institute of Public Health, University of Copenhagen, Øster Farimagsgade 5, Building 5, 2nd floor, PO Box 2099, DK-1014 Copenhagen K, Denmark

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 106 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.

* To whom correspondence should be addressed. Tel: +45 3532 7654; Fax: +45 3525 7686; Email: p.moller{at}pubhealth.ku.dk

Received on September 10, 2007; revised on January 18, 2008; accepted on January 18, 2008.


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