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

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 Wistar rats. The crude mixture consists of five nitrophenanthrene carboxylic acid derivatives with aristolochic acid I [AA I; 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxyli c 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 I. Using the 32P-postlabeling assay, we could show that AA I forms covalent DNA adducts upon metabolic activation in vitro and in vivo in different organs in the rat. Xanthine oxidase, a mammalian nitroreductase, 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 AA I formed by in vitro reaction of AA I 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 fluorescence spectroscopy. Their structures were elucidated as 7-(deoxyguanosin-N2-yl)-aristolactam I and 7-(deoxyadenosin-N6-yl)-aristolactam I. 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.

Liquid chromatography—tandem mass spectrometry analysis of the DNA adducts of aristolochic acids

“…This fragmentation provided hints at where the carcinogen was attached. Consistent with the reported NMR data, the targeted site for AA binding was determined as the exocyclic°amino°group°in°purines° [5].…”

“…DNA-AA adducts were detected in laboratory rodents [4 -12] and in patients suffering from AAN [16 -18]. The DNA-AA adducts were reported as 7-(deoxyadenosine-N 6 -yl)-aristolactam I (dA-AAI), 7-(deoxyguanosine-N 6 -yl)-aristolactam I (dG-AAI), 7-(deoxyadenosine-N 6 -yl)-aristolactam II (dA-AAII), and 7-(deoxyguanosine-N 6 -yl)-aristolactam II (dG-AAII) [5].…”

“…Electrophilic attack of the aristolactam-nitrenium ion by its C7 position to the exocyclic amino group in the base led to the formation of major adducts. The metabolic activation of AA to the corresponding aristolactam and DNA adducts is illustrated in Scheme 1 [5,11]. DNA-AA adScheme 1.…”

Scientific Opinion on the risks for public and animal health related to the presence of citrinin in food and feed

“…in vitro were identified by us as the aristolactam structures 32 P-post-labelling analyses of further biopsy samples of one bound to the exocyclic amino groups of deoxyguanosine and patient showed the known adduct pattern of AA exposure deoxyadenosine (16,17). not only in the kidney, but also in the ureter, whereas in Recently, we demonstrated that AA had indeed been ingested skin and muscle tissue no adduct spots were detectable.…”

Selective Toxicity of Aristolochic Acids I and II

“…This intermediate leads to the formation of purine adducts bound to the exocyclic amino groups of deoxyadenosine and deoxyguanosine residues in DNA ( Fig. 1) (Schmeiser et al, 1988;Pfau et al, 1990aPfau et al, , 1991. The major DNA adducts detected in rodents and humans include 7-(deoxyadenosin-N 6 -yl) aristolactam I (dA-AL-I), 7-(deoxyguanosin-N 2 -yl) aristolactam I (dG-AL-I), 7-(deoxyadenosin-N 6 -yl) aristolactam II (dA-AL-II), and 7-(deoxyguanosin-N 2 -yl) aristolactam II (dG-AL-II).…”