The effect of D-penicillamine on human myeloperoxidase, a mechanism for the efficacy of the drug in rheumatoid arthritis

We investigated the effect of D-penicillamine on the ability of myeloperoxidase, purified from human leukocytes, to catalyse the oxidation of chloride ions to hypochlorite (HOCl) in the presence of H2O2. It is shown that, due to the interaction of D-penicillamine with both myeloperoxidase itself and HOCl, the chlorinating activity of myeloperoxidase in the presence of H2O2 and chloride ions is prevented. A concentration of 100 microM D-penicillamine inhibits the chlorinating activity of myeloperoxidase completely, which Is due to the stabilization of Compound II, an inactive form of the enzyme. In addition, HOCl reacts directly with D-penicillamine. Analysis of the reaction products of D-penicillamine and HOCl showed that D-penicillamine was oxidized to penicillamine disulphide and penicillamine sulphinic acid, and eventually deaminated (indicated by the release of ammonia). Lower concentrations of D-penicillamine (10 microM) inhibited myeloperoxidase less, but still acted as effective scavengers of HOCl. In very low concentrations (1 microM), D-penicillamine did not scavenge HOCl effectively, but rather stimulated the chlorinating activity of myeloperoxidase. However, when instead of D-penicillamine a comparable amount of ascorbate was added, a similar but even larger stimulation was observed. Since the concentration of free D-penicillamine in serum from rheumatoid patients treated with this drug is about 20 microM (Saetre, R. and Rabenstein, D.L. (1978) Anal. Chem. 50, 276-280), the therapeutic effect of D-penicillamine may be due to the protection of tissues against the reactive HOCl released by activated granulocytes at inflammation sites.     

8-Chloroadenine: a novel product formed from hypochlorous acid-induced damage to calf thymus DNA

Hypochlorous acid (HOCl) is formed by the action of the enzyme myeloperoxidase on hydrogen peroxide and chloride ions. It has been shown to be highly bactericidal and cytotoxic by a variety of mechanisms, one of which, may be the modification of DNA. Previously we have demonstrated by GC-MS analysis that exposure of calf thymus DNA to HOCl causes extensive pyrimidine modification, including 5-chlorocytosine formation. Using GC-MS analysis, we now demonstrate the formation of an additional chlorinated base product, 8-Cl adenine. The addition of 50 μM HOCl was sufficient to produce a significant increase in this product. The reaction of HOCl with adenine in calf thymus DNA was shown to be rapid with the reaction complete after 1 min. pH-dependence studies suggest HOCl rather than its conjugate base (OCl-) to be responsible for 8-Cl adenine formation. Other commercially available chlorinated base products, 6-Cl guanine or 2-Cl adenine were not detected. Therefore, 8-Cl adenine might prove a useful biomarker for studying the role of reactive chlorine species (RCS) during inflammatory processes.     

Modeling the reactions of superoxide and myeloperoxidase in the neutrophil phagosome: implications for microbial killing

Neutrophils kill bacteria by ingesting them into phagosomes where superoxide and cytoplasmic granule constituents, including myeloperoxidase, are released. Myeloperoxidase converts chloride and hydrogen peroxide to hypochlorous acid (HOCl), which is strongly microbicidal. However, the role of oxidants in killing and the species responsible are poorly understood and the subject of current debate. To assess what oxidative mechanisms are likely to operate in the narrow confines of the phagosome, we have used a kinetic model to examine the fate of superoxide and its interactions with myeloperoxidase. Known rate constants for reactions of myeloperoxidase have been used and substrate concentrations estimated from neutrophil morphology. In the model, superoxide is generated at several mm/s. Most react with myeloperoxidase, which is present at millimolar concentrations, and rapidly convert the enzyme to compound III. Compound III turnover by superoxide is essential to maintain enzyme activity. Superoxide stabilizes at approximately 25 microM and hydrogen peroxide in the low micromolar range. HOCl production is efficient if there is adequate chloride supply, but further knowledge on chloride concentrations and transport mechanisms is needed to assess whether this is the case. Low myeloperoxidase concentrations also limit HOCl production by allowing more hydrogen peroxide to escape from the phagosome. In the absence of myeloperoxidase, superoxide increases to >100 microM but hydrogen peroxide to only approximately 30 microM. Most of the HOCl reacts with released granule proteins before reaching the bacterium, and chloramine products may be effectors of its antimicrobial activity. Hydroxyl radicals should form only after all susceptible protein targets are consumed.     

