Reagent HOCl Research Articles

An improved method for the detection of myeloperoxidase chlorinating activity in biological systems using the redox probe hydroethidine

Conversion of the redox probe hydroethidine (HE) to 2-chloroethidium (2-Cl-E+) by myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) provides comparable specificity and superior sensitivity to measurement of 3-chlorotyrosine (3-Cl-Tyr), the gold standard biomarker for MPO chlorinating activity in biological systems. However, a limitation of the former method is the complex mixture of products formed by the reaction of HE with reagent HOCl, coupled with the difficult purification of 2-Cl-E+ from this mixture for analytical purposes. This limitation prompted us to test whether 2-Cl-E+ could be formed by reaction of HE with the strong and widely used chlorinating agent, N-chlorosuccinimide (NCS). Unexpectedly, such reaction yielded 2-chlorohydroethidine (2-Cl-HE) as the major product in addition to 2-Cl-E+, as assessed by high performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR). 2-Cl-HE was also observed to be the major chlorination product formed from HE with both reagent and enzymatically generated HOCl, just as it was formed ex vivo in different healthy and diseased mouse and human tissues upon incubation with glucose/glucose oxidase to generate a flux of hydrogen peroxide (H2O2). Quantification of 2-Cl-HE plus 2-Cl-E+ improved the sensitivity of the HE-based method compared with measurement of only 2-Cl-E+. Moreover, 2-chlorodimidium (2-Cl-D+) was developed as a practical internal standard instead of the previously used internal standard, deuterated 2-Cl-E+ (d5-2-Cl-E+). Overall, the present study describes an improved method for the detection of MPO/chlorinating activity in biological systems of health and disease.

Mycobacterium smegmatis Resists the Bactericidal Activity of Hypochlorous Acid Produced in Neutrophil Phagosomes.

Neutrophils are often the major leukocyte at sites of mycobacterial infection, yet little is known about their ability to kill mycobacteria. In this study we have investigated whether the potent antibacterial oxidant hypochlorous acid (HOCl) contributes to killing of Mycobacterium smegmatis when this bacterium is phagocytosed by human neutrophils. We found that M. smegmatis were ingested by neutrophils into intracellular phagosomes but were killed slowly. We measured a t 1/2 of 30 min for the survival of M. smegmatis inside neutrophils, which is 5 times longer than that reported for Staphylococcus aureus and 15 times longer than Escherichia coli Live-cell imaging indicated that neutrophils generated HOCl in phagosomes containing M. smegmatis; however, inhibition of HOCl production did not alter the rate of bacterial killing. Also, the doses of HOCl that are likely to be produced inside phagosomes failed to kill isolated bacteria. Lethal doses of reagent HOCl caused oxidation of mycothiol, the main low-m.w. thiol in this bacterium. In contrast, phagocytosed M. smegmatis maintained their original level of reduced mycothiol. Collectively, these findings suggest that M. smegmatis can cope with the HOCl that is produced inside neutrophil phagosomes. A mycothiol-deficient mutant was killed by neutrophils at the same rate as wild-type bacteria, indicating that mycothiol itself is not the main driver of M. smegmatis resistance. Understanding how M. smegmatis avoids killing by phagosomal HOCl could provide new opportunities to sensitize pathogenic mycobacteria to destruction by the innate immune system.

Evaluating the bactericidal action of hypochlorous acid in culture media

The bactericidal activity of the physiological oxidant hypochlorous acid (HOCl) is commonly studied in a variety of laboratory media. Reactive with numerous targets, HOCl will rapidly lose its toxicity via reduction or be converted to chloramines and other less toxic species. The objective of this study was to test the influence of various media, temperature and reaction time on the toxicity of HOCl. After incubating bacteria in media dosed with reagent HOCl, the bactericidal outcome was measured by colony forming ability. In parallel, we determined the HOCl and chloramine content after dosing media alone. Our results showed that more reagent HOCl was required to kill bacteria in culture media than in aqueous buffer, and this corresponded to the lower concentration of reactive chlorine species achieved in the media. RPMI and MOPS minimal medium retained significant oxidising equivalents after HOCl-dosing, but more nutrient-rich media such as MEM, DMEM, LB and TSB, had higher scavenging capacity. Other factors that lowered the bactericidal strength of HOCl were longer lag-times and raised temperature when pre-dosing media, and insufficient incubation time of cells with the HOCl-treated media. In summary, we demonstrate that the choice of media as well as procedural details within experiments crucially impact the cellular toxicity of HOCl. These factors influence the nature and concentration of oxidants generated, and therefore are critical in affecting cellular responses.

