Godschalk R W, Maas L M, Kleinjans J C, Van Schooten F J
Department of Health Risk Analysis and Toxicology, University of Maastricht, The Netherlands.
Environ Mol Mutagen. 1998;32(4):344-50.
32P-Postlabeling is a widely applied assay for the analysis of carcinogen-DNA adducts. Optimization of most steps in this assay has been given attention, but influences of DNA isolation and DNA purity on adduct quantitation have not been investigated systematically. In this study, DNA was isolated from human lymphocytes exposed to benzo[a]pyrene (B[a]P, 10 microM) for 18 hr and from liver of rats i.p.-treated with B[a]P (10 mg/kg body weight) using two different DNA isolation methods: a phenol-extraction and a salting-out procedure. Subsequently, DNA was analysed by nuclease P1 (NP1) or butanol-enriched 32P-postlabeling. Influences of RNA contamination were studied by labeling RNA isolated from in vitro exposed lymphocytes. In the in vitro experiment, DNA adduct levels were significantly higher using the salting-out procedure (63.2 +/- 13.7 adducts per 10(8) nucleotides, n = 9) as compared with the phenol-extraction (14.3 +/- 0.8). RNA was approximately 4 times less efficiently labeled as compared to DNA. Nonetheless, RNA contamination of DNA samples may result in an overestimation of DNA adduct levels when butanol enrichment is used, because RNA adduct levels seemed to be substantially higher than DNA adduct levels in the same cells. DNA adduct analysis by nuclease P1 enrichment is probably less affected, since RNA adducts appeared to be NP1 sensitive. In vivo, three different adducts were found by NP1 enriched 32P-postlabeling in the liver of B[a]P-exposed rats. Again, DNA adduct levels were significantly higher using salting out as compared to phenol extraction for the adduct which comigrated with the BPDE-DNA adduct standard (adduct 1) and an unknown adduct (adduct 2). However, the results were the opposite for another B[a]P-derived DNA adduct (adduct 3). Our results suggest that differences in DNA isolation procedures as well as RNA contamination influence quantitative DNA adduct analysis by 32P-postlabeling.
32P后标记法是一种广泛应用于分析致癌物-DNA加合物的检测方法。该检测方法中大多数步骤的优化已受到关注，但DNA分离和DNA纯度对加合物定量的影响尚未得到系统研究。在本研究中，使用两种不同的DNA分离方法，即酚抽提法和盐析法，从暴露于苯并[a]芘（B[a]P，10微摩尔）18小时的人淋巴细胞以及经腹腔注射B[a]P（10毫克/千克体重）的大鼠肝脏中分离DNA。随后，通过核酸酶P1（NP1）或丁醇富集的32P后标记法对DNA进行分析。通过标记从体外暴露的淋巴细胞中分离的RNA来研究RNA污染的影响。在体外实验中，与酚抽提法（14.3±0.8）相比，盐析法的DNA加合物水平显著更高（每10^8个核苷酸63.2±13.7个加合物，n = 9）。与DNA相比，RNA的标记效率约低4倍。然而，当使用丁醇富集时，DNA样品中的RNA污染可能导致DNA加合物水平的高估，因为在同一细胞中RNA加合物水平似乎明显高于DNA加合物水平。通过核酸酶P1富集进行DNA加合物分析可能受影响较小，因为RNA加合物似乎对NP1敏感。在体内，通过NP1富集的32P后标记法在暴露于B[a]P的大鼠肝脏中发现了三种不同的加合物。同样，对于与BPDE-DNA加合物标准品共迁移的加合物（加合物1）和一种未知加合物（加合物2），盐析法的DNA加合物水平显著高于酚抽提法。然而，对于另一种B[a]P衍生的DNA加合物（加合物3），结果则相反。我们的结果表明，DNA分离程序的差异以及RNA污染会影响32P后标记法对DNA加合物的定量分析。
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