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dc.contributor.authorAllione, Alessandra
dc.contributor.authorPorcedda, Paola
dc.contributor.authorRusso, Alessia
dc.contributor.authorRicceri, Fulvio
dc.contributor.authorSimonelli, Valeria
dc.contributor.authorMinoprio, Anna
dc.contributor.authorGuarrera, Simonetta
dc.contributor.authorPardini, Barbara
dc.contributor.authorMazzei, Filomena
dc.contributor.authorDogliotti, Eugenia
dc.contributor.authorGiachino, Claudia
dc.contributor.authorMatullo, Giuseppe
dc.date.accessioned2013-07-24T10:05:08Z
dc.date.available2013-07-24T10:05:08Z
dc.date.issued2013-05-30
dc.identifier.citationMutat. Res. 2013,en_GB
dc.identifier.issn0027-5107
dc.identifier.pmid23727398
dc.identifier.doi10.1016/j.mrfmmm.2013.05.001
dc.identifier.urihttp://hdl.handle.net/10146/296981
dc.description.abstractDue to the great number of genes involved in DNA repair and the interactions among the pathways responsible for the repair of different types of DNA damage, there is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacity (DRC). The use of peripheral blood mononuclear cells (PBMCs) in DRC assays is particularly useful for human monitoring studies. However, in such studies it is not always possible to collect and process samples on the same day as the blood is taken. We performed a genotype-phenotype correlation study on DRC on 225 healthy subjects. Due to the large number of blood samples to be processed, PBMCs were either isolated and cryopreserved on the same day of blood collection (day 1) or on the following day after 24h blood storage at room temperature (day 2-RT). Samples processed in different days showed a significant difference in the DRC evaluated as 8-oxoguanine glycosylase activity (OGG assay) in cell extracts (p<0.0001) and as benzo[a]pyrene diol epoxide (BPDE)-induced damage repair by the comet assay (p=0.05). No apparent effect of the blood storage conditions on the outcome of γ-ray induced H2AX phosphorylation assay was reported. These results prompted us to further analyze the effects of blood storage conditions by performing a validation study. Three blood samples were simultaneously taken from ten healthy donors, PBMCs were isolated and cryopreserved as follows: immediately after blood collection (day 1); on the following day, after blood storage at RT (day 2-RT); or after blood storage at 4°C (day 2-4°C). DRC was then evaluated using phenotypic assays. The γ-ray induced H2AX phosphorylation assay has been confirmed as the only assay that showed good reproducibility independently of the blood storage conditions. The measurement of OGG assay was most affected by the blood storage conditions.
dc.description.sponsorshipThis work was supported by a grant of the Associazione Ital-iana per la Ricerca sul Cancro (Italy; G.M.), of the Progetto IntegratoOncologia, Regione Toscana – Ministero della Salute “Identificationof population risk profiles as an approach to cancer prevention”and of the Environmental Cancer Risk Nutrition and Individual Sus-ceptibility project (G.M.), a network of excellence operating withinthe European Union Sixth Framework Program, Priority 5: ‘FoodQuality and Safety’ (Contract No. 513943).en_GB
dc.languageENG
dc.language.isoenen
dc.relation.urlhttp://www.sciencedirect.com/science/article/pii/S0027510713000754
dc.rightsArchived with thanks to Mutation researchen_GB
dc.subjectDNA damageen_GB
dc.subjectDNA repairen_GB
dc.subjectBlood storageen_GB
dc.subjectComet assayen_GB
dc.subjectH2AX phosphorylationen_GB
dc.titleEffect of blood storage conditions on DNA repair capacity measurements in peripheral blood mononuclear cells.en
dc.typeArticleen
dc.identifier.journalMutation Researchen_GB
html.description.abstractDue to the great number of genes involved in DNA repair and the interactions among the pathways responsible for the repair of different types of DNA damage, there is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacity (DRC). The use of peripheral blood mononuclear cells (PBMCs) in DRC assays is particularly useful for human monitoring studies. However, in such studies it is not always possible to collect and process samples on the same day as the blood is taken. We performed a genotype-phenotype correlation study on DRC on 225 healthy subjects. Due to the large number of blood samples to be processed, PBMCs were either isolated and cryopreserved on the same day of blood collection (day 1) or on the following day after 24h blood storage at room temperature (day 2-RT). Samples processed in different days showed a significant difference in the DRC evaluated as 8-oxoguanine glycosylase activity (OGG assay) in cell extracts (p<0.0001) and as benzo[a]pyrene diol epoxide (BPDE)-induced damage repair by the comet assay (p=0.05). No apparent effect of the blood storage conditions on the outcome of γ-ray induced H2AX phosphorylation assay was reported. These results prompted us to further analyze the effects of blood storage conditions by performing a validation study. Three blood samples were simultaneously taken from ten healthy donors, PBMCs were isolated and cryopreserved as follows: immediately after blood collection (day 1); on the following day, after blood storage at RT (day 2-RT); or after blood storage at 4°C (day 2-4°C). DRC was then evaluated using phenotypic assays. The γ-ray induced H2AX phosphorylation assay has been confirmed as the only assay that showed good reproducibility independently of the blood storage conditions. The measurement of OGG assay was most affected by the blood storage conditions.


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