Aristolochic Acid-dna Interactions

Aristolochic acid (AA) is a known nephrotoxin and potential carcinogen, which can form covalent DNA adducts after metabolic activation in vivo and in vitro. Since the AA-DNA adducts standards are still not commercially available and required to be prepared in individual laboratories, comprehensive researches on mutagenesis and carcinogenesis of AA have been restricted. In this study, a simple method for preparation and characterization of aristolochic acid-DNA adducts was developed. Four AA-adducts were synthesized by a direct reaction of AAI/AAII with 2'-deoxynucleosides. The reaction mixture was first cleaned-up and pre-concentrated using solid phase extraction (SPE), and further purified by a reversed-phase HPLC. By application of developed SPE procedure, matrices and byproducts in reaction mixture could be greatly reduced and the adducts of high purity (more than 94% as indicated by HPLC) were obtained. The purified AA-DNA adducts were identified and characterized with LC-ESI-Q-TOF-MS/MS and LC-DAD-fluorescence analysis. This work provides a robust tool for possible large-scale preparation of AA-DNA adduct standards.A sensitive and accurate high-performance liquid chromatography (HPLC) and fluorescence (FL) method was developed for simultaneous quantitative determination of three AA-DNA adducts (dG-AAI, dA-AAII and dA-AAI) by taking advantage of their native fluorescence. In this work, a HPLC column that has comparable separating efficiency as ultra-performance liquid chromatography (UPLC) column was used, providing a fast separating of three adducts (within 5 min). A wide linear range over three magnitudes is obtained for the three adducts. The correlation coefficients of all the calibration curves are found to be higher than 0.9980. The detection limits (LODs) were 11.75 fmol, 0.23 fmol and 0.32 fmol for dG-AAI, dA-AAII and dA-AAI at a sign-to-noise ratio (S/N) of 3, respectively. Intra- and inter-day precisions were also investigated. The relative standard deviations (RSDs) are less than 1.68% for peak areas and 6.46% for retention times. This highly sensitive method was successfully used for detection of DNA digest products of in vitro cells systems and calf thymus DNA with AA.