Production of brominating intermediates by myeloperoxidase. A transhalogenation pathway for generating mutagenic nucleobases during inflammation

The existence of interhalogen compounds was proposed more than a century ago, but no biological roles have been attributed to these highly oxidizing intermediates. In this study, we determined whether the peroxidases of white blood cells can generate the interhalogen gas bromine chloride (BrCl). Myeloperoxidase, the heme enzyme secreted by activated neutrophils and monocytes, uses H2O2 and Cl(-) to produce HOCl, a chlorinating intermediate. In contrast, eosinophil peroxidase preferentially converts Br(-) to HOBr. Remarkably, both myeloperoxidase and eosinophil peroxidase were able to brominate deoxycytidine, a nucleoside, and uracil, a nucleobase, at plasma concentrations of Br(-) (100 microM) and Cl(-) (100 mM). The two enzymes used different reaction pathways, however. When HOCl brominated deoxycytidine, the reaction required Br(-) and was inhibited by taurine. In contrast, bromination by HOBr was independent of Br(-) and unaffected by taurine. Moreover, taurine inhibited 5-bromodeoxycytidine production by the myeloperoxidase-H2O2-Cl(-)- Br(-) system but not by the eosinophil peroxidase-H2O2-Cl(-)-Br(-) system, indicating that bromination by myeloperoxidase involves the initial production of HOCl. Both HOCl-Br(-) and the myeloperoxidase-H2O2-Cl(-)-Br(-) system generated a gas that converted cyclohexene into 1-bromo-2-chlorocyclohexane, implicating BrCl in the reaction. Moreover, human neutrophils used myeloperoxidase, H2O2, and Br(-) to brominate deoxycytidine by a taurine-sensitive pathway, suggesting that transhalogenation reactions may be physiologically relevant. 5-Bromouracil incorporated into nuclear DNA is a well known mutagen. Our observations therefore raise the possibility that transhalogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation.     

Eosinophil peroxidase catalyzes bromination of free nucleosides and double-stranded DNA

Chronic parasitic infections are a major risk factor for cancer development in many underdeveloped countries. Oxidative damage of DNA may provide a mechanism linking these processes. Eosinophil recruitment is a hallmark of parasitic infections and many forms of cancer, and eosinophil peroxidase (EPO), a secreted hemoprotein, plays a central role in oxidant production by these cells. However, mechanisms through which EPO may facilitate DNA oxidation have not been fully characterized. Here, we show that EPO effectively uses plasma levels of bromide as a cosubstrate to brominate bases in nucleotides and double-stranded DNA, forming several stable novel brominated adducts. Products were characterized by HPLC with on-line UV spectroscopy and electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). Ring assignments for brominated purine bases as their 8-bromo adducts were identified by NMR spectroscopy. Using stable isotope dilution LC/ESI/MS/MS, we show that while guanine is the preferred purine targeted for bromination as a free nucleobase, 8-bromoadenine is the major purine oxidation product generated following exposure of double-stranded DNA to either HOBr or the EPO/H(2)O(2)/Br(-) system. Bromination of nucleobases was inhibited by scavengers of hypohalous acids such as the thioether methionine, but not by a large molar excess of primary amines. Subsequently, N-monobromoamines were demonstrated to be effective brominating agents for both free nucleobases and adenine within intact DNA. A rationale for selective modification of adenine, but not guanine, in double-stranded DNA based upon stereochemical criteria is presented. Collectively, these results suggest that specific brominated DNA bases may serve as novel markers for monitoring oxidative damage of DNA and the nucleotide pool by brominating oxidants.     

Phagocytes produce 5-chlorouracil and 5-bromouracil,two mutagenic products of myeloperoxidase,in human inflammatory tissue

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 approximately 5, 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 H2O2 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.     

Synthesis and anticancer evaluation of certain alpha-methylene-gamma-(4-substituted phenyl)-gamma-butyrolactone bearing thymine, uracil, and 5-bromouracil

Certain alpha-methylene-gamma-(4-substituted phenyl)-gamma-butyrolactone bearing thymine, uracil, and 5-bromouracil were synthesized and evaluated for their anticancer activity. These compounds demonstrated a strong growth inhibitory activity against leukemia cell lines. The anticancer potency for the substituents of the lactone C(gamma)-phenyl is in an order of 4-Ph > 4-Cl, 4-Br > 4-Me, 4-NO2 > 4-F. For the pyrimidine portion, 5-bromouracil is more potent than uracil and thymine.     