Myeloperoxidase-derived damage to human plasma fibronectin: Modulation by protein binding and thiocyanate ions (SCN−)

Endothelial cell dysfunction is an early event in cardiovascular disease and atherosclerosis. The origin of this dysfunction is unresolved, but accumulating evidence implicates damaging oxidants, including hypochlorous acid (HOCl), a major oxidant produced by myeloperoxidase (MPO), during chronic inflammation. MPO is released extracellularly by activated leukocytes and binds to extracellular molecules including fibronectin, a major matrix glycoprotein involved in endothelial cell binding. We hypothesized that MPO binding might influence the modifications induced on fibronectin, when compared to reagent HOCl, with this including alterations to the extent of damage to protein side-chains, modified structural integrity, changes to functional domains, and impact on naïve human coronary artery endothelial cell (HCAEC) adhesion and metabolic activity. The effect of increasing concentrations of the alternative MPO substrate thiocyanate (SCN−), which might decrease HOCl formation were also examined. Exposure of fibronectin to MPO/H2O2/Cl− is shown to result in damage to the functionally important cell-binding and heparin-binding fragments, gross structural changes to the protein, and altered HCAEC adhesion and activity. Differences were observed between stoichiometric, and above-stoichiometric MPO concentrations consistent with an effect of MPO binding to fibronectin. In contrast, MPO/H2O2/SCN− induced much less marked changes and limited protein damage. Addition of increasing SCN− concentrations to the MPO/H2O2/Cl− system provided protection, with 20 μM of this anion rescuing damage to functionally-important domains, decreasing chemical modification, and maintaining normal HCAEC behavior. Modulating MPO binding to fibronectin, or enhancing SCN− levels at sites of inflammation may therefore limit MPO-mediated damage, and be of therapeutic value.

Exposure of Pseudomonas aeruginosa to bactericidal hypochlorous acid during neutrophil phagocytosis is compromised in cystic fibrosis

Myeloperoxidase is a major neutrophil antimicrobial protein, but its role in immunity is often overlooked because individuals deficient in this enzyme are usually in good health. Within neutrophil phagosomes, myeloperoxidase uses superoxide generated by the NADPH oxidase to oxidize chloride to the potent bactericidal oxidant hypochlorous acid (HOCl). In this study, using phagocytosis assays and LC-MS analyses, we monitored GSH oxidation in Pseudomonas aeruginosa to gauge their exposure to HOCl inside phagosomes. Doses of reagent HOCl that killed most of the bacteria oxidized half the cells' GSH, producing mainly glutathione disulfide (GSSG) and other low-molecular-weight disulfides. Glutathione sulfonamide (GSA), a HOCl-specific product, was also formed. When neutrophils phagocytosed P. aeruginosa, half of the bacterial GSH was lost. Bacterial GSA production indicated that HOCl had reacted with the bacterial cells, oxidized their GSH, and was sufficient to be solely responsible for bacterial killing. Inhibition of NADPH oxidase and myeloperoxidase lowered GSA formation in the bacterial cells, but the bacteria were still killed, presumably by compensatory nonoxidative mechanisms. Of note, bacterial GSA formation in neutrophils from patients with cystic fibrosis (CF) was normal during early phagocytosis, but it was diminished at later time points, which was mirrored by a small decrease in bacterial killing. In conclusion, myeloperoxidase generates sufficient HOCl within neutrophil phagosomes to kill ingested bacteria. The unusual kinetics of phagosomal HOCl production in CF neutrophils confirm a role for the cystic fibrosis transmembrane conductance regulator in maintaining HOCl production in neutrophil phagosomes.

