Schmeiser H H, Frei E, Wiessler M, Stiborova M
Department of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany.
Carcinogenesis. 1997 May;18(5):1055-62. doi: 10.1093/carcin/18.5.1055.
Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of the carcinogenic plant extract aristolochic acid (AA), are known to be mutagenic and to form DNA adducts in vivo. According to the structures of the major DNA adducts identified in animals and humans, nitroreduction is the crucial pathway in the metabolic activation of these naturally occurring nitroarenes to their ultimate carcinogenic species. Using the nuclease P1-enhanced version of the 32P-post-labelling assay we investigated the formation of DNA adducts by AAI and AAII in different in vitro activation systems in order to determine the most suitable in vitro system mimicking target tissue activation. Although DNA adducts resulting from oxidative activation of AAs have not yet been identified both reductive and oxidative in vitro systems were employed. In vitro incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and chemical reduction by zinc. Enzymatic incubations were performed under aerobic and anaerobic conditions. A combination of two independent chromatographic systems (ion-exchange chromatography and reversed-phase HPLC) with reference compounds was used for the identification of DNA adducts detected by the 32P-post-labelling assay. The two known major adducts of AAI or AAII found in vivo were generated by all in vitro systems except for incubations with AAII and horseradish peroxidase where two unknown adducts predominated. Irrespective of the in vitro activation system used, the majority of adduct spots obtained were identified as the previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and dG-AAII. This indicates that both reductive and peroxidative activation of AAI or AAII resulted in chromatographically indistinguishable DNA adducts. Thus, peroxidase mediated activation of AAs led to the formation of the same adducts that had been observed in vivo and upon reductive activation in several in vitro systems. Quantitative analyses of individual adducts formed in the various in vitro systems revealed relative adduct labelling (RAL) values over a 100,000-fold range from 4 in 10(3) for activation of AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of AAII by lactoperoxidase. The extent of DNA modification by AAI was higher than by AAII in all enzymatic in vitro systems. Only activation by zinc resulted in higher total binding to exogenous DNA by AAII than by AAI. Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA adduct profiles reproducing profiles in target tissue (forestomach) of rats, thus providing the most appropriate activation among the in vitro systems tested.
马兜铃酸I（AAI）和马兜铃酸II（AAII）是致癌植物提取物马兜铃酸（AA）的两种主要成分，已知它们具有致突变性，并能在体内形成DNA加合物。根据在动物和人类中鉴定出的主要DNA加合物的结构，硝基还原是这些天然存在的硝基芳烃代谢活化为其最终致癌物种的关键途径。我们使用核酸酶P1增强版的32P后标记分析法，研究了AAI和AAII在不同体外激活系统中DNA加合物的形成，以确定最适合模拟靶组织激活的体外系统。尽管尚未鉴定出由AA氧化激活产生的DNA加合物，但同时采用了还原和氧化体外系统。体外孵育在标准化条件下进行（0.3 mM AA；4 mM dNp作为小牛胸腺DNA），使用大鼠肝微粒体、黄嘌呤氧化酶（一种哺乳动物硝基还原酶）、辣根过氧化物酶、乳过氧化物酶以及锌进行化学还原。酶促孵育在有氧和无氧条件下进行。结合两个独立的色谱系统（离子交换色谱和反相高效液相色谱）以及参考化合物，用于鉴定通过32P后标记分析法检测到的DNA加合物。除了用AAII和辣根过氧化物酶孵育时两种未知加合物占主导外，所有体外系统都产生了在体内发现的AAI或AAII的两种已知主要加合物。无论使用何种体外激活系统，获得的大多数加合物斑点都被鉴定为先前表征的四种AA-DNA加合物：dA-AAI、dA-AAII、dG-AAI和dG-AAII。这表明AAI或AAII的还原和过氧化激活导致色谱上无法区分的DNA加合物。因此，过氧化物酶介导的AA激活导致形成了与体内观察到的以及在几种体外系统中还原激活时相同的加合物。对各种体外系统中形成的单个加合物的定量分析显示，相对加合物标记（RAL）值范围超过100,000倍，从锌激活AAII生成脱氧腺苷加合物时的4×10³到乳过氧化物酶激活AAII时的仅3×10⁸。在所有酶促体外系统中，AAI对DNA的修饰程度高于AAII。只有锌激活时，AAII与外源DNA的总结合量高于AAI。用大鼠肝微粒体进行的有氧孵育产生的AAI-和AAII-DNA加合物图谱重现了大鼠靶组织（前胃）中的图谱，因此在所测试的体外系统中提供了最合适的激活。
