Schmeiser H H, Janssen J W, Lyons J, Scherf H R, Pfau W, Buchmann A, Bartram C R, Wiessler M
Institute of Toxicology and Chemotherapy, German Cancer Research Center, Heidelberg, Federal Republic of Germany.
Cancer Res. 1990 Sep 1;50(17):5464-9.
Aristolochic acid I (AAI), a nitrophenanthrene derivative, is the major component of the carcinogenic plant extract aristolochic acid, which has been used as a medicine since antiquity. Long term oral administration of AAI to male Wistar rats induces multiple tumors, mainly in the forestomach, ear duct, and small intestine. The presence of activated transforming genes was investigated in various tumors of 18 AAI treated rats, namely in 14 squamous cell carcinomas of the forestomach, 7 squamous cell carcinomas of the ear duct, 8 tumors of the small intestine, 3 tumors of the pancreas, 1 adenocarcinoma of the kidney, 1 lymphoma, and 2 metastases in the lung and the pancreas. By utilizing the tumorigenicity assay and Southern blot analysis, we have detected an activated c-Ha-ras gene in the DNAs of 5 of 5 squamous cell carcinomas of the forestomach. Direct sequencing of amplified material revealed an AT----TA transversion mutation at the second position of codon 61 of the c-Ha-ras gene (CAA to CTA) in all transfectants as well as in the 5 original rat tumors. Enzymatic amplification of ras sequences followed by selective oligonucleotide hybridization detected identical mutations in 93% (13 of 14) of forestomach tumors, in 100% (7 of 7) of ear duct tumors, and in the lung metastasis. Among those tumors tested, we had 4 cases in which the forestomach tumors and the ear duct tumors originated from the same rat, showing the same mutation in both tissues. Moreover, similar mutations were demonstrated at c-Ki-ras codon 61 in 1 of 7 ear duct tumors (CAA to CAT) and in 1 of 8 tumors of the small intestine (CAA to CTA) as well as at c-N-ras 61 (CAA to CTA) in a pancreatic metastasis. Additional transfection experiments of some tumors scoring negative for ras gene mutations in dot blot analyses revealed a CAA to CTA transversion at codon 61 of the c-Ha-ras gene in 1 forestomach tumor as well as at codon 61 of the c-N-ras in 1 hyperplasia of the pancreas and in 1 lymphoma. The apparent selectivity for mutations at adenine residues in AAI induced tumors is consistent with the identification of an N6-deoxyadenosine-AAI adduct formed by reaction of AAI with DNA in vitro, suggesting that carcinogen-deoxyadenosine adducts are the critical lesions in the tumor initiation by aristolochic acid.
马兜铃酸I（AAI）是一种硝基菲衍生物，是致癌植物提取物马兜铃酸的主要成分，自古以来就被用作药物。长期给雄性Wistar大鼠口服AAI会诱发多种肿瘤，主要发生在森林胃、耳道和小肠。在18只接受AAI治疗的大鼠的各种肿瘤中研究了激活的转化基因的存在情况，即14例森林胃鳞状细胞癌、7例耳道鳞状细胞癌、8例小肠肿瘤、3例胰腺肿瘤、1例肾腺癌、1例淋巴瘤以及肺和胰腺中的2个转移瘤。通过利用致瘤性测定和Southern印迹分析，我们在5例森林胃鳞状细胞癌的DNA中检测到了激活的c-Ha-ras基因。对扩增材料的直接测序显示，在所有转染子以及5个原始大鼠肿瘤中，c-Ha-ras基因第61密码子的第二位发生了AT----TA颠换突变（CAA变为CTA）。对ras序列进行酶促扩增，然后进行选择性寡核苷酸杂交，在93%（14例中的13例）的森林胃肿瘤、100%（7例中的7例）的耳道肿瘤和肺转移瘤中检测到相同的突变。在我们测试的那些肿瘤中，有4例森林胃肿瘤和耳道肿瘤来自同一只大鼠，两个组织中显示相同的突变。此外，在7例耳道肿瘤中的1例（CAA变为CAT）、8例小肠肿瘤中的1例（CAA变为CTA）的c-Ki-ras密码子61以及胰腺转移瘤中的c-N-ras 61（CAA变为CTA）处也发现了类似的突变。对一些在斑点印迹分析中ras基因突变检测为阴性的肿瘤进行的额外转染实验显示，1例森林胃肿瘤的c-H-ras基因第61密码子以及胰腺1例增生和1例淋巴瘤中的c-N-ras第61密码子发生了CAA到CTA的颠换。AAI诱导的肿瘤中腺嘌呤残基突变的明显选择性与体外AAI与DNA反应形成的N6-脱氧腺苷-AAI加合物的鉴定一致，这表明致癌物-脱氧腺苷加合物是马兜铃酸引发肿瘤的关键损伤。
