Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA
Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA
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DOI：
10.1006/jmbi.2001.4997
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发表时间：
2001-10-26
影响因子：
5.6
通讯作者：
Castonguay, A
中科院分区：
文献类型：
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作者：
Cloutier, JF;Drouin, R;Castonguay, A
亚硝胺4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)是一种重要的烟草致癌物质，与肺癌相关。其复杂的酶激活，导致甲基和吡啶氧丁基(POB)修饰的DNA，使DNA损伤难以表征和量化。因此，我们使用NNK类似物4-[(乙酰氧甲基)亚硝基]-1-(3-吡啶)-1-丁酮(NNKOAc)来诱导基因组DNA损伤，并使用连接介导的聚合酶链式反应和末端转移酶依赖的聚合酶链式反应(LMPCR和TDPCR)来定位核苷拆分时加合物的位置和频率。NNKOAc以浓度依赖的方式诱导单链断裂。烷基化后处理，包括热哌啶或用大肠杆菌3-甲基腺嘌呤-DNA糖基酶II、甲酰胺嘧啶-DNA糖基酶、大肠杆菌内切酶I11或噬菌体T4 UV内切酶V消化，并不增加NNKOAc处理的DNA的DNA断裂水平。只有当POB-DNA在LMPCR程序之前被5‘-磷酸化时，才能使用LMPCR检测DNA损伤。NNKOAc对所有四种碱基均有损伤作用，损伤程度由大到小依次为鸟嘌呤、腺嘌呤、胞嘧啶和胸腺嘧啶。与NNKOAc损伤分布模式相反，N-亚硝基(乙酰氧甲基)甲胺(一种甲基化NNK类似物)诱导的损伤主要发生在G位，无需磷酸化即可通过酶方法检测到。损伤分布模式分析显示，P53基因第241和245位密码子的损伤频率较高，248位密码子的损伤频率较低。我们用P-32后标记实验或T4DNA聚合酶结合核酸内切酶IV催化的核苷酸交换反应分析了NNKOAc诱导的单链断裂的3‘端。这两种方法都表明单链断裂的3‘末端不是羟基，并且被一种未知的化学结构所阻断，而这种化学结构是不被内切酶IV识别的。这些数据与导致DNA链断裂的POB-磷酸三酯水解是一致的。POB损伤可能是诱变的，因为NNKOAc产生单链断裂，产物是5‘-羟基和3’-封闭基团和链断裂。这些结果代表了确定NNK是否具有与其他模型化合物相似的序列特异性的DNA的第一步。(C)2001年学术出版社。
The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific carcinogen associated with lung cancer. Its complex enzymatic activation, leading to methyl and pyridyloxobutyl (POB)-modified DNA, makes DNA damage difficult to characterize and quantify. Therefore, we use the NNK analogue 4-[(acetoxymethyl)nitrosa mino]-1-(3-pyridyl)-1-butanone (NNKOAc) to induce damage in genomic DNA, and to map the sites and frequency of adducts at nuctide resolution using ligation-mediated polymerase chain reaction and terminal transferase-dependent polymerase chain reactions (LMPCR and TDPCR). NNKOAc induced single-strand breaks in a concentration-dependent manner. Post-alkylation treatments, including hot piperidine or digestion with the enzymes Escherichia coli 3-methyladenine-DNA glycosylase II, formamidopyrimidine-DNA glycosylase, Escherichia coli endonuclease Ill, or phage T4 UV endonuclease V did not increase the level of DNA breaks in NNKOAc-treated DNA. Detection of DNA damage using LMPCR was possible only when POB-DNA was 5'-phosphorylated prior to the LMPCR procedure. NNKOAc generated damage at all four bases with the decreasing order guanine > adenine > cytosine > thymine. In contrast to NNKOAc damage distribution patterns, those induced by N-nitroso(acetoxymethyl)methylamine, a methylating NNK analog, induced damage principally at G positions detectable by enzymatic means that did not require phosphorylation. Analysis of damage distribution patterns, reveals a high frequency of damage in the p53 gene in codons 241 and 245 and a lower frequency of damage in codon 248. We analyzed the 3' termini of the NNKOAc induced single-strand breaks using a P-32-post-labeling assay or a nucleotide exchange reaction at the 3`-termini catalyzed by T4 DNA polymerase combined with endonuclease IV treatment. Both methods indicate that the 3' termini of the single-strand breaks are not hydroxyl groups and are blocked by an unknown chemical structure that is not recognized by endonuclease IV. These data are consistent with POB-phosphotriester hydrolysis leading to strand breaks in DNA. The POB-damage could be mutagenic because NNKOAc produces single-strand breaks with the products being a 5'-hydroxyl group and a 3'-blocking group and strand breaks. These results represent the first step in determining if NNK pyridyloxobutylates DNA with sequence specificity similar to those observed with other model compounds. (C) 2001 Academic Press.