Untargeted DNA Adductomics Identifies Aristolochic Acid III as a Potent DNA-Damaging Agent among 11 Substituted Aromatic Genotoxicants in the Rat Urinary System

An untargeted, data-independent acquisition high-resolution accurate tandem mass spectrometry method using an Orbitrap mass spectrometer was employed to screen for DNA adducts formed from 11 environmental and dietary aromatic or substituted aromatic carcinogens in the kidney, urinary bladder, prostate, pancreas, liver, and the lung of male rats 24 h after treatment. Among the carcinogens investigated, DNA adducts of the structurally related nitrophenanthrenes 3-nitrobenzanthrone (3-NBA), an atmospheric pollutant, and 8-methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid (AA-I), a naturally occurring genotoxicant from Aristolochiaceae plants, were the most abundant across most organs, forming both 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) adducts. In contrast, significantly lower DNA adduct levels were formed with the aromatic amine 4-aminobiphenyl and 2-nitrofluorene, an oxidized derivative of 2-aminofluorene; the heterocyclic aromatic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-α-carboline, and 2-amino-3-methyl-α-carboline; and the polycyclic aromatic hydrocarbon benzo[a]pyrene. DNA adducts of o-toluidine and 2-naphthylamine were not detected. Most notably, 10-methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid (AA-III), an isomer of AA-I, which was later identified as a minor contaminant (5.3%) in the purified herbal extract assayed, exhibited strong organotropism for DNA damage within the urinary system. Dose-adjusted levels of dA and dG adducts of AA-III were 30 to 80 times higher than those of AA-I in the kidney and urinary bladder. This strikingly high organ-specific DNA adduct formation in the urinary system was unique to AA-III and was not observed for the structurally related 3-NBA and AA-I, or the other carcinogens tested. Given that AA-III frequently occurs with AA-I in Aristolochia herbs, these findings underscore the need for further research into the carcinogenic potential of AA-III and its role in renal and urinary bladder cancer associated with traditional herbal medicines.