COMPARISON OF DNA ADDUCT FORMATION BY ARISTOLOCHIC ACIDS IN VARIOUS IN-VITRO ACTIVATION SYSTEMS BY P-32 POST-LABELING - EVIDENCE FOR REDUCTIVE ACTIVATION BY PEROXIDASES

Aristolochic acid I(AAI) and aristolochic acid II (AAII), the two majo r components of the carcinogenic plant extract aristolochic acid (AA), are known to be mutagenic and to form DNA adducts in vivo. According to the structures of the major DNA adducts identified in animals and h umans, nitroreduction is the crucial pathway in the metabolic activati on of these naturally occurring nitroarenes to their ultimate carcinog enic species. Using the nuclease P1-enhanced version of the P-32-post- labelling assay we investigated the formation of DNA adducts by AAI an d AAII in different in vitro activation systems in order to determine the the most suitable in vitro system mimicking target tissue activati on. Although DNA adducts resulting from oxidative activation of AAs ha ve not yet been identified both reductive and oxidative in vitro syste ms were employed. In vitro incubations were conducted under standardiz ed conditions (0.3 mM AAs; 4 mM dNp as calf thymus DNA) using rat live r microsomes, xanthine oxidase (a mammalian nitroreductase), horseradi sh peroxidase, lactoperoxidase and chemical reduction by zinc, Enzymat ic incubations were performed under aerobic and anaerobic conditions. A combination of two independent chromatographic systems (ion-exchange chromatography and reversed-phase HPLC) with reference compounds was used for the identification of DNA adducts detected by the P-32-post-l abelling assay. The two known major adducts of AAI or AAII found in vi vo were generated by all in vitro systems except for incubations with AAII and horseradish peroxidase where two unknown adducts predominated . Irrespective of the in vitro activation system used, the majority of adduct spots obtained were identified as the previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and dG-AAII. This indica tes that both reductive and peroxidative activation of AAI or AAII res ulted in chromatographically indistinguishable DNA adducts. Thus, pero xidase mediated activation of AAs led to the formation of the same add ucts that had been observed in vivo and upon reductive activation in s everal in vitro systems, Quantitative analyses of individual adducts f ormed in the various in vitro systems revealed relative adduct labelli ng (RAL) values over a 100 000-fold range from 4 in 10(3) for activati on of AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for ac tivation of AAII by lactoperoxidase, The extent of DNA modification by AAI was higher than by AAII in all enzymatic in vitro systems, Only a ctivation by zinc resulted in higher total binding to exogenous DNA by AAII than by AAI, Aerobic incubations with rat liver microsomes gener ated AAI- and AAII-DNA adduct profiles reproducing profiles in target tissue (forestomach) of rats, thus providing the most appropriate acti vation among the in vitro systems tested.