3-Bromotyrosine and 3,5-dibromotyrosine are major products of protein oxidation by eosinophil peroxidase: potential markers for eosinophil-dependent tissue injury in vivo

Detection of specific reaction products is a powerful approach for dissecting out pathways that mediate oxidative damage in vivo. Eosinophil peroxidase (EPO), an abundant protein secreted from activated eosinophils, has been implicated in promoting oxidative tissue injury in conditions such as asthma, allergic inflammatory disorders, cancer, and helminthic infections. This unique heme protein amplifies the oxidizing potential of H2O2 by utilizing plasma levels of Br- as a cosubstrate to form potent brominating agents. Brominated products might thus serve as powerful tools for identifying sites of eosinophil-mediated tissue injury in vivo; however, structural identification and characterization of specific brominated products formed during EPO-catalyzed oxidation have not yet been reported. Here we explore the role of EPO and myeloperoxidase (MPO), a related leukocyte protein, in promoting protein oxidative damage through bromination and demonstrate that protein tyrosine residues serve as endogenous traps of reactive brominating species forming stable ring-brominated adducts. Exposure of the amino acid L-tyrosine to EPO, H2O2, and physiological concentrations of halides (100 mM Cl-, 相似文献   

Eosinophil peroxidase-derived reactive brominating species target the vinyl ether bond of plasmalogens generating a novel chemoattractant,alpha-bromo fatty aldehyde

Plasmalogens are a subclass of glycerophospholipids that are enriched in the plasma membrane of many mammalian cells. The vinyl ether bond of plasmalogens renders them susceptible to oxidation. Accordingly, it was hypothesized that reactive brominating species, a unique oxidant formed at the sites of eosinophil activation, such as in asthma, might selectively target plasmalogens for oxidation. Here we show that reactive brominating species produced by the eosinophil peroxidase system of activated eosinophils attack the vinyl ether bond of plasmalogens. Reactive brominating species produced by eosinophil peroxidase target the vinyl ether bond of plasmalogens resulting in the production of a neutral lipid and lysophosphatidylcholine. Chromatographic and mass spectrometric analyses of this neutral lipid demonstrated that it was 2-bromohexadecanal (2-BrHDA). Reactive brominating species produced by eosinophil peroxidase attacked the plasmalogen vinyl ether bond at acidic pH. Bromide was the preferred substrate for eosinophil peroxidase, and chloride was not appreciably used even at a 1000-fold molar excess. Furthermore, 2-BrHDA production elicited by eosinophil peroxidase-derived reactive brominating species in the presence of 100 microM NaBr doubled with the addition of 100 mM NaCl. The potential physiological significance of this pathway was suggested by the demonstration that 2-BrHDA was produced by phorbol myristate acetate-stimulated eosinophils and by the demonstration that 2-BrHDA is a phagocyte chemoattractant. Taken together, the present studies demonstrate the targeting of the vinyl ether bond of plasmalogens by the reactive brominating species produced by eosinophil peroxidase and by activated eosinophils, resulting in the production of brominated fatty aldehydes.     

Bromination and chlorination reactions of myeloperoxidase at physiological concentrations of bromide and chloride

Myeloperoxidase and eosinophil peroxidase use hydrogen peroxide to oxidize halides and thiocyanate to their respective hypohalous acids. Myeloperoxidase produces mainly hypochlorous acid and hypothiocyanite. Hypobromous acid and hypothiocyanite are the major products of eosinophil peroxidase. We have investigated the ability of myeloperoxidase to produce hypobromous acid in the presence of physiological concentrations of chloride and bromide. In accord with previous studies, between pH 5 and 7, myeloperoxidase converted about 90% of available hydrogen peroxide to hypochlorous acid and the remainder to hypobromous acid. Above pH 7, there was an abrupt rise in the yield of hypobromous acid. At pH 7.8, it accounted for 40% of the hydrogen peroxide. Bromide, at physiological concentrations, promoted a dramatic increase in bromination of human serum albumin catalyzed by myeloperoxidase. The level of 3-bromotyrosine increased to 16-fold greater than that for 3-chlorotyrosine. Chlorination of tyrosyl residues was not affected by bromide. With reagent hypohalous acids, bromination of tyrosyl residues was considerably more facile than chlorination. Hypochlorous acid promoted bromination to only a limited extent, which ruled out transhalogenation as a substantive route to 3-bromotyrosine. Chloramines and bromamines were also formed on albumin. Bromamines decayed much faster than chloramines and rapidly gave rise to protein carbonyls. We conclude that at physiological concentrations of chloride and bromide, hypobromous acid can be a major oxidant produced by myeloperoxidase. Its production in vivo will depend on pH and the concentration of bromide. Once produced, hypobromous acid will react with proteins to form bromamines, carbonyls, and brominated tyrosine residues. Consequently, 3-bromotyrosine should be considered as an oxidative product of myeloperoxidase and cannot be used as a specific biomarker for eosinophil peroxidase.     

