Matter Brock, Guza Rebecca, Zhao Jianwei, Li Zhong-ze, Jones Roger, Tretyakova Natalia
University of Minnesota Cancer Center and Department of Medicinal Chemistry, Minneapolis 55455, USA.
Chem Res Toxicol. 2007 Oct;20(10):1379-87. doi: 10.1021/tx7001146. Epub 2007 Sep 15.
Acetaldehyde (AA) is the major metabolite of ethanol and may be responsible for an increased gastrointestinal cancer risk associated with alcohol beverage consumption. Furthermore, AA is one of the most abundant carcinogens in tobacco smoke and induces tumors of the respiratory tract in laboratory animals. AA binding to DNA induces Schiff base adducts at the exocyclic amino group of dG, N2-ethylidene-dG, which are reversible on the nucleoside level but can be stabilized by reduction to N2-ethyl-dG. Mutagenesis studies in the HPRT reporter gene and in the p53 tumor suppressor gene have revealed the ability of AA to induce G-->A transitions and A-->T transversions, as well as frameshift and splice mutations. AA-induced point mutations are most prominent at 5'-AGG-3' trinucleotides, possibly a result of sequence specific adduct formation, mispairing, and/or repair. However, DNA sequence preferences for the formation of acetaldehyde adducts have not been previously examined. In the present work, we employed a stable isotope labeling-HPLC-ESI+-MS/MS approach developed in our laboratory to analyze the distribution of acetaldehyde-derived N2-ethyl-dG adducts along double-stranded oligodeoxynucleotides representing two prominent lung cancer mutational "hotspots" and their surrounding DNA sequences. 1,7,NH 2-(15)N-2-(13)C-dG was placed at defined positions within DNA duplexes derived from the K-ras protooncogene and the p53 tumor suppressor gene, followed by AA treatment and NaBH 3CN reduction to convert N2-ethylidene-dG to N2-ethyl-dG. Capillary HPLC-ESI+-MS/MS was used to quantify N2-ethyl-dG adducts originating from the isotopically labeled and unlabeled guanine nucleobases and to map adduct formation along DNA duplexes. We found that the formation of N2-ethyl-dG adducts was only weakly affected by the local sequence context and was slightly increased in the presence of 5-methylcytosine within CG dinucleotides. These results are in contrast with sequence-selective formation of other tobacco carcinogen-DNA adducts along K-ras- and p53-derived duplexes and the preferential modification of endogenously methylated CG dinucleotides by benzo[a]pyrene diol epoxide and acrolein.
乙醛（AA）是乙醇的主要代谢产物，可能与饮酒导致的胃肠道癌症风险增加有关。此外，AA是烟草烟雾中含量最丰富的致癌物之一，可在实验动物中诱发呼吸道肿瘤。AA与DNA结合会在dG的环外氨基处诱导席夫碱加合物N2-亚乙基-dG的形成，这些加合物在核苷水平上是可逆的，但可通过还原为N2-乙基-dG而稳定下来。在次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HPRT）报告基因和p53肿瘤抑制基因中进行的诱变研究表明，AA能够诱导G→A转换和A→T颠换，以及移码突变和剪接突变。AA诱导的点突变在5'-AGG-3'三核苷酸处最为突出，这可能是序列特异性加合物形成、错配和/或修复的结果。然而，此前尚未研究乙醛加合物形成的DNA序列偏好性。在本研究中，我们采用了在我们实验室开发的稳定同位素标记-HPLC-ESI+-MS/MS方法，来分析乙醛衍生的N2-乙基-dG加合物在代表两个突出的肺癌突变“热点”及其周围DNA序列的双链寡脱氧核苷酸中的分布情况。将1,7,NH2-(15)N-2-(13)C-dG置于源自K-ras原癌基因和p53肿瘤抑制基因的DNA双链体中的特定位置，随后进行AA处理并用NaBH3CN还原，将N2-亚乙基-dG转化为N2-乙基-dG。使用毛细管HPLC-ESI+-MS/MS来定量源自同位素标记和未标记鸟嘌呤碱基的N2-乙基-dG加合物，并绘制加合物沿DNA双链体的形成图谱。我们发现，N2-乙基-dG加合物的形成仅受到局部序列背景的微弱影响，并且在CG二核苷酸中存在5-甲基胞嘧啶时略有增加。这些结果与其他烟草致癌物-DNA加合物沿K-ras和p53衍生双链体的序列选择性形成，以及苯并[a]芘二醇环氧化物和丙烯醛对内源性甲基化CG二核苷酸的优先修饰形成对比。
