Aristolochic acid (AA) is a strong cytotoxic nephrotoxin and carcinogen, which induces forestomach and kidney tumors in mice and is associated with development of urothelial cancer in humans. This study sought to gain mechanistic insight into AAI-induced carcinogenesis through analysis of a tumor-relevant endpoint. Female Hupki mice were treated daily with 5 mg AAI/kg body weight by gavage for 3, 12, or 21 days. Histopathology and DNA adduct analysis confirmed kidney and forestomach as target tissues for AAI-induced toxicity. H-ras codon 61 CAA→CTA mutations were measured in mouse kidney and forestomach, as well as liver and glandular stomach (nontarget organs) by allele-specific competitive blocker-PCR (ACB-PCR), because A→T transversion is the predominant mutation induced by AA and this particular mutation was found previously in AA-induced rodent forestomach tumors. Treatment-related differences were observed, with the H-ras mutant fraction (MF) of mouse kidney and forestomach exposed to 5 mg AAI/kg body weight for 21 days significantly higher than that of vehicle-treated controls (Fisher's exact test, P < 0.05). Statistically significant correlations between dA-AAI adduct levels (measured previously in the same animals) and induced H-ras MFs were evident in forestomach of mice treated for 21 days (linear regression, P < 0.05). The significant increase in H-ras MF in kidney and forestomach, along with the correlation between DNA adducts, histopathology, and oncogene mutation, provide definitive evidence that AA induces tumors through a directly mutagenic mode of action. Thus, measurement of tumor-associated mutations is a useful tool for elucidating the mechanisms underlying the tissue specificity of carcinogenesis.
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