title: Quantitative high-performance liquid chromatography-electrospray ionization tandem mass spectrometry analysis of bis-N7-guanine DNA-DNA cross-links in white blood cells of cancer patients receiving cyclophosphamide therapy.
abstract: Cyclophosphamide (CPA) is a DNA alkylating agent widely used in cancer chemotherapy. CPA undergoes metabolic activation to phosphoramide mustard and nornitrogen mustard (NOR) which alkylate the N-7 position of guanine in DNA to produce N-[2-(N7-guaninyl) ethyl]-N-[2-hydroxyethyl]-amine (G-NOR-OH) monoadducts and N,N-bis[2-(N7-guaninyl) ethyl] amine cross-links (G-NOR-G). G-NOR-G cross-links are strongly cytotoxic and are thought to be responsible for the biological activity of CPA. In the present work, an isotope dilution high-performance liquid chromatography-electrospray ionization (positive ion) tandem mass spectrometry (HPLC-ESI(+)-MS/MS) methodology was developed to accurately quantify G-NOR-G adducts in human blood. In our approach, DNA extracted from white blood cells (5-20 microg) is spiked with an internal standard of [(15)N(10)]-G-NOR-G and subjected to thermal hydrolysis to release G-NOR-G adducts from the DNA backbone. Following solid phase extraction, G-NOR-G conjugates are quantified by capillary HPLC-ESI-MS/MS in the selected reaction monitoring mode. The application of the new methodology is demonstrated for DNA extracted from blood of three cancer patients receiving 50-60 mg/kg of intravenous CPA. The highest numbers of G-NOR-G adduct (up to 18 adducts per 10(6) normal nucleotides) were observed 4-8 h following CPA administration, followed by a gradual decrease over time, probably due to adduct hydrolysis, DNA repair, and white blood cell death. This methodology will be useful for future investigations of the interindividual differences for CPA-induced DNA-DNA cross-linking.
authors.affiliation: Departments of Medicinal Chemistry, Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Bhaskar
authors.initials: B
authors.lastname: Malayappan
authors.forename: L'aurelle
authors.initials: L
authors.lastname: Johnson
authors.forename: Bei
authors.initials: B
authors.lastname: Nie
authors.forename: Dolly
authors.initials: D
authors.lastname: Panchal
authors.forename: Brock
authors.initials: B
authors.lastname: Matter
authors.forename: Pamala
authors.initials: P
authors.lastname: Jacobson
authors.forename: Natalia
authors.initials: N
authors.lastname: Tretyakova
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry; D003520:Cyclophosphamide / Q000494:pharmacology; D004247:DNA / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry*; D006801:Humans; D007962:Leukocytes / Q000737:chemistry*; D015394:Molecular Structure; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D006147:Guanine; D003520:Cyclophosphamide; D004247:DNA