32P-Post-labelling method improvements for aromatic compound-related molecular epidemiology studies
32P-Post-labelling method improvements for aromatic compound-related molecular epidemiology studies
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DOI：
10.1093/mutage/gem030
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发表时间：
2007-11-01
期刊：
影响因子：
2.7
通讯作者：
Peluso, Marco
中科院分区：
文献类型：
--
作者：
Munnia, Armelle;Saletta, Federica;Peluso, Marco
P-32后标记分析已经成为检测暴露于致癌物，特别是芳香族化合物的受试者中大量DNA加合物的主要工具。然而，P-32后标记方案仍然需要使用高剂量的放射性，即每个样本25-50微米的Ci，这一障碍限制了其在大型研究中的使用。所测量的DNA加合物的特征也是有限的。方法的改进和DNA加合物表征的增加是必要的，以使该分析能够达到更高的吞吐量。测试了一种新的方案，以确保有效的水解，减少放射性物质的使用，并获得更高的层析分辨率。不同的基于高尿素或氢氧化铵体系的层析体系也被用来表征被测的加合物。通过重新分析意大利热那亚一群警察和城市居民的DNA加合物来测试改进。致癌物修饰的DNA标准品的分析也包括在研究中，以进行定性和定量比较。用微球菌核酸酶和两种固定浓度的脾磷酸二酯酶的混合物对DNA进行有效的酶解。与未修改的方案相比，用于标记的放射性总量减少了72%，而不会损失DNA加合物的敏感性。通过减少色谱图上斑点的样品体积，提高了层析分辨率。较低的点样体积可以减少样品的扩散和薄层色谱板上未分辨斑点的形成。使用单批无载体[伽马-P-32]三磷酸腺苷的产量增加了约3.5倍。用高尿素或异丙醇-氢氧化铵体系在白细胞中观察到DNA加合物的复杂模式，这两种技术在检测芳香DNA加合物方面是有效的。上述观察表明，所测量的DNA加合物很可能是由芳香族化合物诱导的。
The P-32-post-labelling assay has emerged as a major tool for detecting bulky DNA adducts in subjects exposed to carcinogens, especially aromatic compounds. However, the P-32-post-labelling protocol still requires the use of high amounts of radioactivity, i.e. 25-50 mu Ci per sample, an obstacle that limits its use in large studies. The characterization of the DNA adducts measured is also limited. Methodological improvements and increased DNA adduct characterization are necessary to make this assay capable of achieving higher throughput. A new protocol was tested to ensure efficient hydrolysis to reduce the use of radioactive material and to obtain higher chromatography resolution. Different chromatography systems based on high-urea or ammonium hydroxide systems were also employed to characterize the adducts being measured. Improvements were tested by re-analysing DNA adducts in a group of police officers and urban residents in Genoa, Italy. The analysis of carcinogen-modified DNA standards was also included in the study for qualitative and quantitative comparison. An efficient DNA digestion was obtained using a method involving hydrolysis by micrococcal nuclease and a mixture of two spleen phosphodiesterases at fixed concentrations. A 72% reduction of the amount of radioactivity used for labelling was achieved in respect to the non-modified protocol without loss of DNA adduct sensitivity. An improved chromatography resolution was obtained by reducing the volume of sample to be spotted on the chromatogram. Lower volume of spotting sample can decrease sample diffusion and the formation of unresolved spots on the thin-layer chromatography plate. The amount of output produced using a single batch of carrier-free [gamma-P-32]ATP was increased by about 3.5-fold. A complex pattern of DNA adducts was observed in leukocytes using both high-urea or isopropanol-ammonium hydroxide systems, two techniques effective in the detection of aromatic DNA adducts. The above observations indicate that DNA adducts being measured are likely to have been induced by aromatic compounds.