Arlt V M, Wiessler M, Schmeiser H H
Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Carcinogenesis. 2000 Feb;21(2):235-42. doi: 10.1093/carcin/21.2.235.
The distribution of DNA adducts formed by the two main components, aristolochic acid I (AAI) and aristolochic acid II (AAII), of the carcinogenic plant extract aristolochic acid (AA) was examined in a plasmid containing exon 2 of the mouse c-H-ras gene by a polymerase arrest assay. AAI and AAII were reacted with plasmid DNA by reductive activation and the resulting DNA adducts were identified as the previously characterized adenine adducts (dA-AAI and dA-AAII) and guanine adducts (dG-AAI and dG-AAII) by the (32)P-post-labeling method. In addition, a structurally unknown adduct was detected in AAII-modified DNA and shown to be derived from reaction with cytosine (dC-AAII). Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra showed a preference for reaction with purine bases in the mouse H-ras gene for both activated compounds, consistent with previous results that purine adducts are the principal reaction products of AAI and AAII with DNA. Despite the structural similarities among AAI-DNA and AAII-DNA adducts, however, the polymerase arrest spectra produced by the AAs were different. According to the (32)P-post-labeling analyses reductively activated AAI showed a strong preference for reacting with guanine residues in plasmid DNA, however, the polymerase arrest assay revealed arrest sites preferentially at adenine residues. In contrast, activated AAII reacted preferentially with adenine rather than guanine residues and to a lesser extent with cytosine but DNA polymerase was arrested at guanine as well as adenine and cytosine residues with nearly the same average relative intensity. Thus, the polymerase arrest spectra obtained with the AA-adducted ras sequence do not reflect the DNA adduct distribution in plasmid DNA as determined by (32)P-post-labeling. Arrest sites of DNA polymerase associated with cytosine residues confirmed the presence of a cytosine adduct in DNA modified by AAII. For both compounds adduct distribution was not random; instead, regions with adduct hot spots and cold spots were observed. Results from nearest neighbor binding analysis indicated that flanking pyrimidines displayed the greatest effect on polymerase arrest and therefore on DNA binding by AA.
通过聚合酶阻滞试验，研究了致癌植物提取物马兜铃酸（AA）的两种主要成分马兜铃酸I（AAI）和马兜铃酸II（AAII）在含有小鼠c-H-ras基因外显子2的质粒中形成的DNA加合物的分布情况。通过还原激活使AAI和AAII与质粒DNA反应，并通过³²P后标记法将所得的DNA加合物鉴定为先前已表征的腺嘌呤加合物（dA-AAI和dA-AAII）和鸟嘌呤加合物（dG-AAI和dG-AAII）。此外，在AAII修饰的DNA中检测到一种结构未知的加合物，并证明其源自与胞嘧啶的反应（dC-AAII）。假定DNA聚合酶沿模板前进受阻的位点位于加合物3'端的核苷酸处。聚合酶阻滞谱显示，两种活化化合物在小鼠H-ras基因中都更倾向于与嘌呤碱基反应，这与先前嘌呤加合物是AAI和AAII与DNA的主要反应产物的结果一致。然而，尽管AAI-DNA和AAII-DNA加合物在结构上有相似之处，但AA产生的聚合酶阻滞谱却不同。根据³²P后标记分析，还原激活的AAI显示出强烈倾向于与质粒DNA中的鸟嘌呤残基反应，然而，聚合酶阻滞试验显示阻滞位点优先位于腺嘌呤残基处。相比之下，活化的AAII优先与腺嘌呤而不是鸟嘌呤残基反应，与胞嘧啶的反应程度较小，但DNA聚合酶在鸟嘌呤以及腺嘌呤和胞嘧啶残基处均被阻滞，平均相对强度几乎相同。因此，用AA加合的ras序列获得的聚合酶阻滞谱不能反映³²P后标记法测定的质粒DNA中的DNA加合物分布情况。与胞嘧啶残基相关的DNA聚合酶阻滞位点证实了在AAII修饰的DNA中存在胞嘧啶加合物。对于这两种化合物，加合物分布都不是随机的；相反，观察到有加合物热点和冷点的区域。最近邻结合分析的结果表明，侧翼嘧啶对聚合酶阻滞进而对AA与DNA的结合影响最大。
