Albert van der Vliet

Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite. A potential additional mechanism of nitric oxide-dependent toxicity.

Involvement of peroxynitrite (ONOO−) in inflammatory diseases has been implicated by detection of 3-nitrotyrosine, an allegedly characteristic protein oxidation product, in various inflamed tissues. We show here that nitrite (NO2−), the primary metabolic end product of nitric oxide (NO·), can be oxidized by the heme peroxidases horseradish peroxidase, myeloperoxidase (MPO), and lactoperoxidase (LPO), in the presence of hydrogen peroxide (H2O2), to most likely form NO·2, which can also contribute to tyrosine nitration during inflammatory processes. Phenolic nitration by MPO-catalyzed NO2− oxidation is only partially inhibited by chloride (Cl−), the presumed major physiological substrate for MPO. In fact, low concentrations of NO2− (2-10 μM) catalyze MPO-mediated oxidation of Cl−, indicated by increased chlorination of monochlorodimedon or 4-hydroxyphenylacetic acid, most likely via reduction of MPO compound II. Peroxidase-catalyzed oxidation of NO2−, as indicated by phenolic nitration, was also observed in the presence of thiocyanate (SCN−), an alternative physiological substrate for mammalian peroxidases. Collectively, our results suggest that NO2−, at physiological or pathological levels, is a substrate for the mammalian peroxidases MPO and lactoperoxidase and that formation of NO2· via peroxidase-catalyzed oxidation of NO2− may provide an additional pathway contributing to cytotoxicity or host defense associated with increased NO· production.

Formation of Nitrating and Chlorinating Species by Reaction of Nitrite with Hypochlorous Acid A NOVEL MECHANISM FOR NITRIC OXIDE-MEDIATED PROTEIN MODIFICATION

Detection of 3-nitrotyrosine has served as an in vivo marker for the production of the cytotoxic species peroxynitrite (ONOO−). We show here that reaction of nitrite (NO−2), the autoxidation product of nitric oxide (·NO), with hypochlorous acid (HOCl) forms reactive intermediate species that are also capable of nitrating phenolic substrates such as tyrosine and 4-hydroxyphenylacetic acid, with maximum yields obtained at physiological pH. Monitoring the reaction of NO−2 with HOCl by continuous flow photodiode array spectrophotometry indicates the formation of a transient species with spectral characteristics similar to those of nitryl chloride (Cl-NO2). Reaction of synthetic Cl-NO2 with N-acetyl-L-tyrosine results in the formation of 3-chlorotyrosine and 3-nitrotyrosine in ratios that are similar to those obtained by the NO−2/HOCl reaction (4:1). Tyrosine residues in bovine serum albumin are also nitrated and chlorinated by NO−2/HOCl and synthetic Cl-NO2. The reaction of N-acetyl-L-tyrosine with NO−2/HOCl or authentic Cl-NO2 also produces dityrosine, suggesting that free radical intermediates are involved in the reaction mechanism. Our data indicate that while chlorination reactions of Cl-NO2 are mediated by direct electrophilic addition to the aromatic ring, a free radical mechanism appears to be operative in nitrations mediated by NO−2/HOCl or Cl-NO2, probably involving the combination of nitrogen dioxide (·NO2) and tyrosyl radical. We propose that NO−2 reacts with HOCl by Cl+ transfer to form both cis- and trans-chlorine nitrite (Cl-ONO) and Cl-NO2 as intermediates that modify tyrosine by either direct reaction or after decomposition to reactive free and solvent-caged Cl· and ·NO2 as reactive species. Formation of Cl-NO2 and/or Cl-ONO in vivo may represent previously unrecognized mediators of inflammation-mediated protein modification and tissue injury, and offers an additional mechanism of tyrosine nitration independent of ONOO−.

