Aristolochic acid binds covalently to the exocyclic amino group of purine nucleotides in DNA

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Abstract
The plant extract aristolochic acid (AA) has been used as a herbal drug in many cultures since antiquity. In 1982 AA was shown to be mutagenic and a strong carcinogen in Wistarrats. The crude mixture consists of five nitrophenanthrene carboxylic acid derivatives with aristolochic acid I [AA I; 8-methoxy-6-nitro-phenanthro-(3, 4-d)-l, 3-dioxolo-5-carboxylic acid] being the major component. The isolated compound has been found to be mutagenic in the Ames assay. The major metabolite of AA I formed under anaerobic conditions in vitro and excreted in vivo in several species including man, is the reduction product aristolactam Il. Using the 32P-postlabeling assay, we could show that AA Il forms covalent DNA adducts upon metabolic activation in vitro and in vivo in different organs in the rat. Xanthine oxidase, a mammalian nitro reductase, has served as a sufficient model system mimicking the reductive route of in vivo activation of carcinogenic nitroarenes. This paper reports on two major fluorescent adducts of AAll formed by in vitro reaction of AA Il with xanthine oxidase and deoxyguanosine or deoxyadenosine. After isolation and purification by preparative HPLC the adducts were characterized by 1H-NMR, FAB mass, UV/Vis and fluorecence spectroscopy. Their structures were elucidated as 7-(deoxyguanosin-N2-yl)-aristolactam Il and 7-(deoxyadenosin -N6-yl)-aristolactam Il. These findings are in marked contrast to the results reported for other nitroaromatic carcinogens, where C8-modified deoxyguanosine adducts predominate and N2-substituted deoxyguanosine derivatives are found as minor reaction products. Our results suggest a cyclic N-acylnitrenium ion with delocalized positive charge as the ultimate carcinogenic species, binding preferentially to the exocyclic amino group of purine nucleotides in DNA.