Gupta R C, Earley K
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030.
Carcinogenesis. 1988 Sep;9(9):1687-93. doi: 10.1093/carcin/9.9.1687.
A 32P-adduct assay for the measurement of low levels (1 adduct per 10(7) nucleotides) 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 [gamma-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 to 32P-labeling, however, increased the sensitivity of detection for many adducts to a level of 1 per 10(9-10) nucleotides, although adduct recovery particularly in the latter assay depended on the chemical nature of adducts. We have now compared recoveries for greater than 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 and N'-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 amine moiety (such as 2-acetylaminofluorene, 2-acetylamino-phenanthrene and methyl-4-azoaminophenyl) rendered similar recovery. In contrast, aromatic amines (2-amino-phenanthrene, 2-acetylaminophenanthrene, 2-acetylaminofluorene and methyl-4-azoaminobenzene) and polycyclic aromatic hydrocarbons (benzo[a]pyrene, bromomethylbenzanthracene 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 the 32P-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.
此前已报道过一种用于测量致癌物与DNA低水平结合（每10⁷个核苷酸1个加合物）的³²P加合物测定法。在此方法中，DNA经酶解为正常核苷和加合物的3'-单磷酸，然后通过T4多核苷酸激酶和[γ-³²P]ATP进行5'-³²P标记。标记的加合物通过薄层层析进行分离。然而，在³²P标记之前，用正丁醇萃取[古普塔，R.C.（1985年）《癌症研究》，45，5656]或用核酸酶P1消化[雷迪，M.V.和兰德拉斯，K.（1986年）《致癌作用》，7，1543]来富集加合物，可将许多加合物的检测灵敏度提高到每10⁹ - 10个核苷酸1个加合物的水平，不过加合物的回收率，尤其是后一种测定法中的回收率，取决于加合物的化学性质。我们现在比较了在两种富集方法以及一种用多核苷酸激酶替代核酸酶P1的新方法中，70多种已知和未知化学性质的致癌物 - DNA加合物的回收率。与正丁醇萃取法相比，与鸟嘌呤C8位结合的芳胺（如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）在核酸酶P1介导的测定法中几乎完全损失（回收率为0.2 - 4%），但芳胺部分存在极性基团（如2-乙酰氨基芴、2-乙酰氨基菲和甲基-4-偶氮氨基苯）时，回收率相似。相反，与鸟嘌呤或腺嘌呤外环位置结合的芳胺（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[a]芘、溴甲基苯并蒽和苯并蒽）在核酸酶P1法中的回收率与萃取法一样高或完全相同。一些推测为极性的未知加合物在正丁醇法中的回收率（30 - 70%）低于核酸酶P1富集法。在多核苷酸激酶富集测定法中检测的大多数加合物的回收率模式与核酸酶P1介导的测定法基本相同，只是总体值较低。我们的数据表明，对于给定的DNA样本，应通过³²P加合物测定法的不同版本进行分析，特别是对于暴露于低水平未知致癌物的人类标本的DNA。加合物的化学结构可能对其在酶介导和萃取介导的富集过程中的回收率产生不利影响这一观察结果，可作为未知致癌物加合物结构表征的一种探针。
