Recovery of bulky DNA adducts by the regular and a modified 32P-postlabelling assay; influence of the DNA-isolation method.
Cast your vote
You can rate an item by clicking the amount of stars they wish to award to this item.
When enough users have cast their vote on this item, the average rating will also be shown.
Your vote was cast
Thank you for your feedback
Thank you for your feedback
MetadataShow full item record
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.
CitationMutat. Res. 2011, 721 (1):95-100
SponsorsThis 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.
- An improved fluorometric assay for dosimetry of benzo(a)pyrene diol-epoxide-DNA adducts in smokers' lung: comparisons with total bulky adducts and aryl hydrocarbon hydroxylase activity.
- Authors: Alexandrov K, Rojas M, Geneste O, Castegnaro M, Camus AM, Petruzzelli S, Giuntini C, Bartsch H
- Issue date: 1992 Nov 15
- DNA adducts in tumour, normal peripheral lung and bronchus, and peripheral blood lymphocytes from smoking and non-smoking lung cancer patients: correlations between tissues and detection by 32P-postlabelling and immunoassay.
- Authors: Gyorffy E, Anna L, Gyori Z, Segesdi J, Minárovits J, Soltész I, Kostic S, Csekeo A, Poirier MC, Schoket B
- Issue date: 2004 Jul
- Combined micropreparative techniques with synchronous fluorescence spectroscopy or 32P-postlabelling assay for carcinogen-DNA adduct determination.
- Authors: Shields PG, Kato S, Bowman ED, Petruzzelli S, Cooper DP, Povey AC, Weston A
- Issue date: 1993
- The use of benzo[a]pyrene diolepoxide-modified DNA standards for adduct quantification in 32P-postlabelling to assess exposure to polycyclic aromatic hydrocarbons: application in a biomonitoring study.
- Authors: Baan RA, Steenwinkel MJ, van Asten S, Roggeband R, van Delft JH
- Issue date: 1997 Aug 1
- Influences of DNA isolation and RNA contamination on carcinogen-DNA adduct analysis by 32P-postlabeling.
- Authors: Godschalk RW, Maas LM, Kleinjans JC, Van Schooten FJ
- Issue date: 1998