An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay.
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.
Johansson
Clara
C
Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 57 Huddinge, Sweden.
Møller
Peter
P
Forchhammer
Lykke
L
Loft
Steffen
S
Godschalk
Roger W L
RW
Langie
Sabine A S
SA
Lumeij
Stijn
S
Jones
George D D
GD
Kwok
Rachel W L
RW
Azqueta
Amaya
A
Phillips
David H
DH
Sozeri
Osman
O
Routledge
Michael N
MN
Charlton
Alexander J
AJ
Riso
Patrizia
P
Porrini
Marisa
M
Allione
Alessandra
A
Matullo
Giuseppe
G
Palus
Jadwiga
J
Stepnik
Maciej
M
Collins
Andrew R
AR
Möller
Lennart
L
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
C024829:8-hydroxyguanine; D006147:Guanine; D043382:DNA-Formamidopyrimidine Glycosylase