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.