Dong Huan, Suzuki Naomi, Torres Maria C, Bonala Radha R, Johnson Francis, Grollman Arthur P, Shibutani Shinya
Department of Pharmacological Sciences, State University of New York at Stony Brook, NY, USA.
Drug Metab Dispos. 2006 Jul;34(7):1122-7. doi: 10.1124/dmd.105.008706. Epub 2006 Apr 12.
Aristolochic acids (AA) are nephrotoxic and carcinogenic nitroaromatic compounds produced by the Aristolochiaceae family of plants. Ingestion of these phytotoxins by humans results in a syndrome known as AA nephropathy, characterized by renal tubulointerstitial fibrosis and upper urothelial cancer. After activation by cellular enzymes, AA I and II react with DNA to form covalent adducts and as such represent potential biomarkers for studies of AA toxicity. Using site-specifically modified oligodeoxynucleotides as standards, we have developed a method for quantifying 7-(deoxyadenosin-N(6)-yl) aristolactam-DNA or 7-(deoxyguanosin-N(2)-yl) aristolactam-DNA adducts in tissues of Wistar rats using an assay in which (32)P-postlabeling techniques are coupled with nondenaturing polyacrylamide gel electrophoresis. The limit of detection with this technique is five adducts in 10(9) nucleotides for a 5-microg DNA sample. In contrast to previous reports, we find that the levels of AA adducts in renal tissues of Wistar rats treated p.o. with AA for 1 week with 5 mg/kg/day of AA I or AA II were much higher than that in the forestomach. Highest adduct levels were observed in rats treated with AA II, suggesting that this compound may be more genotoxic than AA I. Treatment of rats with aristolactam I, an end-product of AA I metabolism, resulted in a much lower level of adduction. This study establishes the feasibility of using AA-DNA adducts as intermediate biomarkers of exposure in studies of AA nephropathy and its associated urothelial cancer.
马兜铃酸（AA）是马兜铃科植物产生的具有肾毒性和致癌性的硝基芳香族化合物。人类摄入这些植物毒素会导致一种名为马兜铃酸肾病的综合征，其特征为肾小管间质纤维化和上尿路尿路上皮癌。在被细胞酶激活后，AA I和II与DNA反应形成共价加合物，因此可作为研究AA毒性的潜在生物标志物。我们以位点特异性修饰的寡脱氧核苷酸为标准品，开发了一种方法，利用（32）P后标记技术与非变性聚丙烯酰胺凝胶电泳相结合的检测方法，定量检测Wistar大鼠组织中的7 -（脱氧腺苷 - N（6）-基）马兜铃内酰胺 - DNA或7 -（脱氧鸟苷 - N（2）-基）马兜铃内酰胺 - DNA加合物。对于5μg DNA样品，该技术的检测限为每10（9）个核苷酸中有5个加合物。与之前的报道不同，我们发现口服给予5mg/kg/天的AA I或AA II，连续1周处理的Wistar大鼠肾组织中AA加合物的水平远高于前胃中的水平。在用AA II处理的大鼠中观察到最高的加合物水平，这表明该化合物可能比AA I具有更强的基因毒性。用AA I代谢终产物马兜铃内酰胺I处理大鼠，导致加合水平低得多。本研究证实了在马兜铃酸肾病及其相关尿路上皮癌的研究中，将AA - DNA加合物用作暴露中间生物标志物的可行性。
