Analysis of the DNA adducts of phenyl glycidyl ether in a calf thymus DNA 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 were concentrated using solid phase extraction (SPE), on a polystyrene divinylbenzene 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 stacking. In addition to the base alkylated 2'-deoxynucleotides present in the DNA-hydrolysate, also phosphate alkylated 2'-deoxynucleotide adducts were identified for TMP and dAMP, An additional adduct, dUMP alkylated 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 phosphodiesterase (SVP), Data are shown indicating that alkylations on the pyrimidine bases were more resistant to enzymatic hydrolysis with nuclease P1 than the purine alkylated products