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.     

Comparison of low-density lipoprotein modification by myeloperoxidase-derived hypochlorous and hypobromous acids.

Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, catalyzes the oxidation of halides to hypohalous acids. At plasma concentrations of halides, hypochlorous acid (HOCl) is the major strong oxidant produced. In contrast, the related enzyme eosinophil peroxidase preferentially generates hypobromous acid (HOBr). Since reagent and MPO-derived HOCl converts low-density lipoprotein (LDL) to a potentially atherogenic form, we investigated the effects of HOBr on LDL modification. Compared to HOCl, HOBr caused 2-3-fold greater oxidation of tryptophan and cysteine residues of the protein moiety (apoB) of LDL and 4-fold greater formation of fatty acid halohydrins from the lipids in LDL. In contrast, HOBr was 2-fold less reactive than HOCl with lysine residues and caused little formation of N-bromamines. Nevertheless, HOBr caused an equivalent increase in the relative electrophoretic mobility of LDL as HOCl, which was not reversed upon subsequent incubation with ascorbate, in contrast to the shift in mobility caused by HOCl. Similar apoB modifications were observed with HOBr generated by MPO/H(2)O(2)/Br(-). In the presence of equivalent concentrations of Cl(-) and Br(-), modifications of LDL by MPO resembled those seen in the presence of Br(-) alone. Interestingly, even at physiological concentrations of the two halides (100 mM Cl(-), 100 microM Br(-)), MPO utilized a portion of the Br(-) to oxidize apoB cysteine residues. MPO also utilized the pseudohalide thiocyanate to oxidize apoB cysteine residues. Our data show that even though HOBr has different reactivities than HOCl with apoB, it is able to alter the charge of LDL, converting it into a potentially atherogenic particle.     

Highly sensitive chemiluminescence method for determining myeloperoxidase in human polymorphonuclear leukocytes.

Myeloperoxidase activity was assayed by a chemiluminescence method, using a cypridina luciferin analog as a chemiluminescence probe, after extraction from peripheral human polymorphonuclear leukocytes. The chemiluminescence method was based on the detection of 1O2 generated by myeloperoxidase-catalyzed HOBr formation followed by the interaction of HOBr with H2O2 at pH 4.5. With this method, myeloperoxidase in less than 100 polymorphonuclear leukocytes could be detected and myeloperoxidase in 10(6) polymorphonuclear leukocytes would be calculated to be 14.4 pmol. Eosinophil extract, which contains eosinophil peroxidase, catalyzed 1O2 generation to a great extent, compared with the polymorphonuclear leukocyte extract at pH 4.5. Myeloperoxidase activity in extract of neutrophil fraction could be greatly influenced by eosinophil contamination.     

The dehalogenation of halouracils by hydroxylamine buffers.

At room temperature, hydroxylamine dehalogenates 5-Br-and 5-I-uracil. 5-Cl-uracil reacts to a much less extent. Reaction with 5-F-uracil yields the 6-hydroxyamino-adduct as a product. Kinetics monitored spectrally indicate that dehalogenation involves the formation of a 5-halo-6-hydroxyamino-5, 6-dihydrouracil intermediate which then slowly dehalogenates. 5-Bromo-6-methoxy-5,6-dihydrothymine, a model for the above intermediate, also dehalogenates yielding thymine as a product.Hydroxylamine (NH₂OH), a mutagenic agent (1,2) reacts with pyrimidine rings promoting such reactions as the formation of 5,6-dihydro-N⁴-hydroxy-6-hydroxyaminocytosine from cytosine (3,4) and both urea and isoxazoles from uracil derivatives (2,5,6). It is believed to be unreactive toward 5-substituted uracil derivatives (2,5,6) but has been reported to cause the dehalogenation of 5-bromouracil derivatives yielding Br^? and uracil as products (2,7,8). The object of this report is to demonstrate the generality of NH₂OH addition to the 5-halouracils with the subsequent dehalogenation of both 5-Br-and 5-I-uracil; reactions which appear to proceed via pathways similar to bisulfite buffer mediated halouracil dehalogenation (9–13). A preliminary report of this work has appeared (14).     

