RESUMEN
Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.
Asunto(s)
Intoxicación por Tetracloruro de Carbono/metabolismo , Tetracloruro de Carbono/toxicidad , ADN/metabolismo , Desoxiguanosina/análogos & derivados , Metabolismo de los Lípidos , Estrés Oxidativo , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Ensayo Cometa , Daño del ADN , Desoxiguanosina/farmacología , Radicales Libres , Cromatografía de Gases y Espectrometría de Masas , Peróxido de Hidrógeno/metabolismo , Inmunoensayo , Immunoblotting , Hígado/metabolismo , Masculino , Malondialdehído/farmacología , Metionina/metabolismo , Oxígeno/metabolismo , Ratas , Ratas Endogámicas F344 , Espectrofotometría , Sustancias Reactivas al Ácido Tiobarbitúrico , Factores de Tiempo , Tirosina/química , Tirosina/metabolismoRESUMEN
Oxidative damage to tissue proteins has been implicated in the pathogenesis of liver disease, but the mechanisms that promote oxidation in vivo are unclear. Hydrogen peroxide is transformed into an array of potentially damaging reactants by the heme protein myeloperoxidase. This proinflammatory enzyme is expressed by circulating neutrophils and monocytes but is generally thought to be absent from tissue macrophages. To determine whether myeloperoxidase is present in Kupffer cells, the fixed-tissue macrophages of liver, Western blot analysis, and immunohistochemistry were performed. Two different antibodies monospecific for myeloperoxidase identified a 60-kd protein, the predicted molecular mass of myeloperoxidase, in human liver extracts. Immunostaining detected the enzyme in sinusoidal lining cells of normal and diseased human livers. Immunofluorescence confocal microscopy demonstrated co-localization of myeloperoxidase and CD68, a monocyte/macrophage marker, in sinusoidal lining cells. Numerous myeloperoxidase-expressing cells were also evident in the fibrous septa of cirrhotic livers. Immunostaining with an antibody to proteins modified by hypochlorous acid, a characteristic product of the enzyme, indicated that myeloperoxidase is enzymatically active in cases of acute liver injury and cirrhosis. These findings identify myeloperoxidase as a component of human Kupffer cells. Oxidative damage resulting from the action of myeloperoxidase may contribute to acute liver injury and hepatic fibrogenesis.
Asunto(s)
Macrófagos del Hígado/enzimología , Hígado/enzimología , Peroxidasa/metabolismo , Animales , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Humanos , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Confocal , Oxidación-Reducción , Peroxidasa/genéticaRESUMEN
The oxidation hypothesis proposes that oxidative modification of low density lipoprotein (LDL) plays a critical role in atherogenesis. This review critically evaluates the various mechanisms proposed for LDL oxidation, focusing on insights derived from chemical analysis of human artery wall and studies of genetically engineered mice. The implications of recent clinical trials of vitamin E for the oxidation hypothesis are also briefly discussed.
Asunto(s)
Arterias/química , Arteriosclerosis/metabolismo , Lipoproteínas LDL/metabolismo , Animales , Arterias/efectos de los fármacos , Arteriosclerosis/prevención & control , Países Desarrollados , Modelos Animales de Enfermedad , Predicción , Humanos , Peroxidación de Lípido/fisiología , Ratones , Oxidación-Reducción , Vitamina E/farmacologíaRESUMEN
Oxidative stress has been implicated in the cardiovascular complications that affect chronic renal failure patients on hemodialysis, though the physiologically relevant pathways mediating oxidative damage are poorly understood. It is known, however, that hemodialysis activates neutrophils, a well-characterized source of hydrogen peroxide and myeloperoxidase. The phagocyte-derived myeloperoxidase-hydrogen peroxide-chloride system generates hypochlorous acid, which reacts with tyrosine residues of proteins to form 3-chlorotyrosine. To explore the role of activated phagocytes in oxidative stress in chronic renal failure, we used 3-chlorotyrosine as a specific marker of myeloperoxidase activity. Utilizing isotope dilution gas chromatography-mass spectrometry, we compared 3-chlorotyrosine levels in plasma proteins of five patients on chronic hemodialysis therapy with those of age- and sex-matched healthy controls. The oxidized amino acid was present in the plasma proteins of 4 of the hemodialysis patients (3.5 +/- 0.8 micromol per mol tyrosine) but was undetectable in the healthy subjects. Therefore, one pathway for oxidative stress in hemodialysis patients appears to involve hypochlorous acid generated by the myeloperoxidase system of activated phagocytes. We also examined intradialytic 3-chlorotyrosine levels using membranes that activate white blood cells and the alternative pathway of complement. Hemodialysis increased plasma myeloperoxidase and the expression of CD11b/CD18 by circulating phagocytes, but failed to demonstrably increase 3-chlorotyrosine levels. 3-chlorotyrosine was detectable in 12 of 19 samples in total, with significant intrasubject variability. Our observations suggest that oxidants generated by myeloperoxidase contribute to the increased oxidative stress observed in renal-failure patients but do not damage plasma proteins during the hemodialysis procedure itself.
