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.

Characterization of Nitrogen Mustard Formamidopyrimidine Adduct Formation of Bis(2-chloroethyl)ethylamine with Calf Thymus DNA and a Human Mammary Cancer Cell Line

“…18, 20, 49-52, 56, 59, 64 Recently, tandem mass spectrometry with stable isotope dilution has been employed to identify and quantitate some of these DNA adducts. 53, 54, 61-63 Collectively, the studies indicate that these electrophiles react predominantly at the N7 atom of dG, although N3-dA modification can also be significant. 66 The toxicity of mustard agents has been attributed to interstrand DNA cross-links.…”

“…One study reported the identification of bis-N7-guanine cross-links in white blood cells of cancer patients receiving cyclophosphamide therapy, 53 and sulfur mustard (bis-(2-chloroethyl)-sulfide, SM) also forms bis[2-(guanin-7-yl)ethyl]sulfide cross-links in tissues of rodents, and urine of rabbits exposed to SM. 62, 63 The potential formation of ring-opened FapyG adducts was not addressed in those studies.…”

“…The labile N7-G and N3-A nitrogen and sulfur mustard adducts and their cross-links are often thermally hydolyzed from DNA under neutral 8-10, 51, 57, 61 or acidic 9, 10, 24, 46, 48, 50, 52, 53, 59 conditions. Alternatively, enzymatic digestion to the nucleosides has also been employed.…”

Effects of Four Chemotherapeutic Agents, Bleomycin, Etoposide, Cisplatin, and Cyclophosphamide, on DNA Damage and Telomeres in a Mouse Spermatogonial Cell Line1

“…Our data reveal that alkylating agents and bleomycin directly damage telomeres. Telomeres and the Terc gene may be at increased risk of the damage induced by alkylating agents, such as guanine DNA-DNA cross-links, because of their G-rich DNA sequences [39,40]. Although the target of bleomycin is still unclear, there is evidence that bleomycin treatment can induce DNA damage to the telomere region [15,36].…”

Medical Applications of Infrared Spectral Imaging of Individual Cells

“…Figure 5.18 shows the scores plot of live cells treated with an anticancer drug, cyclophosphamide monohydrate, and an untreated control group of cells. Cyclophosphamide monohydrate is an alkylating agent that acts on DNA at the seventh position of nitrogen in guanine bases [70][71][72] mutations [70][71][72][73][74]. It is a common chemotherapeutic in the treatment of various cancers [73,74].…”