Hypochlorous acid-modified extracellular matrix contributes to the behavioral switching of human coronary artery smooth muscle cells

The extracellular matrix (ECM) influences the structure and function of the arterial wall and modulates the behavior of vascular cells through ECM-cell interactions. Alterations to the ECM have been implicated in multiple pathological processes, including atherosclerosis which is characterized by low-grade chronic inflammation and the infiltration and proliferation of smooth muscle cells during disease development. Considerable evidence has been presented for a role for inflammation-derived oxidation in atherogenesis, with enzymatically-active myeloperoxidase (MPO), elevated levels of 3-chlorotyrosine (a biomarker of MPO-catalyzed damage) and oxidized ECM materials detected in advanced human atherosclerotic lesions. Whether oxidant-modified ECM contributes to the altered behavior of smooth muscle cells is however unclear. This study therefore investigated the effects of hypochlorous acid (HOCl), a major MPO-derived oxidant, on the structure of the native ECM synthesized by human coronary artery smooth muscle cells (HCAMSCs) and whether modified ECM proteins affected HCASMC adhesion, proliferation and gene expression. Exposure of native HCASMC-derived ECM to reagent HOCl or a MPO-Cl--H2O2 system resulted in extensive ECM modifications as evidenced by the loss of antibody recognition of epitopes on type IV collagen, laminin, versican and fibronectin. Oxidation of HCASMC ECM markedly reduced HCASMC adhesion to matrix components, but facilitated subsequent proliferation in vitro. Multiple genes were upregulated in HCASMCs in response to HOCl-modified HCASMC-ECM including interleukin-6 (IL-6), fibronectin (FN1) and matrix-metalloproteinases (MMPs). These data reveal a mechanism through which inflammation-induced ECM-modification may contribute to the behavioral switching of HCASMCs, a key process in plaque formation during the development of atherosclerosis.

Chlorination and oxidation of the extracellular matrix protein laminin and basement membrane extracts by hypochlorous acid and myeloperoxidase

Basement membranes are specialized extracellular matrices that underlie arterial wall endothelial cells, with laminin being a key structural and biologically-active component. Hypochlorous acid (HOCl), a potent oxidizing and chlorinating agent, is formed in vivo at sites of inflammation via the enzymatic action of myeloperoxidase (MPO), released by activated leukocytes. Considerable data supports a role for MPO-derived oxidants in cardiovascular disease and particularly atherosclerosis. These effects may be mediated via extracellular matrix damage to which MPO binds. Herein we detect and quantify sites of oxidation and chlorination on isolated laminin-111, and laminin in basement membrane extracts (BME), by use of mass spectrometry. Increased modification was detected with increasing oxidant exposure. Mass mapping indicated selectivity in the sites and extent of damage; Met residues were most heavily modified. Fewer modifications were detected with BME, possibly due to the shielding effects. HOCl oxidised 30 (of 56 total) Met and 7 (of 24) Trp residues, and chlorinated 33 (of 99) Tyr residues; 3 Tyr were dichlorinated. An additional 8 Met and 10 Trp oxidations, 14 chlorinations, and 18 dichlorinations were detected with the MPO/H2O2/Cl- system when compared to reagent HOCl. Interestingly, chlorination was detected at Tyr2415 in the integrin-binding region; this may decrease cellular adhesion. Co-localization of MPO-damaged epitopes and laminin was detected in human atherosclerotic lesions. These data indicate that laminin is extensively modified by MPO-derived oxidants, with structural and functional changes. These modifications, and compromised cell-matrix interactions, may promote endothelial cell dysfunction, weaken the structure of atherosclerotic lesions, and enhance lesion rupture.

81 - Chlorination and oxidation of the extracellular matrix protein laminin and basement membrane extracts by hypochlorous acid and myeloperoxidase