Aromatic hydroxylation and nitration of phenylalanine and tyrosine by peroxynitrite: Evidence for hydroxyl radical production from peroxynitrite

Peroxynitrite is a highly reactive species, generated from Superoxide and nitric oxide. Some effects of peroxynitrite are ascribed to the molecule itself, but decomposition products of the protonated form, peroxynitrous acid, may account for much of its reactivity in biological systems. Suggested products include highly‐reactive hydroxyl radicals, but thermodynamic calculations have been used to claim that free hydroxyl radicals cannot be formed from peroxynitrite. We utilized aromatic hydroxylation of phenylalanine as a specific detector of hydroxyl radicals, and found that incubation of phenylalanine with peroxynitrite leads to a small amount of p‐, m‐ and o‐tyrosine, specific products of attack by this radical. Products of nitration of phenylalanine and tyrosine were also detected, as was dityrosine. Peroxynitrite decomposition generates several reactive species, including some that can nitrate aromatic rings. Formation of nitro‐aromatic compounds may be a useful marker of peroxynitrite generation in biological systems.

Lipoic and Dihydrolipoic Acids as Antioxidants. a Critical Evaluation

A detailed evaluation of the antioxidant and pro-oxidant properties of lipoic acid (LA) and dihydrolipoic acid (DHLA) was performed. Both compounds are powerful scavengers of hypochlorous acid, able to protect alpha 1-antiproteinase against inactivation by HOCl. LA was a powerful scavenger of hydroxyl radicals (OH.) and could inhibit both iron-dependent OH. generation and peroxidation of ox-brain phospholipid liposomes in the presence of FeCl3-ascorbate, presumably by binding iron ions and rendering them redox-inactive. By contrast, DHLA accelerated iron-dependent OH. generation and lipid peroxidation, probably by reducing Fe3+ to Fe2+. LA inhibited this pro-oxidant action of DHLA. However, DHLA did not accelerate DNA degradation by a ferric bleomycin complex and slightly inhibited peroxidation of arachidonic acid by the myoglobin-H2O2 system. Under certain circumstances, DHLA accelerated the loss of activity of alpha-antiproteinase exposed to ionizing radiation under a N2O/O2 atmosphere and also the loss of creatine kinase activity in human plasma exposed to gas-phase cigarette smoke. Neither LA nor DHLA reacted with superoxide radical (O.2-) or H2O2 at significant rates, but both were good scavengers of trichloromethylperoxyl radical (CCl3O2.). We conclude that LA and DHLA have powerful antioxidant properties. However, DHLA can also exert pro-oxidant properties, both by its iron ion-reducing ability and probably by its ability to generate reactive sulphur-containing radicals that can damage certain proteins, such as alpha 1-antiproteinase and creatine kinase.

Determination of low-molecular-mass antioxidant concentrations in human respiratory tract lining fluids

Antioxidants present within lung epithelial lining fluids (ELFs) constitute an initial line of defense against inhaled environmental oxidants such as ozone, nitrogen oxides, and tobacco smoke, but the antioxidant composition of human ELFs is still incompletely characterized. We analyzed ELF concentrations of the low-molecular-mass antioxidants ascorbate, urate, glutathione (GSH), and α-tocopherol by obtaining bronchoalveolar lavage (BAL) and nasal lavage fluids from healthy nonsmoking volunteers and compared two different BAL procedures. ELF dilution by the lavage procedures was estimated by measurement of urea in recovered BAL fluids in comparison with those in blood plasma from the same subjects. The results indicated that a recently developed single-cycle BAL procedure minimizes influx of non-ELF urea into the instilled fluid and thus allows for a more accurate determination of ELF antioxidant concentrations. Using this procedure, we determined that bronchoalveolar ELF contains 40 ± 18 (SD) μM ascorbate, 207 ± 167 μM urate, 109 ± 64 μM GSH, and 0.7 ± 0.3 μM α-tocopherol ( n = 12 subjects). Similar analysis of nasal lavage fluid yielded nasal ELF levels of 28 ± 19 μM ascorbate and 225 ± 105 μM urate ( n = 12 subjects), whereas GSH was undetectable (<0.5 μM). Our results demonstrate that ascorbate and urate are major low-molecular-mass ELF antioxidants in both the upper and lower respiratory tract, whereas GSH is present at significant concentrations only in bronchoalveolar ELF.Antioxidants present within lung epithelial lining fluids (ELFs) constitute an initial line of defense against inhaled environmental oxidants such as ozone, nitrogen oxides, and tobacco smoke, but the antioxidant composition of human ELFs is still incompletely characterized. We analyzed ELF concentrations of the low-molecular-mass antioxidants ascorbate, urate, glutathione (GSH), and alpha-tocopherol by obtaining bronchoalveolar lavage (BAL) and nasal lavage fluids from healthy nonsmoking volunteers and compared two different BAL procedures. ELF dilution by the lavage procedures was estimated by measurement of urea in recovered BAL fluids in comparison with those in blood plasma from the same subjects. The results indicated that a recently developed single-cycle BAL procedure minimizes influx of non-ELF urea into the instilled fluid and thus allows for a more accurate determination of ELF antioxidant concentrations. Using this procedure, we determined that bronchoalveolar ELF contains 40 +/- 18 (SD) microM ascorbate, 207 +/- 167 microM urate, 109 +/- 64 microM GSH, and 0.7 +/- 0.3 microM alpha-tocopherol (n = 12 subjects). Similar analysis of nasal lavage fluid yielded nasal ELF levels of 28 +/- 19 microM ascorbate and 225 +/- 105 microM urate (n = 12 subjects), whereas GSH was undetectable (<0.5 microM). Our results demonstrate that ascorbate and urate are major low-molecular-mass ELF antioxidants in both the upper and lower respiratory tract, whereas GSH is present at significant concentrations only in bronchoalveolar ELF.