Enzymatic and Bactericidal Activity of Monomeric and Dimeric Forms of Myeloperoxidase

Enzymatic and bactericidal activities of mature, dimeric myeloperoxidase (MPO) and its monomeric form have been compared. Dimeric MPO was isolated from HL-60 cells. Hemi-MPO obtained from dimeric MPO by reductive cleavage of a disulfide bond between protomeric subunits was used as the monomeric form. Both peroxidase and halogenating (chlorinating) activities of MPO were assayed, each by two methods. Bactericidal activity of the MPO/Н₂О₂/Cl￣ system was tested using the Escherichia coli laboratory strain DH5α. No difference in the enzymatic and bactericidal activity between dimeric MPO and hemi- MPO was found. Both forms of the enzyme also did not differ in the resistance to HOCl, the main product of MPO. HOCl caused a dose-dependent decrease in peroxidase and chlorinating activity, and the pattern of this decrease was identical for dimeric MPO and hemi-MPO. At the equal heme concentration, the hemi- MPO/Н₂О₂/Cl￣ system demonstrated a somewhat higher bactericidal effect than the dimeric MPO/Н₂О₂/Cl￣ system. This is most likely explained by higher probability of contacts between the bacterial surface and hemi-MPO molecules, since at the same heme concentration the number of hemi-MPO molecules is 2-fold higher than that of dimeric MPO molecules. Using Western-blotting with antibodies to MPO, we have shown, for the first time, that the dimeric molecule of MPO could be cleaved into two monomeric subunits by HOCl, most probably due to oxidation of the disulfide bond between these subunits. This suggests that appearance in blood of MPO with mass corresponding to its monomer may result from the damage of dimeric MPO by reactive halogen species, especially upon their overproduction inducing oxidative/halogenative stress in inflammatory diseases.     

8-Chloroadenine: a novel product formed from hypochlorous acid-induced damage to calf thymus DNA

Hypochlorous acid (HOCl) is formed by the action of the enzyme myeloperoxidase on hydrogen peroxide and chloride ions. It has been shown to be highly bactericidal and cytotoxic by a variety of mechanisms, one of which, may be the modification of DNA. Previously we have demonstrated by GC-MS analysis that exposure of calf thymus DNA to HOCl causes extensive pyrimidine modification, including 5-chlorocytosine formation. Using GC-MS analysis, we now demonstrate the formation of an additional chlorinated base product, 8-Cl adenine. The addition of 50 μM HOCl was sufficient to produce a significant increase in this product. The reaction of HOCl with adenine in calf thymus DNA was shown to be rapid with the reaction complete after 1 min. pH-dependence studies suggest HOCl rather than its conjugate base (OCl-) to be responsible for 8-Cl adenine formation. Other commercially available chlorinated base products, 6-Cl guanine or 2-Cl adenine were not detected. Therefore, 8-Cl adenine might prove a useful biomarker for studying the role of reactive chlorine species (RCS) during inflammatory processes.     

