Department of Molecular Environmental Epidemiology, National Institute of Environmental Health, Gyáli út 2-6, 1097 Budapest, Hungary.
Mutat Res. 2011 Mar 18;721(1):95-100. doi: 10.1016/j.mrgentox.2010.12.012. Epub 2011 Jan 13.
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.
大体积 DNA 加合物被广泛用作人体暴露于包括多环芳烃在内的环境遗传毒物复杂混合物的生物标志物。32P-后标记法对大体积 DNA 加合物的检测非常敏感，但由于其相对较低的通量，限制了其在大规模分子流行病学研究中的应用。本研究的目的是比较使用商业 DNA 分离试剂盒或经典酚提取程序对 32P-后标记法测量大体积 DNA 加合物的影响，并通过减少每个样品的放射性同位素用量来提高 32P-后标记法的通量——同时保持放射安全性。测试 DNA 样品来自用苯并[a]芘处理的 MCF-7 细胞和人外周血淋巴细胞、白细胞层和外周肺组织。改良的 32P-后标记程序在 DNA 酶消化后包括蒸发至干燥步骤，与常规方法相比，在减少反应体积的情况下，用减少量的[γ-32P]ATP 底物进行放射性标记。与溶剂提取相比，试剂盒分离后的 MCF-7 细胞和肺组织样品中 DNA 加合物水平更高。试剂盒分离的白细胞层 DNA 样品中的加合物水平比酚提取程序高七倍（p<0.001）。在改良的 32P-后标记程序中，每个样品的[γ-32P]ATP 用量从 50 μCi 减少至 25 μCi（>6000 Ci/mmol 比放射性），适用于两种 DNA 分离方法制备的所有测试样品，而不会损失加合物回收。两种 DNA 分离程序产生的大体积 DNA 加合物水平之间的差异需要进一步系统研究。改良的 32P-后标记程序允许每个样品的放射性同位素用量减少 50%，从而在保持放射安全性的同时提高了检测的通量。
