DNA adduct formation of aristolochic acid I and II in vitro and in vivo

Abstract

Aristolochic acid I (AA I) and aristolochic acid II (AA II), the two main ingredients of the carcinogenic plant extract aristolochic acid (AA), are metabolized to reactive intermediates which bind covalently to DNA in vitro and in vivo. DNA adduct formation was analysed by the ³²P-postlabelling assay. In in vitro incubations with rat liver 9000 g supernatant (S9) and calf thymus DNA (CT-DNA), AA I showed an identical pattern of DNA adducts on thin-layer chromatograms under aerobic and anaerobic conditions, whereas AA II gave rise to DNA adduct formation only anaerobically. The anaerobically obtained DNA adduct pattern by AA II in vitro was similar to the AA I adduct patterns. Aristolactams I and II, the metabolites of AA I and AA II formed under anaerobic conditions, did not form DNA adducts in the presence of S9 mix and CT-DNA. Incubations with xanthine oxidase, known to enzymatically reduce aromatic nitro groups, also activated AA I and AA II to reactive intermediates, producing almost identical adduct patterns as obtained by S9 mix-mediated metabolism. Activation of AA I by S9 mix in the presence of poly(dG) resulted in the formation of two adducts, one of which was shown to be chromatographically indistinguishable from an adduct obtained by reaction with CT-DNA. For the in vivo studies AA I and AA II were administered orally to male Wistar rats, and DNA from liver, brain, oesophagus, stomach lining, forestomach lining, kidney and bladder was analysed for DNA adducts by ³²P-postlabelling. The adduct patterns in DNA from forestomach and kidney—target tissues of AA—and DNA from non-target tissues like stomach lining and liver were similar to the patterns obtained from the in vitro incubations. In the bladder (also a target tissue) only AA II gave rise to DNA adduct formation. These findings suggest that DNA adduct formation by AA I and AA II does not directly correlate with the initiation of the carcinogenic process and subsequent tumour formation in target tissues in the rat.