Characterization of DNA adducts formed by aristolochic acids in the target organ (forestomach) of rats by 32P-postlabelling 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), 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.