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

Clara Johansson; Peter Møller; Lykke Forchhammer; Steffen Loft; Roger W L Godschalk; Sabine A S Langie; Stijn Lumeij; George D D Jones; Rachel W L Kwok; Amaya Azqueta; David H Phillips; Osman Sozeri; Michael N Routledge; Alexander J Charlton; Patrizia Riso; Marisa Porrini; Alessandra Allione; Giuseppe Matullo; Jadwiga Palus; Maciej Stepnik; Andrew R Collins; Lennart Möller

doi:10.1093/mutage/gep055

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

Clara Johansson, Peter Møller, Lykke Forchhammer, Steffen Loft, Roger W L Godschalk, Sabine A S Langie, Stijn Lumeij, George D D Jones, Rachel W L Kwok, Amaya Azqueta, David H Phillips, Osman Sozeri, Michael N Routledge, Alexander J Charlton, Patrizia Riso, Marisa Porrini, Alessandra Allione, Giuseppe Matullo, Jadwiga Palus, Maciej StepnikAndrew R Collins, Lennart Möller

84 Citations (Scopus)

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/10⁶ 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/10⁶ 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.

Original language	English
Journal	Mutagenesis
Volume	25
Issue number	2
Pages (from-to)	125-32
Number of pages	7
ISSN	0267-8357
DOIs	https://doi.org/10.1093/mutage/gep055
Publication status	Published - Mar 2010

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Johansson, C., Møller, P., Forchhammer, L., Loft, S., Godschalk, R. W. L., Langie, S. A. S., Lumeij, S., Jones, G. D. D., Kwok, R. W. L., Azqueta, A., Phillips, D. H., Sozeri, O., Routledge, M. N., Charlton, A. J., Riso, P., Porrini, M., Allione, A., Matullo, G., Palus, J., ... Möller, L. (2010). An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay. Mutagenesis, 25(2), 125-32. https://doi.org/10.1093/mutage/gep055

Johansson, C, Møller, P, Forchhammer, L, Loft, S, Godschalk, RWL, Langie, SAS, Lumeij, S, Jones, GDD, Kwok, RWL, Azqueta, A, Phillips, DH, Sozeri, O, Routledge, MN, Charlton, AJ, Riso, P, Porrini, M, Allione, A, Matullo, G, Palus, J, Stepnik, M, Collins, AR & Möller, L 2010, 'An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay', Mutagenesis, vol. 25, no. 2, pp. 125-32. https://doi.org/10.1093/mutage/gep055

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title = "An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay",

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/106 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/106 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.",

author = "Clara Johansson and Peter M{\o}ller and Lykke Forchhammer and Steffen Loft and Godschalk, {Roger W L} and Langie, {Sabine A S} and Stijn Lumeij and Jones, {George D D} and Kwok, {Rachel W L} and Amaya Azqueta and Phillips, {David H} and Osman Sozeri and Routledge, {Michael N} and Charlton, {Alexander J} and Patrizia Riso and Marisa Porrini and Alessandra Allione and Giuseppe Matullo and Jadwiga Palus and Maciej Stepnik and Collins, {Andrew R} and Lennart M{\"o}ller",

year = "2010",

month = mar,

doi = "10.1093/mutage/gep055",

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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 - 2010/3

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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/106 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/106 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/106 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/106 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

SN - 0267-8357

VL - 25

SP - 125

EP - 132

JO - Mutagenesis

JF - Mutagenesis

IS - 2

ER -

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

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