32P-adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures.
32P-adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures.
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32P-加合物测定：不同增强程序中结构多样的 DNA 加合物的比较回收率。
DOI：
10.1093/carcin/9.9.1687
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发表时间：
1988
期刊：
影响因子：
4.7
通讯作者：
Earley,K
中科院分区：
文献类型：
--
作者：
Gupta,RC;Earley,K
用于测量低水平（每107个核苷酸1个加合物）致癌物与DNA结合的A32 P-加合物测定法先前已有报道。在该过程中，DNA被酶促水解为正常核苷的3 '-单磷酸和加合物，其被T4多核苷酸激酶和（γ-32 P）ATP标记为5'-32 P。通过TLC分离标记的加合物。通过在1-丁醇中萃取来富集加合物（Gupta，R.C.（1985）癌症研究，45，5656）或用核酸酶P1消化（Reddy，M.V和Randerath，K.（1986）Carcinogenesis，7，1543），然而，在32 P-标记之前，将许多加合物的检测灵敏度提高到每109- 10个核苷酸1个的水平，尽管加合物的回收，特别是在后一种测定中，取决于加合物的化学性质。我们现在已经比较了在两种富集方法以及用多核苷酸激酶取代核酸酶P1的新方法中，>70种已知和未知化学性质的不同致癌物-DNA加合物的回收率。当与丁醇萃取程序比较时，与鸟嘌呤C8位结合的2-氨基芴、2-氨基菲、2-萘胺、4-氨基联苯、4-偶氮氨基苯和N ′-乙酰联苯胺几乎完全丧失（0.2-4%回收率），但芳香胺部分中存在极性基团（如2-乙酰氨基芴、2-乙酰氨基菲和甲基-4-偶氮氨基苯基）得到类似的回收率。相反，结合到鸟嘌呤或腺嘌呤的环外位置的芳香胺（2-氨基菲、2-乙酰氨基菲、2-乙酰氨基芴和甲基-4-偶氮氨基苯）和多环芳烃（苯并（α）芘、溴甲基苯并蒽和苯并蒽）在核酸酶P1程序中显示出与提取程序一样的广泛或完全回收。与核酸酶P1富集相比，丁醇程序中一些未知的假定极性加合物显示出较低的回收率（30-70%）。在多核苷酸激酶富集试验中检查的大多数加合物的回收模式与核酸酶P1介导的试验中发现的基本相同，除了获得总体较低的值。我们的数据表明，一个给定的DNA样本，应分析不同版本的32 P-加合物测定，特别是，DNA的人类标本暴露于低水平的未知致癌物。观察到加合物的化学结构可能是有害的，在其回收的酶和提取介导的富集过程中，可以作为一个探针在未知致癌物的加合物的结构表征。
A32P-adduct assay for the measurement of low levels (1 adduct per 107nucleotides) of binding of carcinogens to DNA has been reported previously. In this procedure, DNA is enzymatically hydrolyzed to 3'-monophosphates of normal nucleosides and adducts, which are 5'-32P-labeled by T4 polynucleotide kinase and (γ-32P)ATP. Labeled adducts are resolved by TLC. Enrichment of adducts by extraction in 1-butanol (Gupta, R.C. (1985)Cancer Res., 45, 5656) or digestion with nuclease P1 (Reddy, M.V and Randerath, K. (1986)Carcinogenesis, 7, 1543) prior to32P-labeling, however, increased the sensitivity of detection for many adducts to a level of 1 per 109-10nucleotides, although adduct recovery particularly in the latter assay depended on the chemical nature of adducts. We have now compared recoveries for >70, different carcinogen-DNA adducts of known and unknown chemical nature in the two enrichment procedures as well as in a new procedure in which polynucleotide kinase is substituted for nuclease P1. When compared with the butanol extraction procedure, arylamines (such as 2-aminofluorene, 2-aminophenanthrene, 2-naphthylamine, 4-aminobiphenyl, 4-azoaminobenzene andN'-acetylbenzidine) bound to the C8 position of guanine were lost almost completely (0.2-4% recovery) in the nuclease P1-mediated assay, but the presence of a polar group in the aromatic aminemoiety (such as 2-acetylaminofluorene, 2-acetylaminophenanthrene and methyl-4-azoaminophenyl) rendered similar recovery. In contrast, aromatic amines (2-aminophenanthrene, 2-acetylaminophenanthrene, 2-acetylaminofluorene and methyl-4-azoaminobenzene) and polycyclic aromatic hydrocarbons (benzo(α)pyrene, bromomethylbenz anthracene and benzanthracene) bound to the exocyclic positions of guanine or adenine showed extensive or as complete recovery in the nuclease P1 procedure as in the extraction procedure. Some of the unknown presumably polar adducts showed a lower recovery (30-70%) in the butanol procedure as compared to the nuclease P1 enrichment. The recovery pattern of most adducts examined in the polynucleotide kinase-enrichment assay was essentially the same as found in nuclease P1-mediated assay, except that overall lower values were obtained. Our data suggest that a given DNA sample should be analyzed by different versions of the32P-adduct assay, particularly, DNA of specimens of humans exposed to low levels of unknown carcinogens. The observation that chemical structure of an adduct may be detrimental in its recovery in the enzyme- and extraction-mediated enrichment procedures may serve as a probe in the structural characterization of adducts of unknown carcinogens.