title: Mapping DNA adducts of mitomycin C and decarbamoyl mitomycin C in cell lines using liquid chromatography/ electrospray tandem mass spectrometry.
abstract: The antitumor antibiotic and cancer chemotherapeutic agent mitomycin C (MC) alkylates and crosslinks DNA, forming six major MC-deoxyguanosine adducts of known structures in vitro and in vivo. Two of these adducts are derived from 2,7-diaminomitosene (2,7-DAM), a nontoxic reductive metabolite of MC formed in cells in situ. Several methods have been used for the analysis of MC-DNA adducts in the past; however, a need exists for a safer, more comprehensive and direct assay of the six-adduct complex. Development of an assay, based on mass spectrometry, is described. DNA from EMT6 mouse mammary tumor cells, Fanconi Anemia-A fibroblasts, normal human fibroblasts, and MCF-7 human breast cancer cells was isolated after MC or 10-decarbamoyl mitomycin C (DMC) treatment of the cells, digested to nucleosides, and submitted to liquid chromatography electrospray-tandem mass spectrometry. Two fragments of each parent ion were monitored ("multiple reaction monitoring"). Identification and quantitative analysis were based on a standard mixture of six adducts, the preparation of which is described here in detail. The lower limit of detection of adducts is estimated as 0.25 pmol. Three initial applications of this method are reported as follows: (i) differential kinetics of adduct repair in EMT6 cells, (ii) analysis of adducts in MC- or DMC-treated Fanconi Anemia cells, and (iii) comparison of the adducts generated by treatment of MCF-7 breast cancer cells with MC and DMC. Notable results are the following: Repair removal of the DNA interstrand cross-link and of the two adducts of 2,7-DAM is relatively slow; both MC and DMC generate DNA interstrand cross-links in human fibroblasts, Fanconi Anemia-A fibroblasts, and MCF-7 cells as well as EMT6 cells; and DMC shows a stereochemical preference of linkage to the guanine-2-amino group opposite from that of MC.
authors.affiliation: Department of Chemistry, Hunter College, City University of New York, New York 10021, USA.
authors.forename: Manuel M
authors.initials: MM
authors.lastname: Paz
authors.forename: Sweta
authors.initials: S
authors.lastname: Ladwa
authors.forename: Elise
authors.initials: E
authors.lastname: Champeil
authors.forename: Yanfeng
authors.initials: Y
authors.lastname: Liu
authors.forename: Sara
authors.initials: S
authors.lastname: Rockwell
authors.forename: Ernest K
authors.initials: EK
authors.lastname: Boamah
authors.forename: Jill
authors.initials: J
authors.lastname: Bargonetti
authors.forename: John
authors.initials: J
authors.lastname: Callahan
authors.forename: John
authors.initials: J
authors.lastname: Roach
authors.forename: Maria
authors.initials: M
authors.lastname: Tomasz
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D018736:DNA Adducts / Q000096:biosynthesis* / Q000737:chemistry; D004249:DNA Damage; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000737:chemistry / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015674:Mammary Neoplasms, Animal / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016685:Mitomycin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008937:Mitomycins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D003432:Cross-Linking Reagents; D018736:DNA Adducts; D004273:DNA, Neoplasm; D008937:Mitomycins; C067795:10-decarbamoylmitomycin C; D016685:Mitomycin