DNA-repair measurements by use of the modified comet assay: An inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG).

2.50
Hdl Handle:
http://hdl.handle.net/10146/296744
Title:
DNA-repair measurements by use of the modified comet assay: An inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG).
Authors:
Godschalk, Roger W. L.; Ersson, Clara; Riso, Patrizia; Porrini, Marisa; Langie, Sabine A. S.; van Schooten, Frederik-Jan; Azqueta, Amaya; Collins, Andrew R.; Jones, George D. D.; Kwok, Rachel W. L.; Phillips, David H.; Sozeri, Osman; Allione, Alessandra; Matullo, Giuseppe; Möller, Lennart; Forchhammer, Lykke; Loft, Steffen; Møller, Peter
Abstract:
The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories were also less consistent in ordering the repair activity of the other two cell lines, probably because the DNA-repair activity by extracts from these cell lines were very similar (on average approximately 60-65% of the cell line with the highest repair capacity). A significant correlation was observed between the repair activity found in the provided and the self-made cell extracts (r=0.71, P<0.001), which indicates that the predominant source for inter-laboratory variation is derived from the incubation of the extract with substrate cells embedded in the gel. Overall, we conclude that the incubation step of cell extracts with the substrate cells can be identified as a major source of inter-laboratory variation in the modified comet assay for base-excision repair.
Citation:
Mutat. Res. 2013, 757(1): 60-67
Journal:
Mutation Research
Issue Date:
Sep-2013
URI:
http://hdl.handle.net/10146/296744
DOI:
10.1016/j.mrgentox.2013.06.020
PubMed ID:
23830929
Additional Links:
http://www.sciencedirect.com/science/article/pii/S1383571813001903
Type:
Article
ISSN:
0027-5107
Sponsors:
ECNIS2 Towards the establishment of a virtual European Centre for Research and Education on Cancer, Environment and Food (ECRECEF), European Commission FP7-KBBE-2010-4 Grant No. 266198. Ro19-8022 was a gift from F. Hoffmann-La Roche (Basel, Switzerland).
Appears in Collections:
Articles

Full metadata record

DC FieldValue Language
dc.contributor.authorGodschalk, Roger W. L.en_GB
dc.contributor.authorErsson, Claraen_GB
dc.contributor.authorRiso, Patriziaen_GB
dc.contributor.authorPorrini, Marisaen_GB
dc.contributor.authorLangie, Sabine A. S.en_GB
dc.contributor.authorvan Schooten, Frederik-Janen_GB
dc.contributor.authorAzqueta, Amayaen_GB
dc.contributor.authorCollins, Andrew R.en_GB
dc.contributor.authorJones, George D. D.en_GB
dc.contributor.authorKwok, Rachel W. L.en_GB
dc.contributor.authorPhillips, David H.en_GB
dc.contributor.authorSozeri, Osmanen_GB
dc.contributor.authorAllione, Alessandraen_GB
dc.contributor.authorMatullo, Giuseppeen_GB
dc.contributor.authorMöller, Lennarten_GB
dc.contributor.authorForchhammer, Lykkeen_GB
dc.contributor.authorLoft, Steffenen_GB
dc.contributor.authorMøller, Peteren_GB
dc.date.accessioned2013-07-22T07:24:01Z-
dc.date.available2013-07-22T07:24:01Z-
dc.date.issued2013-09-
dc.identifier.citationMutat. Res. 2013, 757(1): 60-67en_GB
dc.identifier.issn0027-5107-
dc.identifier.pmid23830929-
dc.identifier.doi10.1016/j.mrgentox.2013.06.020-
dc.identifier.urihttp://hdl.handle.net/10146/296744-
dc.description.abstractThe measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories were also less consistent in ordering the repair activity of the other two cell lines, probably because the DNA-repair activity by extracts from these cell lines were very similar (on average approximately 60-65% of the cell line with the highest repair capacity). A significant correlation was observed between the repair activity found in the provided and the self-made cell extracts (r=0.71, P<0.001), which indicates that the predominant source for inter-laboratory variation is derived from the incubation of the extract with substrate cells embedded in the gel. Overall, we conclude that the incubation step of cell extracts with the substrate cells can be identified as a major source of inter-laboratory variation in the modified comet assay for base-excision repair.en_GB
dc.description.sponsorshipECNIS2 Towards the establishment of a virtual European Centre for Research and Education on Cancer, Environment and Food (ECRECEF), European Commission FP7-KBBE-2010-4 Grant No. 266198. Ro19-8022 was a gift from F. Hoffmann-La Roche (Basel, Switzerland).en_GB
dc.languageENG-
dc.relation.urlhttp://www.sciencedirect.com/science/article/pii/S1383571813001903-
dc.rightsArchived with thanks to Mutation researchen_GB
dc.subjectBiomonitoringen_GB
dc.subjectDNA damageen_GB
dc.subject8-Oxo-7,9-dihydroguanineen_GB
dc.subjectComet assayen_GB
dc.subjectDNA repairen_GB
dc.subjectInter-laboratory validationen_GB
dc.subjectValidationen_GB
dc.titleDNA-repair measurements by use of the modified comet assay: An inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG).-
dc.typeArticleen
dc.identifier.journalMutation Researchen_GB

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