A myeloperoxidase-specific assay based upon bromide-dependent chemiluminescence of luminol.

Measurement of myeloperoxidase (MPO; EC 1.11.1.7) activity is often used as a marker of neutrophil infiltration into tissues. However, most enzymatic assays for MPO are susceptible to interference from other peroxidases (including eosinophil peroxidase, EPX) and hemoproteins (such as hemoglobin and myoglobin) present in the tissues. In this report, we describe a bromide-dependent chemiluminescence (Br-CL) assay that uses luminol as a chemiluminescence probe. The assay can distinguish between MPO and nonspecific peroxidase reactions. The MPO-specific reaction is believed to proceed in two steps: (i) the enzymatic generation of hypobromous acid (HOBr) from KBr and H(2)O(2) at pH 5 and (ii) the spontaneous reaction of HOBr and H(2)O(2) with luminol to give a Br-CL signal. The assay is sufficiently sensitive to allow detection of MPO in <100 human neutrophils. Other peroxidases and hemoproteins do not interfere with the Br-CL signal. Although EPX can also oxidize bromide to generate HOBr, activities of MPO and EPX can be distinguished at different pHs. As a demonstration of the utility of the Br-CL assay, MPO activity was measured in murine tumors known to be infiltrated with neutrophils. A statistically significant correlation was seen between MPO activity and histological neutrophil counts in the tumors (r = 0.69, P < 0.01, n = 14). The assay should have wide application for measuring the neutrophil content of tissues.     

Degradation of extracellular matrix and its components by hypobromous acid

EPO (eosinophil peroxidase) and MPO (myeloperoxidase) are highly basic haem enzymes that can catalyse the production of HOBr (hypobromous acid). They are released extracellularly by activated leucocytes and their binding to the polyanionic glycosa-minoglycan components of extracellular matrix (proteoglycans and hyaluronan) may localize the production of HOBr to these materials. It is shown in the present paper that the reaction of HOBr with glycosaminoglycans (heparan sulfate, heparin, chondroitin sulfate and hyaluronan) generates polymer-derived N-bromo derivatives (bromamines, dibromamines, N-bromosulfon-amides and bromamides). Decomposition of these species, which can occur spontaneously and/or via one-electron reduction by low-valent transition metal ions (Cu+ and Fe2+), results in polymer fragmentation and modification. One-electron reduction of the N-bromo derivatives generates radicals that have been detected by EPR spin trapping. The species detected are consistent with metal ion-dependent polymer fragmentation and modification being initiated by the formation of nitrogen-centred (aminyl, N-bromoaminyl, sulfonamidyl and amidyl) radicals. Previous studies have shown that the reaction of HOBr with proteins generates N-bromo derivatives and results in fragmentation of the polypeptide backbone. The reaction of HOBr with extracellular matrix synthesized by smooth muscle cells in vitro induces the release of carbohydrate and protein components in a time-dependent manner, which is consistent with fragmentation of these materials via the formation of N-bromo derivatives. The degradation of extracellular matrix glycosaminoglycans and proteins by HOBr may contribute to tissue damage associated with inflammatory diseases such as asthma.     

The role of bisulfite in the debromination of 5-bromouracil. The debromination of 5-bromo-5,6-dihydrouracil-6-sulfonate

5-Bromouracil is dehalogenated in the presence of bisulfite buffers to yield uracil which subsequently adds bisulfite to form 5,6-dihydrouracil-6-sulfonate. Presumably, 5-bromo-5,6-dihydrouracil-6-sulfonate is an intermediate in uracil formation. Kinetic data indicate that the disappearance of 5-bromouracil in the presence of bisulfite buffers is second order with respect to total bisulfite concentration, thus indicating the participation of 2 moles of either sulfite or bisulfite in the overall reaction, Iodometric titrations of total bisulfite combined with spectral analysis of the various pyrimidine and dihydropyrimidine species present indicate that, in addition to the total bisulfite required to form 5,6-dihydrouracil-6-sulfonate, an additional mole of sulfite is consumed per mole of 5-bromouracil dehalogenated. These data combined with the finding that sulfate is generated during dehalogenation are indicative of a pathway for the dehalogenation of the intermediate 5-bromo-5,6-dihydro-uracil-6-sulfonate which involves the attack of sulfite either directly or via an intervening molecule of water to yield uracil. Subsequent reactions of halogen-containing intermediates yield sulfate and bromide as final products of the reaction.     