Production of glutathione sulfonamide and dehydroglutathione from GSH by myeloperoxidase-derived oxidants and detection using a novel LC-MS/MS method

GSH is rapidly oxidized by HOCl (hypochlorous acid), which is produced physiologically by the neutrophil enzyme myeloperoxidase. It is converted into, mainly, oxidized glutathione. Glutathione sulfonamide is an additional product that is proposed to be covalently bonded between the cysteinyl thiol and amino group of the gamma-glutamyl residue of GSH. We have developed a sensitive liquid chromatography-tandem MS assay for the detection and quantification of glutathione sulfonamide as well as GSH and GSSG. The assay was used to determine whether glutathione sulfonamide is a major product of the reaction between GSH and HOCl, and whether it is formed by other two-electron oxidants. At sub-stoichiometric ratios of HOCl to GSH, glutathione sulfonamide accounted for up to 32% of the GSH that was oxidized. It was also formed when HOCl was generated by myeloperoxidase and its yield increased with the flux of oxidant. Of the other oxidants tested, only hypobromous acid and peroxynitrite produced substantial amounts of glutathione sulfonamide, but much less than with HOCl. Chloramines were able to generate detectable levels only when at a stoichiometric excess over GSH. We conclude that glutathione sulfonamide is sufficiently selective for HOCl to be useful as a biomarker for myeloperoxidase activity in biological systems. We have also identified a novel oxidation product of GSH with a molecular weight two mass units less than GSH, which we have consequently named dehydroglutathione. Dehydroglutathione represented a few percent of the total products and was formed with all of the oxidants except H2O2.     

The novel HOCl fluorescent probe CAN induced A549 apoptosis by inhibiting chlorination activity of MPO

Hypochlorous acid (HOCl) is an important signaling molecule for cell survival. However, it has been reported that excessive HOCl contributes to a variety of diseases such as cancers. And in cancer cells, the level of HOCl is much higher than that in normal cells. Here a coumarin-based fluorescent probe 7-Diethylamino-3-(2,3-dihydro-1H-perimidin-2-yl)-chromen-2-one (CAN) was successfully developed for HOCl detection. The probe could be oxidized by HOCl to induce significant change in its fluorescence profile, which made it feasible for ratiometric detecting HOCl. CAN (below 1 µM) did not affect cell viability and had good capacity in ratiometric detection of HOCl in RAW 264.7 cells. CAN induced A549 apoptosis and inhibited tumor growth in vitro and in vivo. And CAN could decrease the chlorination activity of myeloperoxidase (MPO) in A549. These findings suggested that CAN (below 1 µM) would develop into a HOCl probe. High activity of MPO and level of HOCl might be helpful for A549 survival. A549 could be induced apoptosis by reducing the HOCl level by CAN. It implies a new anticancer strategy by targeting HOCl.     

The Fluorescein-derived Dye Aminophenyl Fluorescein Is a Suitable Tool to Detect Hypobromous Acid (HOBr)-producing Activity in Eosinophils

The specific detection of peroxidase activity in human granulocytes is essential to elucidate their role in innate immune responses, immune regulation, and inflammatory diseases. The halogenating activity of myeloperoxidase in neutrophils can be determined by the novel fluorescent probe aminophenyl fluorescein (APF). Thereby non-fluorescent APF is oxidized by HOCl to form fluorescein. We successfully verified that APF equally detects the hypobromous acid (HOBr)-producing activity of eosinophil granulocytes. This was revealed by three different approaches. First, we investigated the conversion of non-fluorescent APF into fluorescein by HOCl and HOBr by means of fluorescence and mass spectrometry approaches. Thereby comparable chemical mechanisms were observed for both acids. Furthermore in vitro kinetic studies were used to detect the halogenating activity of myeloperoxidase and eosinophil peroxidase by using APF. Here the dye well reflected the different substrate specificities of myeloperoxidase and eosinophil peroxidase regarding chloride and bromide. Finally, peroxidase activities were successfully detected in phorbol ester-stimulated neutrophils and eosinophils using flow cytometry. Thereby inhibitory studies confirmed the peroxidase-dependent oxidation of APF. To sum up, APF is a promising tool for further evaluation of the halogenating activity of peroxidases in both neutrophils and eosinophils.     