Asunto(s)
Peroxidasa/metabolismo , Fagocitos/metabolismo , Diálisis Renal , Insuficiencia Renal/sangre , Tirosina/análogos & derivados , Tirosina/sangre , Adulto , Anciano , Proteínas Sanguíneas/metabolismo , Femenino , Cromatografía de Gases y Espectrometría de Masas/métodos , Humanos , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Estrés Oxidativo/fisiología , Insuficiencia Renal/enzimología , Insuficiencia Renal/inmunología , Tirosina/biosíntesisAsunto(s)
Aneurisma de la Aorta Abdominal/etiología , National Institutes of Health (U.S.) , Grupo de Atención al Paciente , Investigación/organización & administración , Aneurisma de la Aorta Abdominal/epidemiología , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/terapia , Fenómenos Biomecánicos , Causalidad , Tejido Conectivo/fisiología , Progresión de la Enfermedad , Doxiciclina/farmacología , Doxiciclina/uso terapéutico , Hemorreología , Humanos , Inflamación , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/fisiología , Biología Molecular , Evaluación de Necesidades , Objetivos Organizacionales , Péptido Hidrolasas/fisiología , Apoyo a la Investigación como Asunto , Estados UnidosRESUMEN
The myeloperoxidase system of neutrophils uses hydrogen peroxide and chloride to generate hypochlorous acid, a potent bactericidal oxidant in vitro. In a mouse model of polymicrobial sepsis, we observed that mice deficient in myeloperoxidase were more likely than wild-type mice to die from infection. Mass spectrometric analysis of peritoneal inflammatory fluid from septic wild-type mice detected elevated concentrations of 3-chlorotyrosine, a characteristic end product of the myeloperoxidase system. Levels of 3-chlorotyrosine did not rise in the septic myeloperoxidase-deficient mice. Thus, myeloperoxidase seems to protect against sepsis in vivo by producing halogenating species. Surprisingly, levels of 3-bromotyrosine also were elevated in peritoneal fluid from septic wild-type mice and were markedly reduced in peritoneal fluid from septic myeloperoxidase-deficient mice. Furthermore, physiologic concentrations of bromide modulated the bactericidal effects of myeloperoxidase in vitro. It seems, therefore, that myeloperoxidase can use bromide as well as chloride to produce oxidants in vivo, even though the extracellular concentration of bromide is at least 1,000-fold lower than that of chloride. Thus, myeloperoxidase plays an important role in host defense against bacterial pathogens, and bromide might be a previously unsuspected component of this system.
Asunto(s)
Infecciones por Klebsiella/enzimología , Klebsiella pneumoniae/patogenicidad , Neutrófilos/enzimología , Oxidantes/metabolismo , Peroxidasa/fisiología , Sepsis/enzimología , Tirosina/análogos & derivados , Tirosina/metabolismo , Animales , Bromo/metabolismo , Cloro/metabolismo , Modelos Animales de Enfermedad , Células HL-60 , Humanos , Ácido Hipocloroso/metabolismo , Iones , Infecciones por Klebsiella/metabolismo , Infecciones por Klebsiella/mortalidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peroxidasa/genética , Peroxidasa/metabolismo , Sepsis/metabolismo , Sepsis/mortalidadRESUMEN
Reactive intermediates generated by phagocytic white blood cells are of central importance in destroying microorganisms, but they may also damage normal tissue at sites of inflammation. To investigate the potential role of such oxidants in tissue injury, we used gas chromatography/mass spectrometry to quantify levels of o,o'-dityrosine in mouse peritoneal neutrophils and urine. In wild-type animals, neutrophils markedly increased their content of protein-bound dityrosine when they were activated in vivo. This increase failed to occur in mice that were deficient in the phagocyte NADPH oxidase. Levels of o,o'-dityrosine in urine mirrored those in neutrophil proteins. When o,o'-[(14)C]dityrosine was injected intravenously into mice, the radiolabel was not metabolized or incorporated into tissue proteins: instead, it was recovered in urine with near-quantitative yield. Patients with sepsis markedly increased their output of o,o'-dityrosine into urine, suggesting that systemic inflammation also may be a potent source of oxidative stress in humans. These observations demonstrate that activated neutrophils produce o,o'-dityrosine cross-links in tissue proteins, which may subsequently be degraded into free amino acids and excreted into urine. Our results indicate that mouse phagocytes use oxidants produced by the NADPH oxidase to create o,o'-dityrosine cross-links in vivo and raise the possibility that reactive intermediates produced by this pathway promote inflammatory tissue damage in humans.
