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: Huachuan Cao, John E Hearst, Laurence Corash, Yinsheng Wang
• Journal: Analytical chemistry (Anal Chem) Vol. 80 Issue 8 Pg. 2932-8 (Apr 15 2008) ISSN: 1520-6882 [Electronic] United States
• PMID: 18324836 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• 8-methoxypsoralen-DNA
• DNA Adducts
• DNA, Neoplasm
• DNA
• Methoxsalen

Topics

• Chromatography, Liquid (methods)
• DNA (chemistry)
• DNA Adducts (analysis, chemistry)
• DNA, Neoplasm (chemistry, genetics)
• Humans
• Melanoma (chemistry, genetics, metabolism, pathology)
• Methoxsalen (analysis, blood, chemistry, metabolism)
• Skin Neoplasms (chemistry, genetics, metabolism, pathology)
• Tandem Mass Spectrometry (methods)
• Tumor Cells, Cultured
• Ultraviolet Rays