Background Basement membranes (BM) are specialized extracellular matrices that underlie arterial wall endothelial cells, with laminin being a key structural and biologically-active component. Hypochlorous acid (HOCl), a potent oxidizing and chlorinating agent, is formed in vivo at sites of inflammation via the enzymatic action of myeloperoxidase (MPO), released by activated leukocytes. Considerable data supports a role for MPO-derived oxidants in cardiovascular disease and particularly atherosclerosis. These effects may be mediated via extracellular matrix damage to which MPO binds avidly. Hypothesis That HOCl and MPO-derived oxidants selectively modify isolated laminin and laminin in basement membrane extracts, and that MPO-mediated damage to laminin occurs in human atherosclerotic lesions. Results Mass spectrometry with peptide mass mapping was used to detect and quantify sites of oxidation and chlorination. Increased modification was detected on isolated laminin-111, and laminin in BM extracts, with increasing oxidant exposure. Damage was detected at multiple Met, Trp and Tyr residues, with Met residues the most heavily modified. Fewer modifications were detected with BME, possibly due to the shielding effects. HOCl oxidized 30 (of 56 total) Met and 7 (of 24) Trp residues, and chlorinated 33 (of 99) Tyr residues; 3 Tyr were also dichlorinated. An additional 8 Met and 10 Trp oxidations, 14 chlorinations, and 18 dichlorinations were detected with MPO/H2O2/Cl- compared to reagent HOCl. Chlorination was detected at Tyr2415 in the integrin-binding region, providing a rationale for decreased cell adhesion to HOCl-modified laminin. Co-localization of MPO-damaged epitopes and laminin was detected in human atherosclerotic lesions. Conclusions These data indicate that laminin is extensively modified by MPO-derived oxidants, and induces structural and functional changes. These modifications, and compromised cell-matrix interactions, may promote endothelial cell dysfunction, weaken the structure of atherosclerotic lesions, and enhance lesion rupture.

The nitroxide 4-methoxy TEMPO inhibits neutrophil-stimulated kinase activation in H9c2 cardiomyocytes

After acute myocardial infarction (AMI), neutrophils are recruited to the affected myocardium. Hypochlorous acid (HOCl) produced by neutrophil myeloperoxidase (MPO) damages cardiomyocytes and potentially expands the primary infarct. Rat cardiomyocyte-like cells were incubated with isolated human neutrophils treated with chemical activators in the absence or presence of nitroxide 4-methoxy-Tempo (MetT; 25 μM) for 4, 6 or 24 h; studies with reagent HOCl served as positive control. Treating cardiomyocytes with activated neutrophils or reagent HOCl resulted in a marked increase in protein tyrosine chlorination and a decline in protein tyrosine phosphatase (PTP) activity. On balance our data also supported an increase in phosphorylation of MAPK p38 and ERK1/2 suggestive of an intracellular hyperphosphorylation status and this was accompanied by decreases in cell viability, as judged by assessing caspases-3/7 activity. For cells exposed to activated neutrophils receptor-mediated uptake of transferrin decreased although total matrix metalloproteinase (MMP) activity was unaffected. Addition of MetT ameliorated protein tyrosine chlorination, decreased MAPK activity and restored receptor-mediated transferrin uptake and PTP activity in cardiomyocytes. Overall, adverse effects of neutrophil-derived HOCl on cultured cardiomyocytes were ameliorated by MetT suggesting that nitroxides may be beneficial to inflammatory pathologies, where neutrophil recruitment/activation is a prominent and early feature.

Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100

Oxidation of LDL by the myeloperoxidase (MPO)-H2O2-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H2O2-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H2O2-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.

Carbamoylation abrogates the antioxidant potential of hydrogen sulfide

Hydrogen sulfide (H2S) has been identified as the third gasotransmitter. Beside its role as signaling molecule in the cardiovascular and nervous system the antioxidant and cyto-protective properties of H2S have gained much attention. In the present study we show that cyanate, an uremic toxin which is found in abundant concentration in sera of patients suffering from chronic kidney disease (CKD), can abrogate the antioxidant and cytoprotective activity of H2S via S-carbamoylation reaction, a reaction that previously has only been shown to have a physiological effect on cysteine groups, but not on H2S. Carbamoylation strongly inhibited the free radical scavenging (ABTS(+·) and alkylperoxyl ROO(·)) properties of H2S. The extent of intracellular ROS formation induced by ROO(·) was diminished by H2S whereas carbamoylation counteracted the protective effect. Reagent HOCl was rapidly inactivated by H2S in contrast to the carbamoylated compound. Protein modification by HOCl was inhibited by H2S but carbamoylation significantly reduced the effect. Thus, S-carbamoylation of low molecular weight thiols by abrogating their antioxidant potential may contribute to the higher oxidative stress observed in CKD.