Hypochlorous acid as a precursor of free radicals in living systems

Hypochlorous acid (HOCl) is produced in the human body by the family of mammalian heme peroxidases, mainly by myeloperoxidase, which is secreted by neutrophils and monocytes at sites of inflammation. This review discusses the reactions that occur between HOCl and the major classes of biologically important molecules (amino acids, proteins, nucleotides, nucleic acids, carbohydrates, lipids, and inorganic substances) to form free radicals. The generation of such free radical intermediates by HOCl and other reactive halogen species is accompanied by the development of halogenative stress, which causes a number of socially important diseases, such as cardiovascular, neurodegenerative, infectious, and other diseases usually associated with inflammatory response and characterized by the appearance of biomarkers of myeloperoxidase and halogenative stress. Investigations aimed at elucidating the mechanisms regulating the activity of enzyme systems that are responsible for the production of reactive halogen species are a crucial step in opening possibilities for control of the development of the body’s inflammatory response.     

Chlorinative stress: An under appreciated mediator of neurodegeneration?

Oxidative stress has been implicated as playing a role in neurodegenerative disorders, such as ischemic stroke, Alzheimer's, Huntington's, and Parkinson's disease. Persuasive evidences have shown that microglial-mediated oxidative stress contributes significantly to cell loss and accompanying cognitive decline characteristic of the diseases. Based on the facts that (i) levels of catalytically active myeloperoxidase are elevated in diseased brains and (ii) myeloperoxidase polymorphism is associated with the risk of developing neurodegenerative disorders, HOCl as a major oxidant produced by activated phagocytes in the presence of myeloperoxidase is therefore suggested to be involved in neurodegeneration. Its association with neurodegeneration is further showed by elevated level of 3-chlorotyrosine (bio-marker of HOCl in vivo) in affected brain regions as well as HOCl scavenging ability of neuroprotectants, desferrioxamine and uric acid. In this review, we will summary the current understanding concerning the association of HOCl and neuronal cell death where production of HOCl will lead to further formation of reactive nitrogen and oxygen species. In addition, HOCl also causes tissue destruction and cellular damage leading cell death.     

A study of the effect of ceruloplasmin and lactoferrin on the chlorination activity of leukocytic myeloperoxidase using the chemiluminescence method

The chlorination activity of free myeloperoxidase and myeloperoxidase bound with ceruloplasmin or with both ceruloplasmin and lactoferrin has been studied by luminal-dependent chemiluminescence. It was shown that the addition of hydrogen peroxide to the "myeloperoxidase + Cl- + luminal" system is accompanied by a fast flash of light emission. In the absence of myeloperoxidase or Cl-, the flash intensity was considerably reduced. The inhibitor of myeloperoxidase NaN3, the HOCl scavengers taurine and methionine, and guaiacol, a substrate for peroxidation cycle of myeloperoxidase, prevented luminescence. These results suggest that the generation of luminescence was due to the halogenating activity of myeloperoxidase, and hence, the flash light sum may serve as a measure of chlorination activity of myeloperoxidase. The activity of myeloperoxidase was suppressed by ceruloplasmin. Lactoferrin exhibited no significant influence on the myeloperoxidase activity, nor did it prevent the inhibitory effect of ceruloplasmin when they both were combined with myeloperoxidase. These data were confirmed using alternative approaches for evaluating the myeloperoxidase activity, namely, the assessment of peroxidation activity and the taurine chlorination assay. It is noteworthy that the inhibitory effect of ceruloplasmin on chlorination and peroxidation activities of myeloperoxidase is seen with the latter, traditional approaches only if ceruloplasmin is present in a large excess relative to myeloperoxidase, whereas the chemiluminescence method allows the detection of the inhibitory effect of ceruloplasmin using lower proportions of the protein with respect to myeloperoxidase, which are close to the stoichiometry of the myeloperoxidase/ceruloplasmin and the myeloperoxidase'ceruloplasmin'lactoferrin complexes.     

