P-32 POST-LABELING ANALYSIS OF DNA-ADDUCTS FORMED BY ARISTOLOCHIC ACID IN TISSUES FROM PATIENTS WITH CHINESE HERBS NEPHROPATHY

Recently, we reported that aristolochic acid (AA) a naturally occurrin g nephrotoxin and carcinogen is implicated in a unique type of renal f ibrosis, designated Chinese herbs nephropathy (CHN). Indeed, we identi fied the principal aristolochic acid-DNA adduct in the kidney of five such patients. We now extend these observations and demonstrate the pr esence of additional AA-DNA adducts by the P-32-post-labelling method not only in the kidneys, but also in a ureter obtained after renal tra nsplantation. Using the nuclease P1 version of the assay not only the major DNA adduct of aristolochic acid, 7-(deoxyadenosin-N-6-yl)-aristo lactam I(dA-AAI), but also the minor adducts, 7-(deoxyguanosin-N-2-yl) -aristolactam I (dG-AAI) and 7-(deoxyadenosin-N-6-yl)-aristolactam II (dA-AAII) were detected, and identified by cochromatographic analyses with TLC and HPLC. Quantitative analyses of six kidneys revealed relat ive adduct levels from 0.7 to 5.3/10(7) for dA-AAI, from 0.02 to 0.12/ 10(7) for dG-AAI and 0.06 to 0.24/ 10(7) nucleotides for dA-AAII. The detection of the dA-AAII adduct is consistent with the occurrence of a ristolochic acid II (AAII) in the herb powder imported under the name of Stephania tetrandra and confirms that the patients had indeed inges ted the natural mixture of AAI and AAII. P-32-post-labelling analyses of further biopsy samples of one patient showed the known adduct patte rn of AA exposure not only in the kidney, but also in the ureter, wher eas in skin and muscle tissue no adduct spots were detectable. In an a ttempt to explain the higher level of the dA-AAI adduct compared to th e dG-AAI adduct level in renal tissue even 44 months after the end of regimen, the persistence of these two purine adducts was investigated in the kidney of rats given a single oral dose of pure AAI. In contras t to the dG-AAI adduct, the dA-AAI adduct exhibited a lifelong persist ence in the kidney of rats. Our data demonstrate that AA forms DNA add ucts in human tissue by the same activation mechanism(s) reported from animal studies. Thus, the carcinogenic/mutagenic activity of AA obser ved in animals could also be responsible for the urothelial cancers ob served in two of the CHN patients.