Synthesis of halohydrines of 5-alkylbicyclo[2.2.1]heptene series using the systems inducing electrophilic reagents

The reactions of induced hydroxyhalogenation of alkylnorbornenes with the use of a mixture of aqueous solutions of hydrochloric or hydrobromic acids (or sodium, potassium and cobalt bromides) and hydrogen peroxide or sodium hypochloriteare are studied. The reaction are established to proceed through the addition at the double bond of the ring of the electrophilic reagent HOBr or HOCl formed in situ yielding of cis-vicinal halohydrines. By dehydrohalogenation of the latter in the presence of alkali are obtained endo-6-alkyl-endo-3-oxatricyclo[3.2.11.5.02.4]octanes and by their oxidation are synthesized respective haloketones in a high yield.

Hypochlorous acid oxidizes methionine and tryptophan residues in myoglobin

After acute myocardial infarction (AMI), infiltrating proinflammatory cells generate two-electron oxidants such as hypochlorous acid (HOCl). Myoglobin (Mb) is present at ∼ 0.3 mM in cardiomyocytes and, therefore, represents a significant target for oxidation. Exposure of horse Mb (50 μM) to reagent HOCl (0–500 μM) or activated human neutrophils (4–40 × 10 6 cells/ml) yielded oxidized Mb (Mb ox) as judged by amino acid analysis and peptide mass mapping. HOCl/Mb ratios of 1–5 mol/mol gave Mb ox with up to four additional oxygen atoms. Hydrolysis of Mb ox followed by amino acid analysis indicated that methionine (Met) and tryptophan (Trp) residues were modified by HOCl. Peptide mass mapping revealed that Met55 was oxidized at a lower HOCl/Mb ratio than Met131 and this preceded Trp7/14 modification (susceptibility Met55 > Met131 > Trp7 > Trp14). Incubation of Mb with activated neutrophils and physiological chloride anion yielded Mb ox with a composition similar to that determined with HOCl/Mb ratios < 2 mol/mol, with oxidation of Met, but not Trp, detected. These data indicate that Mb undergoes site-specific oxidation depending on the HOCl/protein ratio. As Mb is released from necrotic cardiomyocytes into the vasculature after AMI, HOCl-modified Mb may be a useful surrogate marker to gauge the extent of myocardial inflammation.

Hypochlorite and superoxide radicals can act synergistically to induce fragmentation of hyaluronan and chondroitin sulphates.

Activated phagocytes release the haem enzyme MPO (myeloperoxidase) and also generate superoxide radicals (O2*-), and hence H2O2, via an oxidative burst. Reaction of MPO with H2O2 in the presence of chloride ions generates HOCl (the physiological mixture of hypochlorous acid and its anion present at pH 7.4). Exposure of glycosaminoglycans to a MPO-H2O2-Cl- system or reagent HOCl generates long-lived chloramides [R-NCl-C(O)-R'] derived from the glycosamine N-acetyl functions. Decomposition of these species by transition metal ions gives polymer-derived amidyl (nitrogen-centred) radicals [R-N*-C(O)-R'], polymer-derived carbon-centred radicals and site-specific strand scission. In the present study, we have shown that exposure of glycosaminoglycan chloramides to O2*- also promotes chloramide decomposition and glycosaminoglycan fragmentation. These processes are inhibited by superoxide dismutase, metal ion chelators and the metal ion-binding protein BSA, consistent with chloramide decomposition and polymer fragmentation occurring via O2*--dependent one-electron reduction, possibly catalysed by trace metal ions. Polymer fragmentation induced by O2*- [generated by the superoxide thermal source 1, di-(4-carboxybenzyl)hyponitrite] was demonstrated to be entirely chloramide dependent as no fragmentation occurred with the native polymers or when the chloramides were quenched by prior treatment with methionine. EPR spin-trapping experiments using 5,5-dimethyl1-pyrroline-N-oxide and 2-methyl-2-nitrosopropane have provided evidence for both O2*- and polymer-derived carbon-centred radicals as intermediates. The results obtained are consistent with a mechanism involving one-electron reduction of the chloramides to yield polymer-derived amidyl radicals, which subsequently undergo intramolecular hydrogen atom abstraction reactions to give carbon-centred radicals. The latter undergo fragmentation reactions in a site-specific manner. This synergistic damage to glycosaminoglycans induced by HOCl and O2*- may be of significance at sites of inflammation where both oxidants are generated concurrently.

