32P-Postlabelling analysis of the DNA adducts formed by aristolochic acid I and II

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Abstract
We report the quantitation of DNA adducts in target and non-target organs of male Wistar rats treated orally with five daily doses (10 mg/kg body wt) aristolochic acid I (AAI) or aristolochic acid II (AAII), the major components of the herbal drug aristolochic acid, a forestomach carcinogen In the rat. DNA adducts were detected and analysed using the nuclease P1-enhanced variation of the Randerath 32 postlabeiling assay. The highest level of DNA adducts formed was by AAI inthe target organ, forestomach (330 ± 30 adducts/108 nucleotides), but high levels were also observed in a non-target tissue, the glandular stomach (180 ± 15). Lower amounts of adducts were detected in liver, kidney and urinary bladder epltheliuin. With AAII the binding Levels were generally lower than the AAII, the highest Level of adducts being detected in kidney (80 ± 20 adducts/108 nucleotides) and lower levels in liver, stomach and urinary bladder epithelia. Adduct patterns similar to those in vivo were observed in two new in vitro assays. Rat faecal bacteria were shown to be able to activate AM and AAII to reactive species, which were trapped with exogenous calf thymus DNA and analysed by postlabelling. llncuhatlon of AM and AAII in explanted rat stomach held in short-term organ culture resulted In DNA adduct formation in the epithelia of both forestomach and glandular stomach. To assign the recently characterized in vitro nucleoside adducts of AII to the bisphosphate derivatives, a new ion-pair H]PLC procedure on a reversed-phase column was developed. By monitoring Cerenkov radiation on-line, a good separation of AII adducts was observed, demonstrating that adducts formed in vivo were chromatographically indistinguishable with those formed in vitro, and previously characterized as an aristolactam moiety bound covalently to the exocydlic amino groups of deoxyadenosine and deoxyguanosine.