5-Chlorouracil, a Marker of DNA Damage from Hypochlorous Acid during Inflammation

Abstract

Hypochlorous acid (HOCl), generated from H 2 O 2 and Cl ؊ by myeloperoxidase in activated neutrophils, causes tissue damage during inflammation. We have developed a simple, sensitive (ϳ0.2 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 carcinogensis associated with chronic inflammation.

Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

“…Our limit of detection is 50 fmol (5×10 −14 mol) of starting material, corresponding to 500 amol (5×10 −16 mol) on the column (data not shown). This compares favorably with the limits of detection observed for other nucleobases analyzed by GC-NCI-MS (27,38), which range from the high zeptomole (zepto = 10 −21 ) to low femtomole (femto = 10 −15 ) levels.…”

“…More recently, investigators have exploited the extreme sensitivity of negative chemical ionization-mass spectrometry methods for the measurement of damaged DNA bases (33,38). These methods require derivatization with halogenated derivatives such as 3,5-bis (trifluoromethyl)benzyl or pentafluorobenzyl bromide.…”

“…Inlet temperature was 250°C and GC-MS interface temperature 300°C. Oven conditions were slightly modified from Jiang et al (38) as follows: 80°C hold 2 min, 20°C/min ramp to 220°C with 3 min hold, 30°C/min ramp to 250°C with 2 min hold, and 40°C/min ramp to 300°C with 3 min hold. EI source temp was 230°C and quad temp 150°C, with electron energy at 70 eV.…”

Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

“…Oligonucleotides were detritylated using 80% aqueous acetic acid at room temperature for 30 min and then purified using a Waters XTerra C-18 column and a gradient of 0 to 20% acetonitrile in water (44). The base composition of the oligonucleotides reported here was verified by formic acid hydrolysis followed by GC/MS analysis (7,10,14,16,21).…”

“…Modified bases can occur in DNA as a consequence of chemical damage as well as incorporation of base analogs, many of which are important drugs (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Synthetic oligonucleotides containing modified bases are commonly used to determine the biological consequences of base substitutions and DNA damage.…”