title: Alkylation of DNA by melphalan: investigation of capillary liquid chromatography-electrospray ionization tandem mass spectrometry in the study of the adducts at the nucleoside level.
abstract: Nitrogen mustards are among the oldest cancer chemotherapeutic agents and remain the drugs of choice for treatment of many human cancers. A serious complication of treatment with nitrogen mustards is the increased risk of a secondary leukaemia in long-term survivors because not all alkylating agent interactions with DNA result in cell death. In an earlier study 2'-deoxy-5'-mononucleotide/melphalan adducts have been analysed by us by LC-ES MSMS. In this work we want to present the first results of the analysis of the corresponding 2'-deoxynucleoside/melphalan adducts from DNA hydrolysates by column switching/capillary LC-ES tandem mass spectrometry. Nucleosides, compared to nucleotides, give better chromatographic results and show a good sensitivity under electrospray (+) [ES(+)] ionisation. Several adducts were identified under ES(+) conditions. Mono-alkylated nucleoside adducts alkylated at the base moiety were identified for dGuo, dCyd and dAdo. Structures were identified by recording the low-energy CAD product ion scans. Also a mono-alkylated nucleotide pdA with alkylation position at the phosphate moiety could be detected. This proves that in the case of phosphate alkylation the enzymatic dephosphorylation reaction was inhibited. A Jurkat cell suspension was treated with melphalan (1 mM) and incubated at 37 degrees C (5% CO(2)). After 6 and 48 h, the DNA was isolated and enzymatically hydrolysed. The corresponding nucleoside pool was evaluated with the developed LC-MS method. In the 48-h experiment, one adduct could be identified as a N-7 alkylated dGuo. In the 6-h experiment, no adducts could be found. Additional experiments were done wherein Jurkat-DNA, isolated from a non-treated cell culture, was treated with melphalan. These results were analogous with the data found in melphalan-treated calf thymus DNA. Additionally, we tried to determine the exact alkylation position by interpreting high-resolution fragmentation spectra.
authors.affiliation: Department of Chemistry, Nucleoside Research and Mass Spectrometry Unit, University of Antwerp, Groenenborgerlaan 171, Belgium.
authors.forename: Bart
authors.initials: B
authors.lastname: Van den Driessche
authors.forename: Filip
authors.initials: F
authors.lastname: Lemière
authors.forename: Walter
authors.initials: W
authors.lastname: Van Dongen
authors.forename: Eddy L
authors.initials: EL
authors.lastname: Esmans
mesh_terms: D000478:Alkylation; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry*; D006801:Humans; D019169:Jurkat Cells; D008558:Melphalan / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D018906:Antineoplastic Agents, Alkylating; D018736:DNA Adducts; D004247:DNA; D008558:Melphalan