Bieler C A, Stiborova M, Wiessler M, Cosyns J P, van Ypersele de Strihou C, Schmeiser H H
Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany.
Carcinogenesis. 1997 May;18(5):1063-7. doi: 10.1093/carcin/18.5.1063.
Recently, we reported that aristolochic acid (AA) a naturally occurring nephrotoxin and carcinogen is implicated in a unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN). Indeed, we identified the principal aristolochic acid-DNA adduct in the kidney of five such patients. We now extend these observations and demonstrate the presence of additional AA-DNA adducts by the 32P-post-labelling method not only in the kidneys, but also in a ureter obtained after renal transplantation. Using the nuclease P1 version of the assay not only the major DNA adduct of aristolochic acid, 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), but also the minor adducts, 7-(deoxyguanosin-N2-yl)-aristolactam I (dG-AAI) and 7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII) were detected, and identified by cochromatographic analyses with TLC and HPLC. Quantitative analyses of six kidneys revealed relative adduct levels from 0.7 to 5.3/10(7) for dA-AAI, from 0.02 to 0.12/10(7) for dG-AAI and 0.06 to 0.24/ 10(7) nucleotides for dA-AAII. The detection of the dA-AAII adduct is consistent with the occurrence of aristolochic acid II (AAII) in the herb powder imported under the name of Stephania tetrandra and confirms that the patients had indeed ingested the natural mixture of AAI and AAII. 32P-post-labelling analyses of further biopsy samples of one patient showed the known adduct pattern of AA exposure not only in the kidney, but also in the ureter, whereas in skin and muscle tissue no adduct spots were detectable. In an attempt to explain the higher level of the dA-AAI adduct compared to the dG-AAI adduct level in renal tissue even 44 months after the end of regimen, the persistence of these two purine adducts was investigated in the kidney of rats given a single oral dose of pure AAI. In contrast to the dG-AAI adduct, the dA-AAI adduct exhibited a lifelong persistence in the kidney of rats. Our data demonstrate that AA forms DNA adducts in human tissue by the same activation mechanism(s) reported from animal studies. Thus, the carcinogenic/mutagenic activity of AA observed in animals could also be responsible for the urothelial cancers observed in two of the CHN patients.
最近，我们报道了马兜铃酸（AA）这种天然存在的肾毒素和致癌物与一种独特类型的肾纤维化有关，即所谓的中草药肾病（CHN）。实际上，我们在五名此类患者的肾脏中鉴定出了主要的马兜铃酸 - DNA加合物。现在，我们扩展这些观察结果，并通过³²P后标记法证明，不仅在肾脏中，而且在肾移植后获取的一条输尿管中也存在其他AA - DNA加合物。使用该检测方法的核酸酶P1版本，不仅检测到了马兜铃酸的主要DNA加合物7 - （脱氧腺苷 - N6 - 基） - 马兜铃内酰胺I（dA - AAI），还检测到了次要加合物7 - （脱氧鸟苷 - N2 - 基） - 马兜铃内酰胺I（dG - AAI）和7 - （脱氧腺苷 - N6 - 基） - 马兜铃内酰胺II（dA - AAII），并通过与薄层色谱（TLC）和高效液相色谱（HPLC）的共色谱分析进行了鉴定。对六个肾脏的定量分析显示，dA - AAI的相对加合物水平为0.7至5.3/10⁷，dG - AAI为0.02至0.12/10⁷，dA - AAII为0.06至0.24/10⁷核苷酸。dA - AAII加合物的检测与以防己之名进口的草药粉中马兜铃酸II（AAII） 的存在情况一致，并证实患者确实摄入了AAI和AAII的天然混合物。对一名患者进一步活检样本的³²P后标记分析表明，已知的AA暴露加合物模式不仅在肾脏中存在，在输尿管中也存在，而在皮肤和肌肉组织中未检测到加合物斑点。为了解释在治疗结束后44个月肾组织中dA - AAI加合物水平仍高于dG - AAI加合物水平的原因，我们在给大鼠单次口服纯AAI后，对其肾脏中这两种嘌呤加合物的持久性进行了研究。与dG - AAI加合物不同，dA - AAI加合物在大鼠肾脏中呈现终身持久性。我们的数据表明，AA通过动物研究报道的相同激活机制在人体组织中形成DNA加合物。因此，在动物中观察到的AA的致癌/致突变活性也可能是两名CHN患者中观察到的尿路上皮癌的原因。