Formation of S-Nitrosothiols via Direct Nucleophilic Nitrosation of Thiols by Peroxynitrite with Elimination of Hydrogen Peroxide

Peroxynitrite (ONOO−), a potent oxidant formed by reaction of nitric oxide (NO⋅) with superoxide anion, can activate guanylyl cyclase and is able to induce vasodilation or inhibit platelet aggregation and leukocyte adhesion, via thiol-dependent formation of NO⋅. Reaction of ONOO− with thiols is thought to proceed through formation of a S-nitrothiol (thionitrate; RSNO2) intermediate and yields low levels of S-nitrosothiols (thionitrites; RSNO), both of which are theoretical sources of NO⋅. Kinetic analysis of NO⋅ production after reaction of ONOO− with GSH established that NO⋅ originates exclusively from the thionitrite GSNO. Further mechanistic investigations indicated that GSNO formation by ONOO– does not occur via one-electron oxidation mechanisms. Nitrosation of GSH could theoretically proceed via intermediate formation of the thionitrate GSNO2, which, after rearrangement to the corresponding sulfenyl nitrite (GSONO), can react with GSH to form GSNO and GSOH. However, no evidence for such a mechanism was found in experiments with NO2 · or with the stable nitrothiol tert-butylthionitrate. Using high performance liquid chromatography with chemiluminescence detection, formation of H2O2 was observed after reaction of ONOO− with GSH under both aerobic and anaerobic conditions, at levels similar to the yield of GSNO, indicative of a direct nucleophilic nitrosation mechanism with elimination of HOO−. Our results indicate that ONOO− may contribute to S-nitrosation in vivo and that direct nitrosation of thiols or other nucleophilic substrates by ONOO– may represent an important and often overlooked component of NO⋅ biochemistry.

Oxidants, nitrosants, and the lung

The respiratory tract is subjected to a variety of environmental stresses, including oxidizing gases, particulates, and airborne microorganisms, that together, may injure structural and functional lung components and thereby jeopardize the primary lung function of gas exchange. To cope with such various environmental threats, the lung has developed elaborate defense mechanisms that include inflammatory-immune pathways as well as several antioxidant systems. These defense systems operate largely in extracellular spaces, thus protecting underlying bronchial and alveolar epithelial cells from injury, although these cells themselves are also active participants in such (inflammatory) defense mechanisms. Although potentially harmful, oxidants are increasingly recognized as pathophysiologic mediators produced primarily by inflammatory-immune cells as a host defense mechanism, but also by various other cell types as an intracellular mediator in various cell responses, thus affecting inflammatory-immune processes or inducing resistance. The molecular mechanisms and signaling pathways involved in such processes are the focus of much current investigation. Nitric oxide, a messenger molecule produced by many lung cell types, also modulates oxidant-mediated processes, thereby giving rise to a new family of reactive nitrogen species (nitrosants) with potentially unique signaling properties. The complex role of oxidants and nitrosants in various pathophysiologic processes in the lung have confounded the design of therapeutic approaches with antioxidant substrates. This review discusses current knowledge regarding extracellular antioxidant defenses in the lung, and oxidant/nitrosant mechanisms operating under inflammatory-immune conditions and their potential contribution to common lung diseases. Finally, some recent developments in antioxidant therapeutic strategies are discussed.

