BIS(HYDROXYPHENYLETHYL)DEOXYGUANOSINE ADDUCTS IDENTIFIED BY [P-32] POSTLABELING AND 4-SECTOR TANDEM MASS-SPECTROMETRY - UNANTICIPATED ADDUCTS FORMED UPON TREATMENT OF DNA WITH STYRENE 7,8-OXIDE

Calf thymus DNA was incubated with [8-C-18]styrene oxide in vitro, and six covalent xenobiotic-DNA adducts were detected using the[P-32]-postlabeling procedure. Adducts 1-3 were purified by HPLC and identified as bis-substituted-2'-deoxyguanosine 3'-phosphate derivatives using four-sector tandem mass spectrometry. These adducts represented less than 2% of the total adducts detected by [C-14]-radioactivity. Adducts 1-3 were also detected when styrene oxide was allowed to react with the mononucleotide, 2'-deoxyguanosine 3'-phosphate only. The elemental compositions of these adducts (C26H30N5O9P) were determined by measurement of their accurate masses by high-resolution mass spectrometry and revealed the unusual incorporation of 2 mol of hydroxyphenylethyl moieties. The structures of these bis(phenylethyl) adducts were established by interpretation of high-energy collision-induced dissociation (CID) mass spectra, together with UV/visible and fluorescence spectrophotometry as N2-(2-hydroxy-1-phenylethyl)-O6-(2-hydroxy-2-phenylethyl)-2'-deoxyguanosine 3'-phosphate (adduct 1), N2-(2-hydroxy-1-phenylethyl)-O6-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine 3'-phosphate (adduct 2), and N1,N2-bis(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine 3'-phosphate (adduct 3). The other most abundant adducts were detected only by [C-14]-radioactivity and represented approximately 65% of the total covalent binding. These were identified as depurinated N7-substituted guanines by tandem mass spectrometry and UV/visible spectroscopy. The combination of advanced techniques of mass spectrometry with the [P-32] -postlabeling assay and spectroscopic techniques is a comprehensive strategy to assure complete structural identification of all xenobiotic-DNA adducts. In this study the [P-32]-postlabeling assay was important in showing the presence of very hydrophobic bis-substituted adducts which were not readily detected by [C-14]-labeling.