Asunto(s)
Inflamación/metabolismo , NADPH Oxidasas/metabolismo , Neutrófilos/metabolismo , Sepsis/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Orina/química , Enfermedad Aguda , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/análisis , Radioisótopos de Carbono , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Inflamación/complicaciones , Inflamación/inmunología , Inyecciones Intravenosas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Persona de Mediana Edad , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , Neutrófilos/inmunología , Estrés Oxidativo/inmunología , Proteínas/metabolismo , Sepsis/complicaciones , Sepsis/inmunología , Espectrometría de Masa por Ionización de Electrospray , Tirosina/administración & dosificación , Tirosina/análisisRESUMEN
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.
Asunto(s)
Arteriosclerosis/metabolismo , Cisteína/metabolismo , Precursores Enzimáticos/metabolismo , Ácido Hipocloroso/metabolismo , Metaloproteinasa 7 de la Matriz/metabolismo , Oxígeno/metabolismo , Peroxidasa/metabolismo , Secuencia de Aminoácidos , Arteriosclerosis/enzimología , Cromatografía Líquida de Alta Presión , Activación Enzimática , Peróxido de Hidrógeno/metabolismo , Metaloproteinasa 7 de la Matriz/química , Datos de Secuencia Molecular , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría UltravioletaRESUMEN
A wealth of evidence indicates that oxidized low density lipoprotein (LDL) may be of central importance in animal models of atherogenesis. In recent clinical trials, however, dietary vitamin E supplements have not consistently prevented cardiac events in humans with established coronary artery disease. Such mixed results have led many to question the role of LDL oxidation in human atherosclerosis, although this interpretation assumes that the doses of vitamin E used in the studies inhibited lipid oxidation in vivo. In fact, there is remarkably little evidence indicating that those particular regimens effectively inhibit lipid peroxidation in healthy humans. Moreover, evidence of increased oxidative stress was not a criterion for inclusion in the trials; therefore, vitamin E may have benefited only a subset of the participants. These uncertainties raise doubts about the ability of vitamin E to augment antioxidant defense mechanisms in vivo and leave many questions about LDL oxidation and atherosclerosis unanswered.
Asunto(s)
Antioxidantes/uso terapéutico , Arteriosclerosis/metabolismo , Arteriosclerosis/prevención & control , Lipoproteínas LDL/metabolismo , Vitamina E/uso terapéutico , Animales , Antioxidantes/farmacología , Enfermedades Cardiovasculares/prevención & control , Ensayos Clínicos como Asunto , Humanos , Peroxidación de Lípido/efectos de los fármacos , Modelos Animales , Estrés Oxidativo/efectos de los fármacos , Vitamina E/farmacologíaRESUMEN
It has been proposed that elevated levels of tissue iron increase the risk for atherosclerosis, perhaps by favoring the formation of pro-atherogenic oxidized LDL. Working with apoE-deficient (apoE(-/-)) mice, which do not require a high-fat diet to develop atherosclerosis, we compared the effects of standard diet (0.02% iron) or a 2% carbonyl iron diet. After 24 weeks, mice fed the 2% carbonyl iron diet had twice as much iron in their plasma, a ninefold increase in bleomycin-detectable free iron in their plasma, and ten times as much iron in their livers as control mice. Dietary iron overload caused a modest (30%) rise in plasma triglyceride and cholesterol. Nevertheless, this regimen did not exacerbate, but rather reduced the severity of atherosclerosis by 50%, and it failed to elevate hepatic levels of heme oxygenase mRNA, which is induced by many different oxidative insults in vitro. Moreover, hepatic levels of protein-bound dityrosine and ortho-tyrosine, two markers of metal-catalyzed oxidative damage in vitro, failed to rise in iron-overloaded animals. Our observations suggest that elevated serum and tissue levels of iron are not atherogenic in apoE(-/-) mice. Moreover, they call into question the hypothesis that elevated levels of tissue iron promote LDL oxidation and oxidative stress in vivo.
