LC−MS/MS for the Detection of DNA Interstrand Cross-Links Formed by 8-Methoxypsoralen and UVA Irradiation in Human Cells
Open accessAbstract
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.
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ESI-MS/MS with HPLC after nuclease P1 digestion overcomes the limitation of fluorescence detection.
“This has been recently overcome by the use of the versatile, specific and accurate electrospray ionization tandem mass spectrometry (ESI-MS ⁄ MS) technique in association with HPLC following nuclease P1 digestion of DNA (169).”
From Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions<sup>†</sup> (2012) · cites this paper as [169]
ICLs induced by UVA activation of 8-MOP in human skin melanoma cells were detected as tetranucleotides with a threshold sensitivity close to 1 ICL per 10^6 normal nucleosides.
“Therefore, ICLs induced by UVA activation of 8-MOP in the DNA of human skin melanoma cells were detected as tetranucleotides with a threshold sensitivity close to 1 ICL per 10 6 normal nucleosides.”
From Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions<sup>†</sup> (2012) · cites this paper as [169]
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Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions<sup>†</sup>
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- Authors
- Cao, H., Hearst, J. E., Corash, L., & Wang, Y.
- Affiliations
- University of California, Riverside (US)
- Corresponding author
- Wang, Yinsheng
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2008-03-07
- Volume
- 80
- Issue
- 8
- Pages
- 2932-2938
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC8812037
- OpenAlex
- W2001556358
- Cited by
- 70
- References
- 36
- Citations by year
- 2025: 2 · 2024: 2 · 2023: 1 · 2022: 5 · 2021: 6 · 2020: 2 · 2019: 4 · 2018: 3 · 2017: 2 · 2016: 5 · 2015: 5 · 2014: 7 · 2013: 7 · 2012: 4
- FWCI
- 1.59
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Cancer Institute (R01CA101864, R01 CA96906, R01CA096906)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Carcinogens and Genotoxicity Assessment
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- interstrand cross-links, 8-methoxypsoralen, UVA irradiation, DNA adduct quantification, melanoma cells, LC-MS/MS assay, UV dose dependence, thymine dimerization, DNA damage detection
- MeSH terms
- Chromatography, Liquid, DNA, DNA, Neoplasm, Humans, Melanoma, Methoxsalen, Skin Neoplasms, Tumor Cells, Cultured, Ultraviolet Rays, DNA Adducts, Tandem Mass Spectrometry
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