Recovery of bulky DNA adducts by the regular and a modified 32P-postlabelling assay; influence of the DNA-isolation method.

2.50
Hdl Handle:
http://hdl.handle.net/10146/232172
Title:
Recovery of bulky DNA adducts by the regular and a modified 32P-postlabelling assay; influence of the DNA-isolation method.
Authors:
Kovacs, Katalin; Anna, Livia; Rudnai, Peter; Schoket, Bernadette
Abstract:
Bulky DNA adducts are widely used as biomarkers of human exposure to complex mixtures of environmental genotoxicants including polycyclic aromatic hydrocarbons. The 32P-postlabelling method is highly sensitive for the detection of bulky DNA adducts, but its relatively low throughput poses limits to its use in large-scale molecular epidemiological studies. The objectives of this study were to compare the impact of DNA-sample preparation with a commercial DNA-isolation kit or with the classical phenol-extraction procedure on the measurement of bulky DNA adducts by 32P-postlabelling, and to increase the throughput of the 32P-postlabelling method--whilst maintaining radio-safety--by reducing the radioisotope requirement per sample. The test DNA samples were prepared from MCF-7 cells treated with benzo[a]pyrene and from human peripheral blood lymphocytes, buffy coat, and peripheral lung tissue. The modified 32P-postlabelling procedure involved an evaporation-to-dryness step after the enzymatic digestions of the DNA, and radio-labelling with a reduced amount of [γ-32P]ATP substrate in a reduced reaction volume compared with the regular method. Higher levels of DNA adducts were measured in the MCF-7 cells and in the lung-tissue samples after isolation with the kit than after solvent extraction. A seven-fold higher level of adducts was detected in the buffy-coat DNA samples isolated with the kit than with the phenol extraction procedure (p<0.001). Reduction of the amount of [γ-32P]ATP from 50 μCi to 25 μCi (>6000 Ci/mmol specific radioactivity) per sample in the modified 32P-postlabelling procedure was generally applicable without loss of adduct recovery for all test samples prepared with both DNA isolation methods. The difference between the bulky DNA-adduct levels resulting from the two DNA-isolation procedures requires further systematic investigation. The modified 32P-postlabelling procedure allows a 50% reduction of radioisotope requirement per sample, which facilitates increased throughput of the assay whilst maintaining radio-safety.
Citation:
Mutat. Res. 2011, 721 (1):95-100
Journal:
Mutation Research
Issue Date:
18-Mar-2011
URI:
http://hdl.handle.net/10146/232172
DOI:
10.1016/j.mrgentox.2010.12.012
PubMed ID:
21237286
Additional Links:
http://www.sciencedirect.com/science/article/pii/S1383571811000064
Type:
Article
Language:
en
ISSN:
0027-5107
Sponsors:
This work was co-financed by the EU Integrated Project NewGeneris (Contract no. FOOD-CT-2005-016320) and EU Network of Excellence ECNIS (Contract no. FOOD-CT-2005-513943), 6th Framework Programme, Priority 5: Food Quality and Safety.
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Full metadata record

DC FieldValue Language
dc.contributor.authorKovacs, Katalinen_GB
dc.contributor.authorAnna, Liviaen_GB
dc.contributor.authorRudnai, Peteren_GB
dc.contributor.authorSchoket, Bernadetteen_GB
dc.date.accessioned2012-07-05T08:14:22Z-
dc.date.available2012-07-05T08:14:22Z-
dc.date.issued2011-03-18-
dc.identifier.citationMutat. Res. 2011, 721 (1):95-100en_GB
dc.identifier.issn0027-5107-
dc.identifier.pmid21237286-
dc.identifier.doi10.1016/j.mrgentox.2010.12.012-
dc.identifier.urihttp://hdl.handle.net/10146/232172-
dc.description.abstractBulky DNA adducts are widely used as biomarkers of human exposure to complex mixtures of environmental genotoxicants including polycyclic aromatic hydrocarbons. The 32P-postlabelling method is highly sensitive for the detection of bulky DNA adducts, but its relatively low throughput poses limits to its use in large-scale molecular epidemiological studies. The objectives of this study were to compare the impact of DNA-sample preparation with a commercial DNA-isolation kit or with the classical phenol-extraction procedure on the measurement of bulky DNA adducts by 32P-postlabelling, and to increase the throughput of the 32P-postlabelling method--whilst maintaining radio-safety--by reducing the radioisotope requirement per sample. The test DNA samples were prepared from MCF-7 cells treated with benzo[a]pyrene and from human peripheral blood lymphocytes, buffy coat, and peripheral lung tissue. The modified 32P-postlabelling procedure involved an evaporation-to-dryness step after the enzymatic digestions of the DNA, and radio-labelling with a reduced amount of [γ-32P]ATP substrate in a reduced reaction volume compared with the regular method. Higher levels of DNA adducts were measured in the MCF-7 cells and in the lung-tissue samples after isolation with the kit than after solvent extraction. A seven-fold higher level of adducts was detected in the buffy-coat DNA samples isolated with the kit than with the phenol extraction procedure (p<0.001). Reduction of the amount of [γ-32P]ATP from 50 μCi to 25 μCi (>6000 Ci/mmol specific radioactivity) per sample in the modified 32P-postlabelling procedure was generally applicable without loss of adduct recovery for all test samples prepared with both DNA isolation methods. The difference between the bulky DNA-adduct levels resulting from the two DNA-isolation procedures requires further systematic investigation. The modified 32P-postlabelling procedure allows a 50% reduction of radioisotope requirement per sample, which facilitates increased throughput of the assay whilst maintaining radio-safety.en_GB
dc.description.sponsorshipThis work was co-financed by the EU Integrated Project NewGeneris (Contract no. FOOD-CT-2005-016320) and EU Network of Excellence ECNIS (Contract no. FOOD-CT-2005-513943), 6th Framework Programme, Priority 5: Food Quality and Safety.en_GB
dc.language.isoenen
dc.relation.urlhttp://www.sciencedirect.com/science/article/pii/S1383571811000064en_GB
dc.rightsArchived with thanks to Mutation researchen_GB
dc.subjectDNA adductsen_GB
dc.subjectCell lineen_GB
dc.subjectIsotope labelingen_GB
dc.subjectLungen_GB
dc.subjectLymphocytesen_GB
dc.subjectHumansen_GB
dc.subject.meshCell Line-
dc.subject.meshDNA-
dc.subject.meshDNA Adducts-
dc.subject.meshHumans-
dc.subject.meshIsotope Labeling-
dc.subject.meshLung-
dc.subject.meshLymphocytes-
dc.subject.meshPhenol-
dc.subject.meshPhosphorus Radioisotopes-
dc.subject.meshReagent Kits, Diagnostic-
dc.titleRecovery of bulky DNA adducts by the regular and a modified 32P-postlabelling assay; influence of the DNA-isolation method.en
dc.typeArticleen
dc.identifier.journalMutation Researchen_GB
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