Asunto(s)
Apolipoproteínas E/genética , Arteriosclerosis/etiología , Sobrecarga de Hierro/complicaciones , Animales , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Colesterol/metabolismo , Femenino , Compuestos de Hierro Carbonilo , Sobrecarga de Hierro/metabolismo , Hígado/metabolismo , Ratones , Ratones Noqueados , Compuestos Organometálicos/farmacología , Oxidación-Reducción , ARN Mensajero/biosíntesis , Triglicéridos/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
The structural integrity of apolipoprotein A-I (apo A-I) is critical to the physiological function of high-density lipoprotein (HDL). Oxidized lipoproteins are thought to be of central importance in atherogenesis, and oxidation products characteristic of myeloperoxidase, a heme protein secreted by activated phagocytes, have been detected in human atherosclerotic tissue. At plasma concentrations of halide ion, hypochlorous acid is a major product of the myeloperoxidase-hydrogen peroxide-chloride system. We therefore investigated the effects of activated human neutrophils, a potent source of myeloperoxidase and hydrogen peroxide, on the protein and lipid components of HDL. Both free and HDL-associated apo A-I exposed to activated human neutrophils underwent extensive degradation as monitored by RP-HPLC and Western blotting with a polyclonal antibody to apo A-I. Replacement of the neutrophils with reagent HOCl resulted in comparable damage (at molar oxidant : HDL subclass 3 ratio = 100) as observed in the presence of activated phagocytes. Apo A-I degradation by activated neutrophils was partially inhibited by the HOCl scavenger methionine, by the heme inhibitor azide, by chloride-free conditions, by the peroxide scavenger catalase, and by a combination of superoxide dismutase (SOD)/catalase, implicating HOCl in the cell-mediated reaction. The addition of a protease inhibitor (3,4-dichloroisocoumarin) further reduced the extent of apo A-I damage. In contrast to the protein moiety, there was little evidence for oxidation of unsaturated fatty acids or cholesterol in HDL3 exposed to activated neutrophils, suggesting that HOCl was selectively damaging apo A-I. Our observations indicate that HOCl generated by myeloperoxidase represents one pathway for protein degradation in HDL3 exposed to activated phagocytes.
Asunto(s)
Cloruros/metabolismo , Peróxido de Hidrógeno/metabolismo , Neutrófilos/metabolismo , Peroxidasa/metabolismo , Western Blotting , Catalasa/metabolismo , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Humanos , Neutrófilos/enzimología , Superóxido Dismutasa/metabolismoRESUMEN
Increased aerobic metabolism during exercise is a potential source of oxidative stress. In muscle, mitochondria are one important source of reactive intermediates that include superoxide (O2*-), hydrogen peroxide (H2O2), and possibly hydroxyl radical (HO*). The recent discovery that mitochondria may generate nitric oxide (NO*) also has implications for oxidant production and mitochondrial function. In this review, we critically examine the concept that production of reactive intermediates increases during exercise. Because the health benefits of regular exercise are well-documented, we also examine adaptations to exercise that may decrease oxidative stress. These include increased antioxidant defenses, reduced basal production of oxidants, and reduction of radical leak during oxidative phosphorylation.
