No 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.

We report the analysis of DNA adducts in the target organ (forestomach) of male Sprague-Dawley rats treated orally with two doses (10 mg/kg body wt) per week for 2 weeks of either aristolochic acid I (AAI), aristolochic acid II (AAII) or the plant extract aristolochic acid (AA). DNA adducts were detected and quantitated using the nuclease P1-enhanced version of the 32P-postlabelling assay. For identification of adducts, reference compounds were prepared by reaction of enzymatically activated AAI and AAII with 3'-purine phosphonucleosides and analysed by the n-butanol enrichment procedure. These reference compounds were assigned to the previously characterized DNA adducts of AAI [7-(deoxyguanosin-N2-yl)-aristolactam I = dG-AAI, 7-(deoxyadenosin-N6-yl)-aristolactam I = dA-AAI] and AAII [7-(deoxyadenosin-N6-yl)-aristolactam II = dA-AAII]. Cross referencing of the carcinogen-modified nucleoside bisphosphates obtained from forestomach DNA with the synthetic standard compounds by ion-exchange chromatography and reversed-phase HPLC demonstrated that the major DNA adducts formed by AAI and AA were identical to dG-AAI and dA-AAI. Likewise, forestomach DNA isolated from AAII-treated rats showed two purine-derived adduct spots, the major one being dA-AAII, the minor one being tentatively identified as 7-(deoxyguanosin-N2-yl)-aristolactam II. A minor adduct detected in forestomach DNA of rats treated with AAI was found to be chromatographically indistinguishable from the adduct identified as dA-AAII, indicating a possible demethoxylation reaction of AAI. Quantitation of DNA adducts revealed that in in vitro reactions with 3'-phosphonucleosides the adduct levels were approximately one order higher for both AAI- and AAII-derived adducts than in forestomach DNA modified with AAI or AAII in vivo. In vitro as well as in vivo adduction by AAI was more efficient than adduction by AAII. The pattern of adduct spots obtained from forestomach DNA of rats treated with the plant extract AA reflected the composition of the extract determined by HPLC analysis. Irrespective of the aristolochic acid used to induce DNA adducts, deoxyadenosine is the major target of modification, pointing to the general importance of deoxyadenosine adducts for chemical carcinogenesis of these naturally occurring products. This study shows that the combination of two independent chromatographic systems considerably enhances the fidelity of identification of DNA adducts with the 32P-postlabelling assay.

We report the quantitation of DNA adducts in target and nontarget organs of male Wistar rats treated orally with five daily doses (10 mg/kg body wt) aristolochic acid I (AAI) or aristolochic acid II (AAII), the major components of the herbal drug aristolochic acid, a forestomach carcinogen in the rat. DNA adducts were detected and analysed using the nuclease P1-enhanced variation of the Randerath 32P-postlabelling assay. The highest level of DNA adducts formed was by AAI in the target organ, forestomach (330 +/- 30 adducts/10(8) nucleotides), but high levels were also observed in a non-target tissue, the glandular stomach (180 +/- 15). Lower amounts of adducts were detected in liver, kidney and urinary bladder epithelium. With AAII the binding levels were generally lower than the AAI, the highest level of adducts being detected in kidney (80 +/- 20 adducts/10(8) nucleotides) and lower levels in liver, stomach and urinary bladder epithelia. Adduct patterns similar to those in vivo were observed in two new in vitro assays. Rat faecal bacteria were shown to be able to activate AAI and AAII to reactive species, which were trapped with exogenous calf thymus DNA and analysed by postlabelling. Incubation of AAI and AAII in explanted rat stomach held in short-term organ culture resulted in DNA adduct formation in the epithelia of both forestomach and glandular stomach. To assign the recently characterized in vitro nucleoside adducts of AAI to the bisphosphate derivatives, a new ion-pair HPLC procedure on a reversed-phase column was developed. By monitoring Cerenkov radiation on-line, a good separation of AAI adducts was observed, demonstrating that adducts formed in vivo were chromatographically indistinguishable with those formed in vitro, and previously characterized as an aristolactam I moiety bound covalently to the exocyclic amino groups of deoxyadenosine and deoxyguanosine.