5-Chlorouracil, a marker of DNA damage from hypochlorous acid during inflammation. A gas chromatography-mass spectrometry assay

• Published in: The Journal of biological chemistry 2003-08, Vol.278 (35), p.32834-32840
• Main Authors: Jiang, Qing, Blount, Ben C, Ames, Bruce N
• Format: Article
• Language: English
• Subjects: Alkaline Phosphatase - metabolism; Animals; Biomarkers; Carrageenan - pharmacology; Cell Line; Chlorine - chemistry; DNA - metabolism; DNA Damage; Dose-Response Relationship, Drug; Electrons; Gas Chromatography-Mass Spectrometry - methods; Hydrogen Peroxide - chemistry; Hydrolysis; Hypochlorous Acid - pharmacology; Inflammation - metabolism; Mice; Models, Chemical; Neutrophils - metabolism; Nucleosides - chemistry; Oxygen - metabolism; Rats; Single-Strand Specific DNA and RNA Endonucleases - metabolism; Thymidine Phosphorylase - metabolism; Time Factors; Uracil - analogs & derivatives; Uracil - chemistry; Uracil - pharmacology
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M304021200

Hypochlorous acid (HOCl), generated from H2O2 and Cl- by myeloperoxidase in activated neutrophils, causes tissue damage during inflammation. We have developed a simple, sensitive (approximately 0.2 fmol on column) and specific GC-MS assay for the detection of 5-chlorouracil (5-ClUra), a signature product of HOCl-mediated damage to nucleobases. In this assay, 5-ClUra is released from isolated DNA by a digestion with nuclease P1, alkaline phosphatase, and thymidine phosphorylase (TP), or from chlorinated nucleosides in biological fluids by TP. The freed 5-ClUra is derivatized with 3, 5-bis-(trifluoromethyl)-benzyl bromide, which is detected by negative chemical ionization mass spectrometry. The assay can be used to simultaneously detect other halogenated uracils including bromouracil. Using this assay, we showed that 5-ClUra is generated by the reaction of low micromolar HOCl with (deoxy)cytidine, (deoxy)uridine, and DNA. In cell cultures, an increase of 5-ClUra was detected in DNA when cells were treated with sublethal doses of HOCl and allowed to proliferate. The elevation of 5-ClUra was markedly accentuated when physiologically relevant concentrations of (deoxy)uridine, (deoxy) cytidine, uracil, or cytosine were present in the medium during HOCl treatment. In the carrageenan-induced inflammation model in rats, chlorinated nucleosides was significantly increased, compared with controls, in the exudate fluid isolated from the inflammation site. Our study provides the direct evidence that chlorinated nucleosides are found in the inflammation site and can be incorporated in DNA during cell/tissue proliferation. These findings may be relevant to the carcinogenesis associated with chronic inflammation.