Asunto(s)
Antioxidantes/metabolismo , Ejercicio Físico/fisiología , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Adaptación Fisiológica/fisiología , Animales , Radicales Libres/análisis , Radicales Libres/metabolismo , Humanos , Modelos AnimalesRESUMEN
Eosinophils use eosinophil peroxidase, hydrogen peroxide (H(2)O(2)), and bromide ion (Br(-)) to generate hypobromous acid (HOBr), a brominating intermediate. This potent oxidant may play a role in host defenses against invading parasites and eosinophil-mediated tissue damage. In this study, we explore the possibility that HOBr generated by eosinophil peroxidase might oxidize nucleic acids. When we exposed uracil, uridine, or deoxyuridine to reagent HOBr, each reaction mixture yielded a single major oxidation product that comigrated on reversed-phase HPLC with the corresponding authentic brominated pyrimidine. The eosinophil peroxidase-H(2)O(2)-Br(-) system also converted uracil into a single major oxidation product, and the yield was near-quantitative. Mass spectrometry, HPLC, UV--visible spectroscopy, and NMR spectroscopy identified the product as 5-bromouracil. Eosinophil peroxidase required H(2)O(2) and Br(-) to produce 5-bromouracil, implicating HOBr as an intermediate in the reaction. Primary and secondary bromamines also brominated uracil, suggesting that long-lived bromamines also might be physiologically relevant brominating intermediates. Human eosinophils used the eosinophil peroxidase-H(2)O(2)-Br(-) system to oxidize uracil. The product was identified as 5-bromouracil by mass spectrometry, HPLC, and UV--visible spectroscopy. Collectively, these results indicate that HOBr generated by eosinophil peroxidase oxidizes uracil to 5-bromouracil. Thymidine phosphorylase, a pyrimidine salvage enzyme, transforms 5-bromouracil to 5-bromodeoxyridine, a mutagenic analogue of thymidine. These findings raise the possibility that halogenated nucleobases generated by eosinophil peroxidase exert cytotoxic and mutagenic effects at eosinophil-rich sites of inflammation.
Asunto(s)
Bromuros/metabolismo , Bromouracilo/metabolismo , Eosinófilos/enzimología , Peróxido de Hidrógeno/metabolismo , Mutágenos/metabolismo , Peroxidasas/metabolismo , Compuestos de Sodio/metabolismo , Bromatos/metabolismo , Bromuros/sangre , Bromo/química , Bromo/metabolismo , Bromodesoxiuridina/metabolismo , Catalasa/antagonistas & inhibidores , Catalasa/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Peroxidasa del Eosinófilo , Eosinófilos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Peroxidasas/antagonistas & inhibidores , Pirimidinas/química , Compuestos de Sodio/sangre , Timidina Fosforilasa/metabolismo , Uracilo/metabolismoRESUMEN
Recent evidence argues strongly that the marked increase in risk for atherosclerotic heart disease seen in diabetics cannot be explained by a generalized increase in oxidative stress. Here, we used streptozotocin to induce hyperglycemia in cynomolgus monkeys for 6 months and tested whether high glucose levels promote localized oxidative damage to artery wall proteins. We focused on three potential agents of oxidative damage: hydroxyl radical, tyrosyl radical, and reactive nitrogen species. To determine which pathways operate in vivo, we quantified four stable end products of these reactants -- ortho-tyrosine, meta-tyrosine, o,o'-dityrosine, and 3-nitrotyrosine -- in aortic proteins. Levels of ortho-tyrosine, meta-tyrosine, and o,o'-dityrosine, but not of 3-nitrotyrosine, were significantly higher in aortic tissue of hyperglycemic animals. Of the oxidative agents we tested, only hydroxyl radical mimicked this pattern of oxidized amino acids. Moreover, tissue levels of ortho-tyrosine and meta-tyrosine correlated strongly with serum levels of glycated hemoglobin, a measure of glycemic control. We conclude that short-term hyperglycemia in primates promotes oxidation of artery wall proteins by a species that resembles hydroxyl radical. Our observations suggest that glycoxidation reactions in the arterial microenvironment contribute to early diabetic vascular disease, raising the possibility that antioxidant therapies might interrupt this process.
