Recovery of bulky DNA adducts by the regular and a modified 32P-postlabelling assay; influence of the DNA-isolation method.
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.
Department of Molecular Environmental Epidemiology, National Institute of Environmental Health, Gyáli út 2-6, 1097 Budapest, Hungary. kovacs.katalin@oki.antsz.hu
Katalin
K
Kovács
Lívia
L
Anna
Péter
P
Rudnai
Bernadette
B
Schoket
D002460:Cell Line; D004247:DNA / Q000302:isolation & purification*; D018736:DNA Adducts / Q000032:analysis*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008168:Lung; D008214:Lymphocytes; D019800:Phenol; D010761:Phosphorus Radioisotopes; D011933:Reagent Kits, Diagnostic
D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; D011933:Reagent Kits, Diagnostic; D019800:Phenol; D004247:DNA