TOBACCO-RELATED DISEASES: Is There a Role for Antioxidant Micronutrient Supplementation?

It is clear that smoking causes an increase in free radicals, reactive nitrogen and oxygen species (RNS and ROS, respectively), and that cigarette smoking is associated with increases in the incidence and severity of several diseases including atherosclerosis, cancer, and chronic obstructive lung disease. Although there is still no unequivocal evidence that oxidative stress is a contributor to these diseases or that an increased intake of antioxidant nutrients is beneficial, the observation that smokers have lower circulating levels of some of these nutrients, raises concern. This article discusses the possible links between the observed oxidant-induced damage related to tobacco smoking, effects on cellular mechanisms, and their potential involvement in the causation and enhancement of disease processes.

Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue.

Protein S-thiolation, the formation of mixed disulphides of cysteine residues in proteins with low-molecular-mass thiols, occurs under conditions associated with oxidative stress and can lead to modification of protein function. In the present study, we examined the site of S-thiolation of the enzyme creatine kinase (CK), an important source of ATP in myocytes. Inactivation of this enzyme is thought to play a critical role in cardiac injury during oxidative stress, such as during reperfusion injury. Reaction of rabbit CK M isoenzyme with GSSG, used to model protein S-thiolation, was found to result in enzyme inactivation that could be reversed by GSH or dithiothreitol. Measurement of GSH that is released during the thiolation reaction indicated that the maximum extent of CK thiolation was approx. 1 mol of GSH/mol of protein, suggesting thiolation on one reactive cysteine residue. Accordingly, matrix-assisted laser-desorption ionization MS confirmed that the molecular mass of CK was increased, consistent with addition of one GSH molecule/molecule of CK. Using trypsin digestion, HPLC and MS analysis, the active-site cysteine residue (Cys(283)) was identified as the site of thiolation. Reversal of thiolation was shown to be rapid when GSH is abundant, rendering dethiolation of CK thermodynamically favoured within the cell. We conclude that S-glutathionylation of CK could be one mechanism to explain temporary reversible loss in activity of CK during ischaemic injury. The maintainance of GSH levels represents an important mechanism for regeneration of active CK from S-glutathionylated CK.

Peroxynitrite Induces Covalent Dimerization of Epidermal Growth Factor Receptors in A431 Epidermoid Carcinoma Cells

Irreversible tyrosine modifications by inflammatory oxidants such as peroxynitrite (ONOO−) can affect signal transduction pathways involving tyrosine phosphorylation. The epidermal growth factor receptor (EGFR), a member of the c-ErbB receptor tyrosine kinase family, is involved in regulation of epithelial cell growth and differentiation, and possible modulation of EGFR-dependent signaling by ONOO− was studied. Exposure of epidermoid carcinoma A431 cells to 0.1–1.0 mm ONOO− resulted in tyrosine nitration on EGFR and other proteins but did not significantly affect EGFR tyrosine autophosphorylation. A high molecular mass tyrosine-phosphorylated protein (∼340 kDa) was detected in A431 cell lysates after exposure to ONOO−, most likely representing a covalently dimerized form of EGFR, based on immunoprecipitation and/or immunoblotting with α-EGFR antibodies and co-migration with ligand-induced EGFR dimers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Covalent EGFR dimerization by ONOO− probably involved intermolecular dityrosine cross-linking and was enhanced after receptor activation with epidermal growth factor. Furthermore, irreversibly cross-linked EGFR was more extensively tyrosine-phosphorylated compared with the monomeric form, indicating that ONOO− preferentially cross-links activated EGFR. Exposure of A431 cells to ONOO− markedly reduced the kinetics of tyrosine phosphorylation of a downstream EGFR substrate, phospholipase C-γ1, which may be related to covalent alterations in EGFR. Alteration of EGFR signaling by covalent EGFR dimerization by inflammatory oxidants such as ONOO− may affect conditions of increased EGFR activation such as epithelial repair or tumorigenesis.