Asunto(s)
Aorta/metabolismo , Arteriosclerosis/metabolismo , Diabetes Mellitus Experimental/metabolismo , Radical Hidroxilo/metabolismo , Tirosina/análogos & derivados , Animales , Arteriosclerosis/etiología , Arteriosclerosis/patología , Glucosa/metabolismo , Hemoglobina Glucada/análisis , Lípidos/sangre , Macaca fascicularis , Masculino , Espectrometría de Masas/métodos , Oxidación-Reducción , Factores de Tiempo , Tirosina/metabolismoRESUMEN
Phagocytic oxidants have been implicated in tissue injury and oncogenesis, and their pathophysiological role in modifying nucleobases and amino acids has been widely explored. Their ability to cross-link proteins and DNA, however, has not been considered, even though reversible DNA-protein interactions are key to gene expression and to DNA replication and repair. In the current studies, we show that hypochlorous acid (HOCl), generated by the myeloperoxidase-hydrogen peroxide-chloride system of phagocytes, cross-links single-stranded DNA-binding protein (SSB) to single-stranded oligonucleotides. Exposure of SSB and a homopolymer of radiolabeled thymidine (dT(40)) to HOCl resulted in the formation of a radiolabeled band with slower mobility than the free oligonucleotide, as determined by denaturing polyacrylamide gel electrophoresis. This radiolabeled band did not appear if the reaction mixture was treated with protease or nuclease, indicating that it represents a covalent complex of DNA and protein. Oligonucleotides of adenosine and cytidine behaved similarly to the thymidine oligonucleotide, demonstrating that they are also capable of participating in the cross-linking reaction. The covalent complex of radiolabeled dT(40) and SSB was also generated by chloramines and the complete myeloperoxidase-hydrogen peroxide-chloride system. The enzymatic reaction required each component of the system and was inhibited by heme poisons and chloride-free conditions, implicating myeloperoxidase and HOCl. DNA-protein cross-links were generated in Escherichia coli exposed to HOCl, suggesting that double-stranded DNA is also a target for the reaction. These results indicate that long-lived chloramines and HOCl generated by myeloperoxidase can generate covalent DNA-protein cross-links that may contribute to the mutagenic and cytotoxic effects of phagocytes on microbial pathogens and host tissue.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Ácido Hipocloroso/química , Ácido Hipocloroso/metabolismo , Peroxidasa/metabolismo , Fagocitos/enzimología , Cloraminas/química , Cloruros/metabolismo , ADN Bacteriano/metabolismo , ADN de Cadena Simple/antagonistas & inhibidores , Desoxirribonucleósidos/metabolismo , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Ácido Hipocloroso/antagonistas & inhibidores , Sustancias Intercalantes/química , Sustancias Intercalantes/metabolismo , Oxidantes/química , Oxidantes/metabolismo , Fagocitos/metabolismo , Polímeros/metabolismo , Albúmina Sérica Bovina/química , Factores de TiempoRESUMEN
Inflammation and oxidative stress contribute to the pathogenesis of many human diseases including atherosclerosis. Advanced human atheroma contains high levels of the enzyme myeloperoxidase that produces the pro-oxidant species, hypochlorous acid (HOCl). This study documents increased numbers of myeloperoxidase-expressing macrophages in eroded or ruptured plaques causing acute coronary syndromes. In contrast, macrophages in human fatty streaks contain little or no myeloperoxidase. Granulocyte macrophage colony-stimulating factor, but not macrophage colony-stimulating factor, selectively regulates the ability of macrophages to express myeloperoxidase and produce HOCl in vitro. Moreover, myeloperoxidase-positive macrophages in plaques co-localized with granulocyte macrophage colony-stimulating factor. Pro-inflammatory stimuli known to be present in human atherosclerotic plaque, including CD40 ligand, lysophosphatidylcholine, or cholesterol crystals, could induce release of myeloperoxidase from HOCl production by macrophages in vitro. HOCl-modified proteins accumulated at ruptured or eroded sites of human coronary atheroma. These results identify granulocyte macrophage colony-stimulating factor as an endogenous regulator of macrophage myeloperoxidase expression in human atherosclerosis and support a particular role for the myeloperoxidase-expressing macrophages in atheroma complication and the acute coronary syndromes.
Asunto(s)
Arteriosclerosis/etiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Macrófagos/enzimología , Infarto del Miocardio/etiología , Peroxidasa/biosíntesis , Arteriosclerosis/enzimología , Arteriosclerosis/patología , Antígenos CD40/farmacología , Diferenciación Celular , Células Cultivadas , Colesterol/farmacología , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Ácido Hipocloroso/metabolismo , Lisofosfatidilcolinas/farmacología , Macrófagos/efectos de los fármacos , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/enzimología , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Síndrome , Túnica Íntima/enzimologíaRESUMEN
Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by eosinophil peroxidase was taken up by cultured cells and incorporated into genomic DNA as 5-bromodeoxyuridine. Although previous studies have focused on oxidation of chromosomal DNA, our observations suggest another mechanism for oxidative damage of DNA. In this scenario, peroxidase-catalyzed halogenation of nucleotide precursors yields products that subsequently can be incorporated into DNA. Because the thymine analog 5-BrUra mispairs with guanine in DNA, generation of brominated pyrimidines by eosinophils might constitute a mechanism for cytotoxicity and mutagenesis at sites of inflammation.
