Comparison of immunoaffinity chromatography enrichment and nuclease P1 procedures for 32P-postlabelling analysis of PAH-DNA adducts
Comparison of immunoaffinity chromatography enrichment and nuclease P1 procedures for 32P-postlabelling analysis of PAH-DNA adducts
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DOI：
10.1016/s0009-2797(98)00003-9
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发表时间：
1998-03-12
影响因子：
5.1
通讯作者：
Watson, WP
中科院分区：
文献类型：
--
作者：
Randerath, K;Sriram, P;Watson, WP
P-32-后标记分析用于检测由多环芳香族化合物形成的DNA加合物是用于评估与这些化合物相关的遗传毒性的最广泛使用的技术之一。在加合物形成极低的情况下，分析中的关键步骤是在放射性标记步骤之前进行加合物的富集程序。核酸酶P1增强程序是这些方法中最成熟和最常用的。类特异性识别的多环芳烃（PAH）-DNA加合物的免疫亲和程序，因此，已与核酸酶P1方法的范围内形成的多环芳烃的DNA加合物进行了比较。用局部用苯并[a]芘、7，12-二甲基-苯并[a]蒽、5-甲基苯并[a]芘或苯并[a]蒽处理的小鼠的皮肤DNA进行评价。固定化的抗体对结构上类似于BPDE-1-脱氧鸟苷加合物（（+/-）-N-2-（7 r，8 t，9 r-三羟基-7，8，9，10-四氢苯并[a]芘-10t-基）-2 '-脱氧鸟苷）的加合物具有最高的亲和力，抗体针对该加合物产生。评价的PAH-修饰的DNA，最大的加合物回收率获得的DNA含有BPDE I-脱氧鸟苷加合物。与DMBA修饰的DNA，从柱回收的加合物的配置文件是相似的，当柱材料处理与DMBA修饰的DNA的消化物或与P-32标记的DMBA加合物。I-化合物（未暴露动物组织DNA中的内源性加合物），具有与PAH-DNA加合物相似的色谱特性，未通过免疫亲和程序富集。与简单的核酸酶P1增强程序相比，免疫亲和方法更快速，更劳动密集。免疫亲和程序的优点包括：特异性，允许选择性检测某类加合物;有效的加合物富集，为其他富集程序提供可行的替代方案;模型研究的足够灵敏度和纯化加合物以进一步表征的潜力。然而，作为检测DNA加合物形成的一般筛选，核酸酶P1程序被视为首选方法，因为它能够检测更广泛的PAH-DNA加合物。(C)1998由Elsevier Science爱尔兰有限公司出版。保留所有权利。
P-32-postlabelling analysis for detecting DNA adducts formed by polycyclic aromatic compounds is one of the most widely used techniques for assessing genotoxicity associated with these compounds. In cases where the formation of adducts is extremely low, a crucial step in the analysis is an enrichment procedure for adducts prior to the radiolabelling step. The nuclease P1 enhancement procedure is the most established and frequently used of these methods. An immunoaffinity procedure developed for class specific recognition for polycyclic aromatic hydrocarbon (PAH)-DNA adducts has therefore been compared with the nuclease P1 method for a range of DNA adducts formed by PAHs. The evaluation was carried out with skin DNA from mice treated topically with benzo[a]pyrene, 7,12-dimethyl-benz[a]anthracene, 5-methylchrysene or chrysene. The immobilised antibody had the highest affinity for adducts structurally similar to the BPDE-I-deoxyguanosine adduct ((+/-)-N-2-(7r,8t,9r-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10t-yl)-2'-deoxyguanosine) against which the antibody had been raised. Of the PAH-modified DNAs evaluated, the maximum adduct recovery was obtained for DNA containing the BPDE I-deoxyguanosine adduct. With DMBA-modified DNA, the profiles of adducts recovered from the column were similar when the column material was treated either with a digest of DMBA-modified DNA or with P-32-labelled DMBA adducts. I-compounds (endogenous adducts in tissue DNA of unexposed animals), which had similar chromatographic properties to PAH-DNA adducts, were not enriched by the immunoaffinity procedure. Compared to the simple nuclease P1 enhancement procedure, the immunoaffinity methods were lengthier and more labour intensive. Advantages of the immunoaffinity procedure include: specificity, allowing the selective detection of a certain class of adducts; efficient adduct enrichment, providing a viable alternative to other enrichment procedures; adequate sensitivity for model studies and the potential to purify adducts for further characterisation. However, as a general screen for detecting the formation of DNA adducts, the nuclease P1 procedure was viewed as the initial method of choice since it was capable of detecting a wider range of PAH-DNA adducts. (C) 1998 Published by Elsevier Science Ireland Ltd. All rights reserved.