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.     

Hypochlorous acid oxygenates the cysteine switch domain of pro-matrilysin (MMP-7). A mechanism for matrix metalloproteinase activation and atherosclerotic plaque rupture by myeloperoxidase

Myeloperoxidase uses hydrogen peroxide (H2O2) to generate hypochlorous acid (HOCl), a potent cytotoxic oxidant. We demonstrate that HOCl regulates the activity of matrix metalloproteinase-7 (MMP-7, matrilysin) in vitro, suggesting that this oxidant activates MMPs in the artery wall. Indeed, both MMP-7 and myeloperoxidase were colocalized to lipid-laden macrophages in human atherosclerotic lesions. A highly conserved domain called the cysteine switch has been proposed to regulate MMP activity. When we exposed a synthetic peptide that mimicked the cysteine switch to HOCl, HPLC analysis showed that the thiol residue reacted rapidly, generating a near-quantitative yield of products. Tandem mass spectrometric analysis identified the products as sulfinic acid, sulfonic acid, and a dimer containing a disulfide bridge. In contrast, the peptide reacted slowly with H2O2, and the only product was the disulfide. Moreover, HOCl markedly activated pro-MMP-7, an MMP expressed at high levels in lipid-laden macrophages in vivo. Tandem mass spectrometric analysis of trypsin digests revealed that the thiol residue of the enzyme's cysteine switch domain had been converted to sulfinic acid. Thiol oxidation was associated with autolytic cleavage of pro-MMP-7, strongly suggesting that oxygenation activates the latent enzyme. In contrast, H2O2 failed to oxidize the thiol residue of the protein or activate the enzyme. Thus, HOCl activates pro-MMP-7 by converting the thiol residue of the cysteine switch to sulfinic acid. This activation mechanism is distinct from the well-studied proteolytic cleavage of MMP pro-enzymes. Our observations raise the possibility that HOCl generated by myeloperoxidase contributes to MMP activation, and therefore to plaque rupture, in the artery wall. HOCl and other oxidants might regulate MMP activity by the same mechanism in a variety of inflammatory conditions.     

Identification of proteins susceptible to thiol oxidation in endothelial cells exposed to hypochlorous acid and N-chloramines

Hypochlorous acid (HOCl) is a potent oxidant produced by the enzyme myeloperoxidase, which is released by neutrophils under inflammatory conditions. Although important in the immune system, HOCl can also damage host tissue, which contributes to the development of disease. HOCl reacts readily with free amino groups to form N-chloramines, which also cause damage in vivo, owing to the extracellular release of myeloperoxidase and production of HOCl. HOCl and N-chloramines react readily with cellular thiols, which causes dysfunction via enzyme inactivation and modulation of redox signaling processes. In this study, the ability of HOCl and model N-chloramines produced on histamine and ammonia at inflammatory sites, to oxidize specific thiol-containing proteins in human coronary artery endothelial cells was investigated. Using a proteomics approach with the thiol-specific probe, 5-iodoacetamidofluorescein, we show that several proteins including peptidylprolyl isomerase A (cyclophilin A), protein disulfide isomerase, glyceraldehyde-3-phosphate dehydrogenase and galectin-1 are particularly sensitive to oxidation by HOCl and N-chloramines formed at inflammatory sites. This will contribute to cellular dysfunction and may play a role in inflammatory disease pathogenesis.     

Inhibition of hyaluronan uptake in lymphatic tissue by chondroitin sulphate proteoglycan.

