Quantitative Determination of Aristolochic Acid-derived Dna Adducts in Rats Using P-postlabeling/polyacrylamide Gel Electrophoresis Analysis

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-yl) aristolactam-DNA or 7-(deoxyguanosin-N-yl) aristolactam-DNA adducts in tissues of Wistar rats using an assay in which P-postlabeling techniques are coupled with nondenaturing polyacrylamide gel electrophoresis. The limit of detection with this technique is five adducts in 10 nucleotides for a 5g 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. The nephrotoxic and carcinogenic effects of aristolochic acids (AA) in animals have been established (Pohl, 1892; Dumić, 1954; Mengs et al., 1982; Mengs, 1987; IARC, 2002). Similar toxicities were observed in humans when a cluster of cases of chronic renal failure reported in Belgium was traced to the ingestion of an herbal preparation containing Aristolochia fangchi (Depierreux et al., 1994; Cosyns et al., 1999; Nortier et al., 2000; Arlt et al., 2002; Cosyns, 2003; Li and Wang, 2004). This syndrome, initially referred to as Chinese herb nephropathy and more recently as AA nephropathy, soon was recognized as a global health problem (IARC, 2002; Cosyns, 2003). The persistence of AA-DNA adducts in renal tissues of the Belgian women cohort is consistent with the postulated role of AA in urothelial cancer (Nortier et al., 2000). AA includes a mixture of structurally related nitrophenanthrene carboxylic acids produced by the Aristolochiaceae family, with 8-methoxy-6-nitrophenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AA I) and its 8-desmethoxylated form (AA II) representing the major constituents of these plants (Kumar et al., 2003). The biotransformation of AA has been investigated in extracts prepared from human and rodent cells (Stiborova et al., 1999, 2001, 2002, 2003, 2005) and, to a limited degree, in humans and experimental animals (Krumbiegel et al., 1987). As shown in Fig. 1, AA I is metabolized along two major pathways: 1) demethylation to form AA Ia, a substrate for phase II conjugation reactions, and 2) reduction of the nitro group to form aristolactam I (L I), which in turn can be converted to L Ia and subsequently esterified (Krumbiegel et al., 1987; Arlt et al., 2002). The N-hydroxy intermediate formed during the reduction process is expected to form an aristolactam nitrenium ion, which in turn can give rise to an isomeric carbonium ion that reacts covalently with DNA. Microsomal enzymes, including CYP1A1 and CYP1A2 (Stiborova et al., 2001), and cytosolic enzymes, including nitroreductases, xanthine oxidase (Schmeiser et al., 1988), and NAD(P)H:quinone oxidoreductase (Stiborova et al., 2002), are believed to be involved in these reactions. The primary route of AA II metabolism appears to be the nitro reduction pathway to form L II (Arlt et al., 2002). Treatment of animals and humans with AA results in the formation of covalent DNA adducts (Fig. 2), identified chromatographically as 7-(deoxyadenosin-N-yl) aristolactam I (dA-AA I), 7-(deoxyguanosin-N-yl) aristolactam I (dG-AA I), and 7-(deoxyadenosin-N-yl) aristolactam II (dA-AA II) (Pfau et al., 1990a,b). After p.o. ingestion of AA, extensive formation of AA-DNA adducts was observed in the forestomach, accompanied by development of tumors (Fernando et This research was supported by a Catacosinos Cancer Scholar Award from the School of Medicine, SUNY Stony Brook and Grant ES04068 from the National Institutes of Environmental Health Sciences. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.008706. ABBREVIATIONS: AA, aristolochic acid(s); AA I, aristolochic acid I; AA II, aristolochic acid II; L I, aristolactam I; dA-AA I, 7-(deoxyadenosin-N-yl) aristolactam I; dG-AA I, 7-(deoxyguanosin-N-yl) aristolactam I; dA-AA II, 7-(deoxyadenosin-N-yl) aristolactam II; PAGE, polyacrylamide gel electrophoresis; HPLC, high-performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight. 0090-9556/06/3407-1122–1127$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 7 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 8706/3120498 DMD 34:1122–1127, 2006 Printed in U.S.A. 1122 at A PE T Jornals on A uust 7, 2017 dm d.aspurnals.org D ow nladed from