title: LC-MS/MS for the detection of DNA interstrand cross-links formed by 8-methoxypsoralen and UVA irradiation in human cells.
abstract: DNA interstrand cross-links (ICLs) are induced by many carcinogens and anitcancer drugs. ICL is a covalent linkage of both strands of DNA, preventing DNA strand separation during transcription and replication; thus, it is extremely cytotoxic in vivo. Psoralen and its derivatives are widely applied for the clinical treatment of several skin diseases and cutaneous T cell lymphoma, and they are also commonly used as model compounds for the study of ICL. Upon UVA photoactivation, 8-methoxypsoralen alkylates both strands of DNA at the 5,6-double bond of thymidines at the 5'-TpA-3' site, generating monoadducts and ICLs. Here we developed a method utilizing HPLC-tandem mass spectrometry, combined with nuclease P1 digestion, to assess the formation of ICL in DNA of human skin melanoma cells exposed to 500 ng/mL 8-methoxypsoralen and UVA irradiation. We were able to quantify ICL, in the form of tetranucleotide, at the level of 1 lesion/10(6) unmodified nucleobases using a low-microgram quantity of DNA. In addition, our results revealed that the formation of ICL increased linearly with the UVA dose. The yield of ICL increased by 15-fold from 4.5 to 76 lesions/10(6) nucleotides when the UV dose was increased from 0.5 to 5 J/cm2. This is the first report of an LC-MS assay for the quantification of DNA interstrand cross-links. The specificity and accuracy of this high-throughput approach are advantageous over other methods for the detection of ICLs formed in vitro and in vivo.
authors.affiliation: Department of Chemistry, University of California, Riverside, California 92521-0403, USA.
authors.forename: Huachuan
authors.initials: H
authors.lastname: Cao
authors.forename: John E
authors.initials: JE
authors.lastname: Hearst
authors.forename: Laurence
authors.initials: L
authors.lastname: Corash
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D004247:DNA / Q000737:chemistry; D018736:DNA Adducts / Q000032:analysis / Q000737:chemistry*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008545:Melanoma / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008730:Methoxsalen / Q000032:analysis / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D012878:Skin Neoplasms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
chemical_list: C057863:8-methoxypsoralen-DNA; D018736:DNA Adducts; D004273:DNA, Neoplasm; D004247:DNA; D008730:Methoxsalen