Stimulated neutrophils discharge large quantities of superoxide (O2.-), which dismutates to form H2O2. In combination with Cl-, H2O2 is converted into the potent oxidant hypochlorous acid (HOCl) by the haem enzyme myeloperoxidase. We have used an H2O2 electrode to monitor H2O2 uptake by myeloperoxidase, and have shown that in the presence of Cl- this accurately represents production of HOCl. Monochlorodimedon, which is routinely used to assay production of HOCl, inhibited H2O2 uptake by 95%. This result confirms that monochlorodimedon inhibits myeloperoxidase, and that the monochlorodimedon assay grossly underestimates the activity of myeloperoxidase. With 10 microM-H2O2 and 100 mM-Cl-, myeloperoxidase had a neutral pH optimum. Increasing the H2O2 concentration to 100 microM lowered the pH optimum to pH 6.5. Above the pH optimum there was a burst of H2O2 uptake that rapidly declined due to accumulation of Compound II. High concentrations of H2O2 inhibited myeloperoxidase and promoted the formation of Compound II. These effects of H2O2 were decreased at higher concentrations of Cl-. We propose that H2O2 competes with Cl- for Compound I and reduces it to Compound II, thereby inhibiting myeloperoxidase. Above pH 6.5, O2.- generated by xanthine oxidase and acetaldehyde prevented H2O2 from inhibiting myeloperoxidase, increasing the initial rate of H2O2 uptake. O2.- allowed myeloperoxidase to function optimally with 100 microM-H2O2 at pH 7.0. This occurred because, as previously demonstrated, O2.- prevents Compound II from accumulating by reducing it to ferric myeloperoxidase. In contrast, at pH 6.0, where Compound II did not accumulate, O2.- retarded the uptake of H2O2. We propose that by generating O2.- neutrophils prevent H2O2 and other one-electron donors from inhibiting myeloperoxidase, and ensure that this enzyme functions optimally at neutral pH.     

Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase

Hypochlorous acid (HOCl) produced via the enzyme myeloperoxidase is a major antibacterial oxidant produced by neutrophils, and Met residues are considered primary amino acid targets of HOCl damage via conversion to Met sulfoxide. Met sulfoxide can be repaired back to Met by methionine sulfoxide reductase (Msr). Catalase is an important antioxidant enzyme; we show it constitutes 4-5% of the total Helicobacter pylori protein levels. msr and katA strains were about 14- and 4-fold, respectively, more susceptible than the parent to killing by the neutrophil cell line HL-60 cells. Catalase activity of an msr strain was much more reduced by HOCl exposure than for the parental strain. Treatment of pure catalase with HOCl caused oxidation of specific MS-identified Met residues, as well as structural changes and activity loss depending on the oxidant dose. Treatment of catalase with HOCl at a level to limit structural perturbation (at a catalase/HOCl molar ratio of 1:60) resulted in oxidation of six identified Met residues. Msr repaired these residues in an in vitro reconstituted system, but no enzyme activity could be recovered. However, addition of GroEL to the Msr repair mixture significantly enhanced catalase activity recovery. Neutrophils produce large amounts of HOCl at inflammation sites, and bacterial catalase may be a prime target of the host inflammatory response; at high concentrations of HOCl (1:100), we observed loss of catalase secondary structure, oligomerization, and carbonylation. The same HOCl-sensitive Met residue oxidation targets in catalase were detected using chloramine-T as a milder oxidant.     

Reactive oxygen species are partially involved in the bacteriocidal action of hypochlorous acid.

Hypochlorous acid (HOCl) is probably the most widely used disinfectant worldwide and has an important role in inflammatory reaction and in human resistance to infection. However, the nature and mechanisms of its bactericidal activity are still poorly understood. Bacteria challenged aerobically with HOCl concentrations ranging from 9.5 to 76 microM exhibit higher ability to form colonies anaerobically than aerobically. Conversely, aerobic plating greatly increased lethality after an anaerobic HOCl challenge, although anaerobic survival did not depend on whether HOCl exposure was aerobic or anaerobic. Even a short transient exposure to air after anaerobic HOCl challenge reduced anaerobic survival, indicative of immediate deleterious effects of oxygen. Exposure to HOCl can cause lethal DNA damage as judged by the fact that recA sensitivity to HOCl was oxygen dependent. Antioxidant defenses such as reduced glutathione and glucose-6-phosphate dehydrogenase were depleted or inactivated at 10 microM HOCl, while other activities, such as superoxide dismutase, dropped only above 57 microM HOCl. Cumulative deficiencies in superoxide dismutase and glucose-6-phosphate dehydrogenase rendered strains hypersensitive to HOCl. This indicates that part of HOCl toxicity on Escherichia coli is mediated by reactive oxygen species during recovery.     

