Validation of the nucleotide excision repair comet assay on cryopreserved PBMCs to measure inter-individual variation in DNA repair capacity.

2.50
Hdl Handle:
http://hdl.handle.net/10146/269553
Title:
Validation of the nucleotide excision repair comet assay on cryopreserved PBMCs to measure inter-individual variation in DNA repair capacity.
Authors:
Allione, Alessandra; Russo, Alessia; Ricceri, Fulvio; Vande Loock, Kim; Guarrera, Simonetta; Voglino, Floriana; Kirsch-Volders, Micheline; Matullo, Giuseppe
Abstract:
Inter-individual susceptibility to mutagens/carcinogens can be assessed by either genotyping DNA repair genes in different pathways or phenotyping DNA repair capacity (DRC) at the molecular or cellular level. Due to the large number of known DNA repair genes, and the interactions between repair pathways, phenotyping is becoming the preferred approach to measure DRC, and reliable assays are therefore increasingly needed. The use of a cellular phenotype comet assay for the nucleotide excision repair (NER) pathway using benzo[a]pyrene diol epoxide (BPDE) has been described in previous papers, but no thorough evaluation of its applicability in large genotype-phenotype studies has been presented. Our aim was to evaluate the possibility of using cryopreserved instead of fresh peripheral blood mononuclear cells (PBMCs) to evaluate intra- and inter-assay variation, and inter-individual variation, for the aphidicolin (APC)-block NER comet assay. Moreover, we measured the variation for the designated internal standard (K562 erythroleukaemia cell line) and we evaluated the feasibility to use lymphoblastoid cell lines (LCLs) as surrogate of PBMCs. Our results showed a low intra-assay [coefficient of variation (CV) 19.9%] and inter-assay (CV 32.3%) variation, with a good inter-individual variation (122 subjects, mean ± standard deviation 7.38 ± 4.99; range 0.66-26.14; CV 67.63%). A significant correlation between results derived from cryopreserved and fresh PBMCs from the same individuals was found (10 subjects, r = 0.62, P = 0.05). Results from LCLs and cryopreserved PBMCs from the same subjects showed an inverse significant correlation (10 subjects, r = -0.712, P = 0.02). K562 cells as internal standard showed low intra-assay variation. In the present study the APC-block NER comet assay on cryopreserved PBMCs seemed to be a reliable method to measure DRC variation in epidemiological studies; LCLs were not a good surrogate in this assay.
Citation:
Mutagenesis 2013, 28 (1):65-70
Journal:
Mutagenesis
Issue Date:
Jan-2013
URI:
http://hdl.handle.net/10146/269553
DOI:
10.1093/mutage/ges054
PubMed ID:
23042048
Additional Links:
http://mutage.oxfordjournals.org/content/28/1/65.long
Type:
Article
Language:
en
ISSN:
1464-3804
Sponsors:
This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (Italy; G.M.), the Progetto Integrato Oncologia, Regione Toscana—Ministero della Salute ‘Identification of population risk profiles as an approach to cancer prevention’ and the Environmental Cancer Risk Nutrition and Individual Susceptibility project (G.M., M.K.V.), a network of excellence operating within the European Union Sixth Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No. 513943).
Appears in Collections:
Articles

Full metadata record

DC FieldValue Language
dc.contributor.authorAllione, Alessandraen_GB
dc.contributor.authorRusso, Alessiaen_GB
dc.contributor.authorRicceri, Fulvioen_GB
dc.contributor.authorVande Loock, Kimen_GB
dc.contributor.authorGuarrera, Simonettaen_GB
dc.contributor.authorVoglino, Florianaen_GB
dc.contributor.authorKirsch-Volders, Michelineen_GB
dc.contributor.authorMatullo, Giuseppeen_GB
dc.date.accessioned2013-02-14T12:57:30Z-
dc.date.available2013-02-14T12:57:30Z-
dc.date.issued2013-01-
dc.identifier.citationMutagenesis 2013, 28 (1):65-70en_GB
dc.identifier.issn1464-3804-
dc.identifier.pmid23042048-
dc.identifier.doi10.1093/mutage/ges054-
dc.identifier.urihttp://hdl.handle.net/10146/269553-
dc.description.abstractInter-individual susceptibility to mutagens/carcinogens can be assessed by either genotyping DNA repair genes in different pathways or phenotyping DNA repair capacity (DRC) at the molecular or cellular level. Due to the large number of known DNA repair genes, and the interactions between repair pathways, phenotyping is becoming the preferred approach to measure DRC, and reliable assays are therefore increasingly needed. The use of a cellular phenotype comet assay for the nucleotide excision repair (NER) pathway using benzo[a]pyrene diol epoxide (BPDE) has been described in previous papers, but no thorough evaluation of its applicability in large genotype-phenotype studies has been presented. Our aim was to evaluate the possibility of using cryopreserved instead of fresh peripheral blood mononuclear cells (PBMCs) to evaluate intra- and inter-assay variation, and inter-individual variation, for the aphidicolin (APC)-block NER comet assay. Moreover, we measured the variation for the designated internal standard (K562 erythroleukaemia cell line) and we evaluated the feasibility to use lymphoblastoid cell lines (LCLs) as surrogate of PBMCs. Our results showed a low intra-assay [coefficient of variation (CV) 19.9%] and inter-assay (CV 32.3%) variation, with a good inter-individual variation (122 subjects, mean ± standard deviation 7.38 ± 4.99; range 0.66-26.14; CV 67.63%). A significant correlation between results derived from cryopreserved and fresh PBMCs from the same individuals was found (10 subjects, r = 0.62, P = 0.05). Results from LCLs and cryopreserved PBMCs from the same subjects showed an inverse significant correlation (10 subjects, r = -0.712, P = 0.02). K562 cells as internal standard showed low intra-assay variation. In the present study the APC-block NER comet assay on cryopreserved PBMCs seemed to be a reliable method to measure DRC variation in epidemiological studies; LCLs were not a good surrogate in this assay.en_GB
dc.description.sponsorshipThis work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (Italy; G.M.), the Progetto Integrato Oncologia, Regione Toscana—Ministero della Salute ‘Identification of population risk profiles as an approach to cancer prevention’ and the Environmental Cancer Risk Nutrition and Individual Susceptibility project (G.M., M.K.V.), a network of excellence operating within the European Union Sixth Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No. 513943).en_GB
dc.language.isoenen
dc.relation.urlhttp://mutage.oxfordjournals.org/content/28/1/65.longen_GB
dc.rightsArchived with thanks to Mutagenesisen_GB
dc.subjectMutagensen_GB
dc.subjectDNA repairen_GB
dc.subjectComet assayen_GB
dc.subject.meshCell Line-
dc.subject.meshComet Assay-
dc.subject.meshCryopreservation-
dc.subject.meshDNA Repair-
dc.subject.meshHumans-
dc.subject.meshLeukocytes, Mononuclear-
dc.titleValidation of the nucleotide excision repair comet assay on cryopreserved PBMCs to measure inter-individual variation in DNA repair capacity.en
dc.typeArticleen
dc.identifier.journalMutagenesisen_GB

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