32P-post-labelling 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 occurringnephrotoxin and carcinogen is implicated in a unique type of renalfibrosis, designated Chinese herbs nephropathy (CHN). Indeed, we identifiedthe principal aristolochic acid-DNA adduct in the kidney of five suchpatients. We now extend these observations and demonstrate the presence ofadditional AA-DNA adducts by the 32P-post-labelling method not only in thekidneys, but also in a ureter obtained after renal transplantation. Usingthe nuclease P1 version of the assay not only the major DNA adduct ofaristolochic acid, 7-(deoxyadenosin-N6-yl)- aristolactam I (dA-AAI), butalso the minor adducts, 7-(deoxyguanosin- N2-yl)-aristolactam I (dG-AAI)and 7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII) were detected, andidentified by cochromatographic analyses with TLC and HPLC. Quantitativeanalyses of six kidneys revealed relative adduct levels from 0.7 to5.3/10(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 isconsistent with the occurrence of aristolochic acid II (AAII) in the herbpowder imported under the name of Stephania tetrandra and confirms that thepatients had indeed ingested the natural mixture of AAI and AAII. 32P-post-labelling analyses of further biopsy samples of one patient showed theknown adduct pattern of AA exposure not only in the kidney, but also in theureter, whereas in skin and muscle tissue no adduct spots were detectable.In an attempt to explain the higher level of the dA-AAI adduct compared tothe dG-AAI adduct level in renal tissue even 44 months after the end ofregimen, the persistence of these two purine adducts was investigated inthe kidney of rats given a single oral dose of pure AAI. In contrast to thedG-AAI adduct, the dA-AAI adduct exhibited a lifelong persistence in thekidney of rats. Our data demonstrate that AA forms DNA adducts in humantissue by the same activation mechanism(s) reported from animal studies.Thus, the carcinogenic/mutagenic activity of AA observed in animals couldalso be responsible for the urothelial cancers observed in two of the CHNpatients.