Comparative study of HOCl-inflicted damage to bacterial DNA ex vivo and within cells

The prospects for using bacterial DNA as an intrinsic probe for HOCl and secondary oxidants/chlorinating agents associated with it has been evaluated using both in vitro and in vivo studies. Single-strand and double-strand breaks occurred in bare plasmid DNA that had been exposed to high levels of HOCl, although these reactions were very inefficient compared to polynucleotide chain cleavage caused by the OH-generating reagent, peroxynitrite. Plasmid nicking was not increased when intact Escherichia coli were exposed to HOCl; rather, the amount of recoverable plasmid diminished in a dose-dependent manner. At concentration levels of HOCl exceeding lethal doses, genomic bacterial DNA underwent extensive fragmentation and the amount of precipitable DNA-protein complexes increased several-fold. The 5-chlorocytosine content of plasmid and genomic DNA isolated from HOCl-exposed E. coli was also slightly elevated above controls, as measured by mass spectrometry of the deaminated product, 5-chlorouracil. However, the yields were not dose-dependent over the bactericidal concentration range. Genomic DNA recovered from E. coli that had been subjected to phagocytosis by human neutrophils occasionally showed small increases in 5-chlorocytosine content when compared to analogous cellular reactions where myeloperoxidase activity was inhibited by azide ion. Overall, the amount of isolable 5-chlorouracil from the HOCl-exposed bacterial cells was far less than the damage manifested in polynucleotide bond cleavage and cross-linking.     

Chlorination of guanosine and other nucleosides by hypochlorous acid and myeloperoxidase of activated human neutrophils. Catalysis by nicotine and trimethylamine

Activated human neutrophils secrete myeloperoxidase, which generates HOCl from H2O2 and Cl(-). We have found that various (2'-deoxy)nucleosides react with HOCl to form chlorinated (2'-deoxy)nucleosides, including novel 8-chloro(2'-deoxy)guanosine, 5-chloro(2'-deoxy)cytidine, and 8-chloro(2'-deoxy)adenosine formed in yields of 1.6, 1.6, and 0.2%, respectively, when 0.5 mM nucleoside reacted with 0.5 mM HOCl at pH 7.4. The relative chlorination, oxidation, and nitration activities of HOCl, myeloperoxidase, and activated human neutrophils in the presence and absence of nitrite were studied by analyzing 8-chloro-, 8-oxo-7,8-dihydro-, and 8-nitro-guanosine, respectively, using guanosine as a probe. 8-Chloroguanosine was always more easily formed than 8-oxo-7,8-dihydro- or 8-nitro-guanosine. Using electrospray ionization tandem mass spectrometry, we show that several chlorinated nucleosides including 8-chloro(2'-deoxy)guanosine are formed following exposure of isolated DNA or RNA to HOCl. Micromolar concentrations of tertiary amines such as nicotine and trimethylamine dramatically enhanced chlorination of free (2'-deoxy)nucleosides and nucleosides in RNA by HOCl. As the G-463A polymorphism of the MPO gene, which strongly reduces myeloperoxidase mRNA expression, is associated with a reduced risk of lung cancer, chlorination damage of DNA /RNA and nucleosides by myeloperoxidase and its enhancement by nicotine may be important in the pathophysiology of human diseases associated with tobacco habits.