Hypobromous acid and bromamine production by neutrophils and modulation by superoxide

MPO (myeloperoxidase) catalyses the oxidation of chloride, bromide and thiocyanate to their respective hypohalous acids. We have investigated the generation of HOBr by human neutrophils in the presence of physiological concentrations of chloride and bromide. HOBr was trapped with taurine and detected by monitoring the bromination of 4-HPAA (4-hydroxyphenylacetic acid). With 100 microM bromide and 140 mM chloride, neutrophils generated HOBr and it accounted for approx. 13% of the hypohalous acids they produced. Addition of SOD (superoxide dismutase) doubled the amount of HOBr detected. Therefore we investigated the reaction of superoxide radicals with a range of bromamines and bromamides and found that superoxide radicals stimulated the decomposition of these species, with this occurring in a time- and dose-dependent manner. The protection afforded by SOD against such decay demonstrates that these processes are superoxide-radical-dependent. These data are consistent with neutrophils generating HOBr at sites of infection and inflammation. Both HOBr and bromamines/bromamides have the potential to react with superoxide radicals to form additional radicals that may contribute to inflammatory tissue damage.     

Effects of oxidation of lysozyme by hypohalous acids and haloamines on enzymatic activity and aggregation

Hen egg white lysozyme (HEL), an antibacterial enzyme, is a prototype protein for studying the physical and chemical events that underlie the formation of amyloid fibril aggregates. Here, we studied alterations in enzymatic activity and aggregation provoked by oxidation of HEL by hypochlorous acid (HOCl), hypobromous acid (HOBr), taurine chloramine (Tau-NHCl), taurine monobromamine (Tau-NHBr), and taurine dibromamine (Tau-NBr(2)). Addition of only 4-fold molar excess of Tau-NHBr or Tau-NBr(2) to HEL caused complete depletion of its intrinsic fluorescence, whereas HOCl and HOBr caused 40%-50% bleaching. Tau-NHCl was unable to oxidize lysozyme. The selective effect of bromamines on tryptophan residues had a direct effect on enzymatic activity; bromamines were about two-fold more effective as inhibitors of lysozyme than the acid precursors. The oxidation of HEL by HOCl and HOBr was more effective regarding the aggregation of the protein, which was evidenced by increased turbidity, Rayleigh scattering, and anisotropy. The aggregates presented spectroscopic properties that suggested the formation of amyloid fibrils, as measured by the thioflavin assay. In conclusion, the capacity of Tau-NHBr and Tau-NBr(2) as inhibitors of the bactericidal activity of HEL could represent a role in the exacerbation of pulmonary infection, since leukocytes are rich sources of both taurine and HOBr. Moreover, the oxidation of HEL by just a small excess of hypohalous acids, a condition that could be found in inflammatory sites, may represent a new pathway for initiation of aggregation.     

Reaction of 3′,5′-di-O-acetyl-2′-deoxyguansoine with hypobromous acid

Hypobromous acid (HOBr) is formed by eosinophil peroxidase and myeloperoxidase in the presence of H₂O₂, Cl^?, and Br^? in the host defense system of humans, protecting against invading bacteria. However, the formed HOBr may cause damage to DNA and its components in the host. When a guanine nucleoside (3′,5′-di-O-acetyl-2′-deoxyguansoine) was treated with HOBr at pH 7.4, spiroiminodihydantoin, guanidinohydantoin/iminoallantoin, dehydro-iminoallantoin, diimino-imidazole, amino-imidazolone, and diamino-oxazolone nucleosides were generated in addition to an 8-bromoguanine nucleoside. The major products were spiroiminodihydantoin under neutral conditions and guanidinohydantoin/iminoallantoin under mildly acidic conditions. All the products were formed in the reaction with HOCl in the presence of Br^?. These products were also produced by eosinophil peroxidase or myeloperoxidase in the presence of H₂O₂, Cl^?, and Br^?. The results suggest that the products other than 8-bromoguanine may also have importance for mutagenesis by the reaction of HOBr with guanine residues in nucleotides and DNA.     

