ANALYSIS OF THE DNA-ADDUCTS OF PHENYL GLYCIDYL ETHER IN A CALF THYMUSDNA HYDROLYSATE BY CAPILLARY-ZONE-ELECTROPHORESIS ELECTROSPRAY MASS-SPECTROMETRY - EVIDENCE FOR PHOSPHATE ALKYLATION

Calf thymus DNA was reacted irt vitro with phenyl glycidyl ether (PGE) and was hydrolysed enzymatically, to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I) and nuclease P1, The adducts we re concentrated using solid phase extraction (SPE), on a polystyrene d ivinylbenzene copolymer in order to remove the unmodified nucleotides. The adducts could be identified using capillary zone electrophoresis- electrospray tandem mass spectrometry (CZE ES-MS/MS), using sample sta cking. In addition to the base alkylated 2'-deoxynucleotides present i n the DNA-hydrolysate, also phosphate alkylated 2'-deoxynucleotide add ucts were identified for TMP and dAMP, An additional adduct, dUMP alky lated on the uridine moiety was found originating from the hydrolytic deamination of dCMP alkylated on N-3 Of the cytosine moiety, Enzymatic hydrolysis using nuclease P1 was incomplete as shown by the presence of dinucleotides alkylated on the base moiety, They were successfully hydrolysed to the corresponding 2'-deoxynucleotides by snake venom pho sphodiesterase (SVP), Data are shown indicating that alkylations on th e pyrimidine bases were more resistant to enzymatic hydrolysis with nu clease P1 than the purine alkylated products.