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.

Formation of a diimino-imidazole nucleoside from 2′-deoxyguanosine by singlet oxygen generated by methylene blue photooxidation

Singlet oxygen ( 1O 2) is capable of inducing genotoxic, carcinogenic and mutagenic effects. It has previously been reported that the reaction of 1O 2 with 2′-deoxyguanosine, which is a major target of 1O 2 among the DNA constituents, leads to formation of various oxidized products including 8-oxo-7,8-dihydro-2′-deoxyguanosine and spiroiminodihydantoin, amino-imidazolone and diamino-oxazolone nucleosides. In addition to these products, we report that a novel diimino-imidazole nucleoside, 2,5-diimino-4-[(2-deoxy-β- d- erythro-pentofuranosyl)amino]-2 H,5 H-imidazole (dD), is formed by reaction of 2′-deoxyguanosine with 1O 2 generated by irradiation with visible light in the presence of methylene blue under aerobic conditions. Its identification is based on identical chromatographic and spectroscopic data with an authentic compound, which we recently isolated and characterised from the reaction mixture of 2′-deoxyguanosine with reagent HOCl and a myeloperoxidase–H 2O 2–Cl − system. The yield of dD was increased by D 2O and decreased by azide. dD was not generated from 8-oxo-7,8-dihydro-2′-deoxyguanosine. These results indicate that dD is generated by 1O 2 directly from 2′-deoxyguanosine, but not via 8-oxo-7,8-dihydro-2′-deoxyguanosine. dD may play a role in the genotoxicity of singlet oxygen in cells.

Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques.

Oxidation is believed to play a role in atherosclerosis. Oxidized lipids, sterols and proteins have been detected in early, intermediate and advanced human lesions at elevated levels. The spectrum of oxidized side-chain products detected on proteins from homogenates of advanced human lesions has been interpreted in terms of the occurrence of two oxidative mechanisms, one involving oxygen-derived radicals catalysed by trace transition metal ions, and a second involving chlorinating species (HOCl or Cl2), generated by the haem enzyme myeloperoxidase (MPO). As MPO is released extracellularly by activated monocytes (and possibly macrophages) and is a highly basic protein, it would be expected to associate with polyanions such as the glycosaminoglycans of the extracellular matrix, and might result in damage being localized at such sites. In this study proteins extracted from extracellular matrix material obtained from advanced human atherosclerotic lesions are shown to contain elevated levels of oxidized amino acids [3,4-dihydroxyphenylalanine (DOPA), di-tyrosine, 2-hydroxyphenylalanine ( o-Tyr)] when compared with healthy (human and pig) arterial tissue. These matrix-derived materials account for 83-96% of the total oxidized protein side-chain products detected in these plaques. Oxidation of matrix components extracted from healthy artery tissue, and model proteins, with reagent HOCl is shown to give rise to a similar pattern of products to those detected in advanced human lesions. The detection of elevated levels of DOPA and o-Tyr, which have been previously attributed to the occurrence of oxygen-radical-mediated reactions, by HOCl treatment, suggests an alternative route to the formation of these materials in plaques. This is believed to involve the formation and subsequent decomposition of protein chloramines.

Identification of products formed by reaction of 3',5'-di-O-acetyl-2'-deoxyguanosine with hypochlorous acid or a myeloperoxidase-H2O2-Cl- system.

