An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay
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An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay. / Johansson, Clara; Møller, 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; Möller, Lennart.
In: Mutagenesis, Vol. 25, No. 2, 2010, p. 125-32.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay
AU - Johansson, Clara
AU - Møller, Peter
AU - Forchhammer, Lykke
AU - Loft, Steffen
AU - Godschalk, Roger W L
AU - Langie, Sabine A S
AU - Lumeij, Stijn
AU - Jones, George D D
AU - Kwok, Rachel W L
AU - Azqueta, Amaya
AU - Phillips, David H
AU - Sozeri, Osman
AU - Routledge, Michael N
AU - Charlton, Alexander J
AU - Riso, Patrizia
AU - Porrini, Marisa
AU - Allione, Alessandra
AU - Matullo, Giuseppe
AU - Palus, Jadwiga
AU - Stepnik, Maciej
AU - Collins, Andrew R
AU - Möller, Lennart
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
U2 - 10.1093/mutage/gep055
DO - 10.1093/mutage/gep055
M3 - Journal article
C2 - 19948595
VL - 25
SP - 125
EP - 132
JO - Mutagenesis
JF - Mutagenesis
SN - 0267-8357
IS - 2
ER -
ID: 18360051