Asunto(s)
Desoxicitidina/metabolismo , Eosinófilos/enzimología , Peroxidasas/metabolismo , Animales , Bromo , Bromodesoxiuridina/metabolismo , Células CHO , Catálisis , Cricetinae , Citosina/metabolismo , ADN/metabolismo , Peroxidasa del Eosinófilo , Humanos , Peróxido de Hidrógeno/farmacología , Mutagénesis , Nucleótidos , PorcinosRESUMEN
Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, generates an array of oxidants proposed to play critical roles in host defense and local tissue damage. Both MPO and its reaction products are present in human atherosclerotic plaque, and it has been proposed that MPO oxidatively modifies targets in the artery wall. We have now generated MPO-deficient mice, and show here that neutrophils from homozygous mutants lack peroxidase and chlorination activity in vitro and fail to generate chlorotyrosine or to kill Candida albicans in vivo. To examine the potential role of MPO in atherosclerosis, we subjected LDL receptor-deficient mice to lethal irradiation, repopulated their marrow with MPO-deficient or wild-type cells, and provided them a high-fat, high-cholesterol diet for 14 weeks. White cell counts and plasma lipoprotein profiles were similar between the two groups at sacrifice. Cross-sectional analysis of the aorta indicated that lesions in MPO-deficient mice were about 50% larger than controls. Similar results were obtained in a genetic cross with LDL receptor-deficient mice. In contrast to advanced human atherosclerotic lesions, the chlorotyrosine content of aortic lesions from wild-type as well as MPO-deficient mice was essentially undetectable. These data suggest an unexpected, protective role for MPO-generated reactive intermediates in murine atherosclerosis. They also identify an important distinction between murine and human atherosclerosis with regard to the potential involvement of MPO in protein oxidation.
Asunto(s)
Arteriosclerosis/etiología , Peroxidasa/fisiología , Tirosina/análogos & derivados , Animales , Candida albicans/inmunología , Humanos , Ácido Hipocloroso/metabolismo , Ratones , Ratones Endogámicos C57BL , Neutrófilos/enzimología , Oxidación-Reducción , Peroxidasa/deficiencia , Peroxidasa/genética , Fagocitos/metabolismo , Tirosina/análisisRESUMEN
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.
Asunto(s)
Bromo/metabolismo , Desoxicitidina/metabolismo , Inflamación/metabolismo , Mutágenos/metabolismo , Peroxidasa/metabolismo , Uracilo/metabolismo , Bromouracilo/metabolismo , Daño del ADN , Humanos , Peróxido de Hidrógeno/metabolismo , Ácido Hipocloroso/metabolismo , Inflamación/complicaciones , Neutrófilos/metabolismo , Oxidación-ReducciónRESUMEN
Reactive aldehydes might have a pivotal role in the pathogenesis of atherosclerosis by covalently modifying low-density lipoprotein (LDL). However, the identities of the aldehyde adducts that form on LDL in vivo are not yet clearly established. We previously demonstrated that the haem protein myeloperoxidase oxidizes proteins in the human artery wall. We also have shown that p-hydroxyphenylacetaldehyde (pHA), the aldehyde that forms when myeloperoxidase oxidizes L-tyrosine, covalently modifies the N(epsilon)-lysine residues of proteins. The resulting Schiff base can be quantified as N(epsilon)-[2-(p-hydroxyphenyl)ethyl]lysine (pHA-lysine) after reduction with NaCNBH(3). Here we demonstrate that pHA-lysine is a marker for LDL that has been modified by myeloperoxidase, and that water-soluble, but not lipid-soluble, antioxidants inhibit the modification of LDL protein. To determine whether myeloperoxidase-generated aldehydes might modify LDL in vivo, we used a combination of isotope-dilution GC-MS to quantify pHA-lysine in aortic tissues at various stages of lesion evolution. We also analysed LDL isolated from atherosclerotic aortic tissue. Comparison of normal and atherosclerotic aortic tissue demonstrated a significant elevation (more than 10-fold) of the reduced Schiff base adduct in fatty streaks, intermediate lesions and advanced lesions compared with normal aortic tissue. Moreover, the level of pHA-lysine in LDL recovered from atherosclerotic aortic intima was 200-fold that in plasma LDL of healthy donors. These results indicate that pHA-lysine, a specific covalent modification of LDL, is generated in human atherosclerotic vascular tissue. They also raise the possibility that reactive aldehydes generated by myeloperoxidase have a role in converting LDL into an atherogenic lipoprotein.