Synthesis And Mass Spectrometric Analysis Of Aristolochic Acid-DNA Adducts

ObjectiveAristolochic acid (AA) was found to be a genotoxic mutagen and a strong carcinogen, which was considered to be associated with aristolochic acid-DNA adducts (AA-DNA adducts). To investigate more about the biochemical characters and biological actions of AA-DNA adducts, it is necessary to develop a powerful tool to detect the AA-DNA adducts simply, rapidly and accurately.This study was designed to synthesize AA-DNA adducts in vitro and develop a novel method for the characterization of the adducts. High-performance liquid chromatography-electrospray ionization/tandem mass spectrometry (HPLC-ESI-MS/MS) and fourier transform-ion cyclotron resonance mass spectrometry (FT-ICRMS) were used for the characterization of the AA-DNA adducts. The AA-DNA adducts were systematically investigated by the two methods. The relationship between their structural features and the corresponding characteristic fragmentation behavior was investigated and the formation of characteristic ions and the factors influencing their intensities were also discussed.MethodsAA was incubated with 2′-deoxyadenosine 5′-monophosphate (dAp) or 2′-deoxyguanosine 5′-monophosphate (dGp) in vitro using either enzymatic activation (by xanthine oxidase) or chemical activation (by zinc) to synthesize AA-DNA adducts, and the reaction conditions were optimized.The AA-DNA adducts were characterized by the liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS). Modes and parameters of LC-ESI-MS/MS were optimized respectively. MS behavior of AA-DNA adducts was analyzed particularly. Based on the analytical methods established previously, crude extracts were analyzed by ESI-MS~n and LC-MS~n techniques to investigate more about the fragmentation rules of AA-DNA adducts in negative mode. Using the high accuracy mass data and isotope pattern of super high resolution FT-ICRMS, the AA-DNA adducts were identified. Based on the data AA-DNA adducts mass spectrometric analytical methods were developed.Results2′-Deoxyadenosine 5′-monophosphate-AAI(dAp-AAI) , 2′- deoxyadenosine 5′-monophosphate-AAII(dAp-AAII), 2′-deoxyguanosine 5′-monophosphate-AAI(dGp-AAI) and 2′-deoxyguanosine 5′-monophosphate-AAlI(dGp-AAII) DNA adducts were prepared by either enzymatic activation or chemical activation method. Full scan spectra were obtained in the negative ion mode and the quasi-molecular ion peaks of the AA-DNA adducts were m/z 621, m/z 591, m/z 637 and m/z 607 respectively. Analysis by electrospray ionization/ tandem mass spectrometry (ESI-MS/MS) provided useful structural information about AA-DNA adducts. The corresponding characteristic fragmentation behavior of the AA-DNA adducts was similar. Using the super high resolution FT-ICRMS , accuracy mass data of AA-DNA adducts were obtained and the molecular formulas of the samples provided by the super high resolution FT-ICRMS matched the AA-DNA adducts. Isotope pattern of super high resolution FT-ICRMS showed that spectra of isotope cluster of the AA-DNA adducts matched the theoretical spectra.ConclusionsAA can bind covalently to the exocyclic amino group of purine nucleotides to form AA-DNA adducts. Analysis by MS can provide useful structural information about AA-DNA adducts. LC/MS/MS is a powerful tool to detect and identify AAI-DNA adducts simply, rapidly and accurately.