An unprecedented free radical mechanism for the formation of DNA adducts by the carcinogenic N-sulfonated metabolite of aristolochic acids.

The carcinogenicity of aristolochic acids (AAs) has been attributed mainly to the formation of stable DNA-aristolactam (DNA-AL) adducts by its reactive N -sulfonated metabolite N-sulfonatooxyaristolactam (N–OSO 3 --AL). The most accepted mechanism for such DNA-AL adduct formation is via the postulated but never unequivocally-confirmed aristolactam nitrenium ion. Here we found that both sulfate radical and two ALI-derived radicals (N-centered and C-centered spin isomers) were produced by N–OSO 3 --ALI, which were detected and unequivocally identified by complementary applications of ESR spin-trapping, HPLC-MS coupled with deuterium-exchange methods. Both the formation of the three radical species and DNA-ALI adducts can be significantly inhibited (up to 90%) by several well-known antioxidants, typical radical scavengers, and spin-trapping agents. Taken together, we propose that N–OSO 3 --ALI decomposes mainly via a new N–O bond homolysis rather than the previously proposed heterolysis pathway, yielding reactive sulfate and ALI-derived radicals, which are together and in concert responsible for forming DNA-ALI adducts. This study presents strong and direct evidence for the production of free radical intermediates during N–OSO 3 --ALI decomposition, providing an unprecedented free radical perspective and conceptual breakthrough, which can better explain and understand the molecular mechanism for the formation of DNA-AA adducts, the carcinogenicity of AAs and their potential prevention. [Display omitted] • First direct ESR evidence of aristolochic acid-related aristolactam radical. • Identification of two isomers of aristolactam radical by ESR and HPLC-MS. • First evidence for sulfate radical formation under physiological conditions. • Synergistic free radical mechanism for forming DNA-aristolactam adducts. • First radical mechanism for DNA damage & carcinogenicity by aristolochic acid. [ABSTRACT FROM AUTHOR]