Hypochlorous acid (HOCl), generated by myeloperoxidase from H(2)O(2) and Cl(-), plays an important role in host defense and inflammatory tissue injury. We have studied the reaction of 3',5'-di-O-acetyl-2'-deoxyguanosine with reagent HOCl and with a human myeloperoxidase-H(2)O(2)-Cl(-) system in order to characterize polar reaction products. When 100 microM 3',5'-di-O-acetyl-2'-deoxyguanosine was reacted with 100 microM HOCl at pH 7.4 and 37 degrees C and the reaction was terminated by N-acetylcysteine, 3',5'-di-O-acetyl derivatives of previously reported products, such as diastereomers of spiroiminodihydantoin nucleoside, a diimino-imidazole nucleoside, an amino-imidazolone nucleoside, and 8-chloro-2'-deoxyguanosine were formed. In addition, we report the formation of 3',5'-di-O-acetyl derivatives of a guanidinohydantoin nucleoside, an iminoallantoin nucleoside, and a diamino-oxazolone nucleoside in this system. The identification of the products was based on their identical ESI-MS and UV spectra and HPLC retention times with authentic compounds synthesized with other oxidation systems. All of these products were also formed in the reaction of 3',5'-di-O-acetyl-2'-deoxyguanosine with the myeloperoxidase-H(2)O(2)-Cl(-) system under mildly acidic conditions. The yields of the products were greatly affected by the pH of the reaction mixture. The total yields of these products formed by HOCl at pH 7.4 and by the myeloperoxidase-H(2)O(2)-Cl(-) system at pH 4.5 were 72 and 43% of the consumed 3',5'-di-O-acetyl-2'-deoxyguanosine, respectively, indicating that nearly half of the consumption of 3',5'-di-O-acetyl-2'-deoxyguanosine by HOCl and the myeloperoxidase-H(2)O(2)-Cl(-) system can be accounted for by the formation of these products.

Distinct Modes of Cell Death Induced by Different Reactive Oxygen Species: AMINO ACYL CHLORAMINES MEDIATE HYPOCHLOROUS ACID-INDUCED APOPTOSIS

Oxidants derived from inflammatory phagocytes compose a key element of the host immune defense system and can kill mammalian cells by one of several different mechanisms. In this report, we compare mechanisms of cell death induced in human B lymphoma cells by the inflammatory oxidants superoxide, H(2)O(2), and HOCl. The results indicate that the mode of cell death induced depends on the nature of the oxidant involved and the medium in which the cells are treated. When human Burkitt's lymphoma cells are exposed to superoxide anion, generated as a flux from xanthine and xanthine oxidase, the cells die by a non-apoptotic mechanism (pyknosis/necrosis) identical to that seen when cells are treated with a bolus of reagent H(2)O(2). Addition of superoxide dismutase has no effect, whereas catalase is completely protective, indicating that exogenously generated superoxide kills cells entirely through its dismutation into H(2)O(2). In contrast, cells treated in culture media with reagent HOCl die largely by apoptosis. HOCl-induced apoptosis is mediated by aminoacyl chloramines generated in the culture media and can be mimicked by treatment of cells with taurine chloramine or with long lived chloramines generated from modified Lys or Arg. The results suggest that in a physiological milieu in which O(2)(-) and H(2)O(2) are the main oxidants being formed, the principal form of cell death may be necrotic, and under inflammatory conditions in which HOCl is generated, apoptotic cell death may predominate.

Selective oxidation in vitro by myeloperoxidase of the N-terminal amine in apolipoprotein B-100

In contrast to the multiple low abundance 2,4-dinitrophenylhydrazine-reactive tryptic peptides formed by oxidation of LDL with reagent HOCl in vitro, myeloperoxidase-catalyzed oxidation produces a dominant product in considerably greater yield and selectivity. This modified peptide had a single amino-terminal sequence corresponding to amino acids 53–66 of apolipoprotein B-100 (apoB-100), but its mass spectra indicated a significantly higher mass than could be reconciled with simple modifications of this peptide. Subsequent studies indicate that this product appears to result from N-chlorination of the N-terminal amino group of apoB-100 and dehydrohalogenation to the corresponding imine, which may form the hydrazone derivative directly, or after hydrolysis to the ketone. The methionine residue is oxidized to the corresponding sulfoxide, and the primary sequence peptide (residues 1–14 of apoB-100) is linked by the intramolecular disulfide bond between C-12 and C-61 to the peptide composed of residues 53–66, as we have observed previously (Yang, C-Y., T. W. Kim, S. A. Weng, B. Lee, M. Yang, and A. M. Gotto, Jr. 1990. Proc. Natl. Acad. Sci. USA. 87: 5523–5527) in unmodified LDL. The selective oxidation by myeloperoxidase of the N-terminal amine suggests strong steric effects in the approach of substrate to the enzyme catalytic site, an effect that may apply to other macromolecules and to cell surface molecules.—Yang, C-y., J. Wang, A. N. Krutchinsky, B. T. Chait, J. D. Morrisett, and C. V. Smith. Selective oxidation in vitro by myeloperoxidase of the N-terminal amine in apolipoprotein B-100.