Activated leukocytes oxidatively damage DNA, RNA, and the nucleotide pool through halide-dependent formation of hydroxyl radical

A variety of chronic inflammatory conditions are associated with an increased risk for the development of cancer. Because of the numerous links between DNA oxidative damage and carcinogenesis, a potential role for leukocyte-generated oxidants in these processes has been suggested. In the present study, we demonstrate a novel free transition metal ion-independent mechanism for hydroxyl radical ((*)OH)-mediated damage of cellular DNA, RNA, and cytosolic nucleotides by activated neutrophils and eosinophils. The mechanism involves reaction of peroxidase-generated hypohalous acid (HOCl or HOBr) with intracellular superoxide (O(2)(*)(-)) forming (*)OH, a reactive oxidant species implicated in carcinogenesis. Incubation of DNA with either isolated myeloperoxidase (MPO) or eosinophil peroxidase (EPO), plasma levels of halides (Cl(-) and Br(-)), and a cell-free O(2)(*)(-) -generating system resulted in DNA oxidative damage. Formation of 8-hydroxyguanine (8-OHG), a mutagenic base which is a marker for (*)OH-mediated DNA damage, required peroxidase and halides and occurred in the presence of transition metal chelators (DTPA +/- desferrioxamine), and was inhibited by catalase, superoxide dismutase (SOD), and scavengers of hypohalous acids. Similarly, exposure of DNA to either neutrophils or eosinophils activated in media containing metal ion chelators resulted in 8-OHG formation through a pathway that was blocked by peroxidase inhibitors, hypohalous acid scavengers, and catalytically active (but not heat-inactivated) catalase and SOD. Formation of 8-OHG in target cells (HA1 fibroblasts) occurred in all guanyl nucleotide-containing pools examined following exposure to both a low continuous flux of HOCl (at sublethal doses, as assessed by [(14)C]adenine release and clonogenic survival), and hyperoxia (to enhance intracellular O(2)(*)(-) levels). Mitochondrial DNA, poly A RNA, and the cytosolic nucleotide pool were the primary targets for oxidation. Moreover, modest but statistically significant increases in the 8-OHG content of nuclear DNA were also noted. These results suggest that the peroxidase-H(2)O(2)-halide system of leukocytes is a potential mechanism contributing to the well-established link between chronic inflammation, DNA damage, and cancer development.     

Mutagenic properties of 5-halogenuracils: correlated quantum chemical ab initio study

The relative stability of all possible 5-bromouracil tautomers was studied theoretically in a gas phase, in a microhydrated environment (with one water molecule), and in bulk water. Tautomer structures were determined by gradient optimization at the correlated ab initio quantum chemical level with an extended basis set of atomic orbitals. The role of water was examined by using a self-consistent reaction field method. The relative stabilization and free energies in the gas phase, the microhydrated environment, and the bulk water clearly support the preference of the canonical keto form of 5-bromouracil in all mentioned environments. An increased abundance of enol tautomers when passing from uracil to 5-bromouracil is not supported by our calculations. Thus, the tautomeric model of the mutagenic activity of 5-bromouracil proposed previously [Hu et al. Biochemistry (2004) 43, 6361] can be refuted. The validity of other mutagenic models was also discussed, and finally a new mechanism for explaining the mutagenic activity of halogenuracils based on their different behaviors in triplet excited states was suggested.     

The role of reactive N-bromo species and radical intermediates in hypobromous acid-induced protein oxidation

Activated eosinophils, and hypobromous acid (HOBr) generated by these cells, have been implicated in the tissue injury in asthma, allergic reactions, and some infections. Proteins are major targets for this oxidant, but limited information is available on the mechanisms of damage and intermediates formed. Reaction of HOBr with proteins is shown to result in the formation of bromamines and bromamides, from side-chain and backbone amines and amides, and 3-bromo- and 3,5-dibromo-Tyr, from Tyr residues; these materials account for ca. 70% of the oxidant consumed. Protein carbonyls, dityrosine, and 3,4-dihydroxyphenylalanine are also formed, though these are minor products (<5% of HOBr added). With BSA, extensive (selective and nonspecific) protein fragmentation and limited aggregation are also observed. The bromamines/bromamides are unstable and induce further oxidation and free radical formation as detected by EPR spin trapping. Evidence was obtained for the generation of nitrogen-centered radicals on side-chain and backbone amide groups of amino acids, peptides, and proteins. These radicals readily undergo rearrangement reactions to give carbon-centered radicals. With proteins, alpha-carbon (backbone) radicals are detected, which may play a role in protein fragmentation. A novel damage transfer pathway from Gln side-chain amide groups to backbone sites was also observed.