Phagocytes Produce 5-Chlorouracil and 5-Bromouracil, Two Mutagenic Products of Myeloperoxidase, in Human Inflammatory Tissue

Abstract

Oxidative damage to DNA has been implicated in carcinogenesis during chronic inflammation. Epidemiological and biochemical studies suggest that one potential mechanism involves myeloperoxidase, a hemeprotein secreted by human phagocytes. In this study, we demonstrate that human neutrophils use myeloperoxidase to oxidize uracil to 5-chlorouracil in vitro. Uracil chlorination by myeloperoxidase or reagent HOCl exhibited an unusual pH dependence, being minimal at pH ϳ5, but increasing markedly under either acidic or mildly basic conditions. This bimodal curve suggests that myeloperoxidase initially produces HOCl, which subsequently chlorinates uracil by acid-or base-catalyzed reactions. Human neutrophils use myeloperoxidase and H 2 O 2 to chlorinate uracil, suggesting that nucleobase halogenation reactions may be physiologically relevant. Using a sensitive and specific mass spectrometric method, we detected two products of myeloperoxidase, 5-chlorouracil and 5-bromouracil, in neutrophil-rich human inflammatory tissue. Myeloperoxidase is the most likely source of 5-chlorouracil in vivo because halogenated uracil is a specific product of the myeloperoxidase system in vitro. In contrast, previous studies have demonstrated that 5-bromouracil could be generated by either eosinophil peroxidase or myeloperoxidase, which preferentially brominates uracil at plasma concentrations of halide and under moderately acidic conditions. These observations indicate that the myeloperoxidase system promotes nucleobase halogenation in vivo. Because 5-chlorouracil and 5-bromouracil can be incorporated into nuclear DNA, and these thymine analogs are well known mutagens, our observations raise the possibility that halogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation.

Neuronal expression of myeloperoxidase is increased in Alzheimer's disease

“…Increased levels of several oxidation products have been found in AD brain including protein carbonyls (Smith et al 1998), o,o¢-dityrosine (Hensley et al 1998), AGEs (Vitek et al 1994;Yan et al 1994), 3-nitrotyrosine , lipid oxidation products Pratico et al 1998), and oxidized DNA (Mecocci et al 1994;Gabbita et al 1998). It is noteworthy that myeloperoxidase is capable of generating all of these products (Heinecke 1999;Henderson et al 2003) and that the localization of myeloperoxidase in the AD brain is similar to the localization of oxidative stress markers reported in the diseased brain: amyloid plaques (Smith et al 1994;Vitek et al 1994;Wong et al 2001), neurofilbrillary tangles (Good et al 1996;Sayre et al 1997;Smith et al 1997), and neuronal cytoplasm Smith et al 1997Smith et al , 1998. Our observation that myeloperoxidase is markedly unregulated in AD raises the possibility that the enzyme is one pathway for oxidative damage in this disorder.…”

Oxidative Cross-linking of Tryptophan to Glycine Restrains Matrix Metalloproteinase Activity

“…Chlorinated amino acids and nucleobases identical to those formed by myeloperoxidase or HOCl in vitro have been detected in inflamed human tissue (22,23), but remarkably little is known about the reaction of HOCl with tryptophan residues in proteins (24 -26). Our studies demonstrate that HOCl targets tryptophan residues adjacent to glycine residues in the primary sequence.…”

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

“…Recent studies have shown that in addition to oxidation, DNA bases can be damaged by inflammation-mediated reactive halogen species (7,8,10) under physiological conditions. The introduction of halogen atoms into a molecule complicates the appearance of the mass spectrum due to the higher abundance of heavy isotopes of chlorine and bromine (Fig.…”

“…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.…”