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

Abstract

Calf thymus DNA was reacted in 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 N3 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.

Liquid chromatography-electrospray ionization-mass spectrometry: the future of DNA adduct detection

“…Due to the presence of the phosphate group negative ESI is used, which is in contrast to the base and 2 0 -deoxynucleoside adducts which are analysed using positive ESI. Siethoff et al showed that phosphotriester adducts as well as base adducts were formed following the reaction of phenyl glycidyl ether, a compound used in the paint and resin industry, with calf thymus DNA (145). The position of the phosphate group on the 2 0 -deoxyribose may affect the extent of fragmentation.…”

“…A similar approach was used by Barry et al to analyse enzymatically hydrolysed calf thymus DNA treated with benzo[a]pyrene diol epoxide for which the authors noted the presence of an adducted thymidine-guanine dinucleotide in addition to a 2 0 -deoxyguanosine 5 0 -monophosphate adduct of benzo[a]pyrene diol epoxide (197). Again the sample stacking approach in combination with CZE-ESI-MS was used by Deforce et al to detect the formation of adducts following the reaction of phenyl glycidyl ether with calf thymus DNA (145). The sample stacking approach is ideally suited for the analysis of 2 0 -deoxynucleotide DNA adducts due to the presence of a negative charge on the phosphate group and is discussed in detail by Wolf and Vorous (1995) (198).…”

The use of biomarkers for improved retrospective exposure assessment in epidemiological studies: summary of an ECETOC workshop

“…There appears to be no enzymatic repair of these adducts in eukaryotic systems. These adducts have been found for N-nitroso compounds (Beranek et al 1980, Singer 1985, cyanoethylene oxide (Yates et al 1994), cyclophosphamide (Maccubbin et al 1991) and phenyl glycidyl ether (Deforce et al 1998). Long lifetimes have already been demonstrated in vivo in human fibroblasts (Bodell et al 1979).…”

Analysis of nucleic acid constituents by on‐line capillary electrophoresis‐mass spectrometry

“…Deforce and co-workers [79][80][81] published detailed studies of the in vitro adduct formation between PGE and 2'-deoxynucleotides or DNA. CZE-ESI-MS and CZE-ESI-MS/MS in conjunction with sample stacking allowed separation and identification of a mixture of alkylated 2'-deoxynucleotides.…”

“…Additional hydrolysis with snake venom phosphodiesterase (SVP) removed the dinucleotides, which could not be completely hydrolyzed by the nuclease P1 enzyme. For dGMP two alkylation products were found, an N-7-and an N 2 -or O 6 -alkylated adduct [80]. The reaction products present in the supernatant (versus the analysis of the pellet so far) after DNA precipitation were analyzed the same way after preconcentration using SPE.…”