CHARACTERIZATION OF DNA-ADDUCTS FORMED BY ARISTOLOCHIC ACIDS IN THE TARGET ORGAN (FORESTOMACH) OF RATS BY P-32 POSTLABELING ANALYSIS USING DIFFERENT CHROMATOGRAPHIC PROCEDURES

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), ari stolochic acid II (AAII) or the plant extract aristolochic acid (AA). DNA adducts were detected and quantitated using the nuclease Pi-enhanc ed version of the P-32-postlabelling assay. For identification of addu cts, reference compounds were prepared by reaction of enzymatically ac tivated AAI and AAII with 3'-purine phosphonucleosides and analysed by the n-butanol enrichment procedure. These reference compounds were as signed to the previously characterized DNA adducts of AAI [7-(deoxygua nosin-N-2-yl)-aristolactam I = dG-AAI, 7-(deoxyadenosin-N-6-yl)-aristo lactam I = dA-AAI] and AAII [7-(deoxyadenosin-N-6-yl)-aristolactam II = dA-AAII]. Cross referencing of the carcinogen-modified nucleoside bi sphosphates obtained from forestomach DNA with the synthetic standard compounds by ion-exchange chromatography and reversed-phase HPLC demon strated that the major DNA adducts formed by AAI and AA were identical to dG-AAI and dA-AAI. Likewise, forestomach DNA isolated from AAII-tr eated rats showed two purine-derived adduct spots, the major one being dA-AAII, the minor one being tentatively identified as 7-(deoxyguanos in-N-2-yl)-aristolactam II.;A minor adduct detected in forestomach DNA of rats treated with AAI was found to be chromatographically indistin guishable from the adduct identified as dA-AAII, indicating a possible demethoxylation reaction of AAI. Quantitation of DNA addicts revealed that in in vitro reactions with 3'-phosphonucleosides the adduct leve ls 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 addu ction by AAII. The pattern of adduct spots obtained from forestomach D NA of rats treated with the plant extract AA reflected the composition of the extract determined by HPLC analysis. Irrespective of the arist olochic acid used to induce DNA adducts, deoxyadenosine is the major t arget of modification, pointing to the general importance of deoxyaden osine adducts for chemical carcinogenesis of these naturally occurring products. This study shows that the combination of two independent ch romatographic systems considerably enhances the fidelity of identifica tion of DNA adducts with the P-32-postlabelling assay.