Carroll E. Cross

Oxygen Radicals and Human Disease

Toxic oxygen free radicals have been implicated as important pathologic mediators in many clinical disorders. We discuss the chemistry of oxygen radical production and the roles of iron and of various antioxidants as well as the diseases that have received active attention in oxy-radical research. Particular attention is focused on cigarette smoke oxidants, ischemia-reperfusion-induced radical production, carcinogenesis, and aging. Such research may well provide a firm foundation for therapeutic breakthroughs.

Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils

Nitric oxide (˙NO) plays a central role in the pathogenesis of diverse inflammatory and infectious disorders,. The toxicity of ˙NO is thought to be engendered, in part, by its reaction with superoxide (O˙−2), yielding the potent oxidant peroxynitrite (ONOO−). However, evidence for a role of ONOO− in vivo is based largely upon detection of 3-nitrotyrosine in injured tissues. We have recently demonstrated that nitrite (NO2−), a major end-product of ˙NO metabolism, readily promotes tyrosine nitration through formation of nitryl chloride (NO2Cl) and nitrogen dioxide (˙NO2) by reaction with the inflammatory mediators hypochlorous acid (HOCl) or myeloperoxidase,. We now show that activated human polymorphonuclear neutrophils convert NO2− into NO2Cl and ˙NO2 through myeloperoxidase-dependent pathways. Polymorphonuclear neutrophil-mediated nitration and chlorination of tyrosine residues or 4-hydroxyphenylacetic acid is enhanced by addition of NO2− or by fluxes of ˙NO. Addition of 15NO2− led to 15N enrichment of nitrated phenolic substrates, confirming its role in polymorphonuclear neutrophil-mediated nitration reactions. Polymorphonuclear neutrophil-mediated inactivation of endothelial cell angiotensin-converting enzyme was exacerbated by NO2−, illustrating the physiological significance of these reaction pathways to cellular dysfunction. Our data reveal that NO2− may regulate inflammatory processes through oxidative mechanisms, perhaps by contributing to the tyrosine nitration and chlorination observed in vivo.

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.

Dietary antioxidants and cigarette smoke-induced biomolecular damage: a complex interaction.

Epidemiologic evidence suggests that cigarette smoking is a major risk factor for chronic obstructive pulmonary diseases such as chronic bronchitis and emphysema, for carcinogenesis, and for cardiovascular disease. However, the precise mechanisms of these effects are incompletely understood. The gas phase of cigarette smoke contains abundant free radicals including nitric oxide. Hence, cigarette smoke may induce some of its damaging effects by free radical mechanisms. We report that exposure of plasma, a model for respiratory tract lining fluids, to gas-phase cigarette smoke causes depletion of antioxidants, including ascorbate, urate, ubiquinol-10, and alpha-tocopherol, and a variety of carotenoids, including beta-carotene. Gas-phase cigarette smoke induced some lipid peroxidation, as measured by cholesteryl linoleate hydroperoxide (18:2OOH) formation. Ascorbate was effective in preventing 18:2OOH formation. In contrast to the low concentrations of lipid hydroperoxides measured (< 1 mumol/L), protein carbonyl formation, a measure of protein modification, increased by approximately 400 mumol/L after nine puffs of cigarette smoke. Reduced glutathione inhibited protein carbonyl formation, whereas other plasma antioxidants, including ascorbate, were ineffective. alpha, beta-Unsaturated aldehydes (acrolein and crotonaldehyde) in cigarette smoke may react with protein -SH and -NH2 groups by a Michael addition reaction that results in a protein-bound aldehyde functional group. Gas-phase cigarette smoke is capable of converting tyrosine to 3-nitrotyrosine and dityrosine, indicating free radical mechanisms of protein damage by nitrogen oxides. Aldehydes and nitrogen oxides in cigarette smoke may be significant contributors to biomolecular damage, and endogenous antioxidants can attenuate some of these adverse effects.

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.

NITROTYROSINE AS BIOMARKER FOR REACTIVE NITROGEN SPECIES

Publisher Summary This chapter discusses the modification of proteins by reactive nitrogen species (RNS) and of tyrosine residues. The mechanisms by which tyrosine is modified by nitrogen oxides (NO), and assay methods for 3-nitrotyrosine, a modified product of tyrosine specific for RNS is also described. The biological systems are commonly exposed to various oxides of nitrogen with the nitrogen atom present in various oxidation states, ranging from +1 to +5. Tyrosine modification by NO has received attention because one of the major products formed (3-nitrotyrosine) is a stable end product, and characteristic of nitrogen oxides. The analysis of modified tyrosine residues is also reviewed. RNS reacts with various amino acid residues in proteins, forming metabolites which may be specific for particular RNS. The analysis of specific products, such as 3-nitrotyrosine, is useful in determining formation of RNS in vivo. Reaction of NO2. with tyrosine is demonstrated to result in formation of phenoxyl radicals that can either dimerize or combine with NO2. to form 3-nitrotyrosine. The peroxynitrite (ONOO–) also oxidizes tyrosine to form both the dimer and 3-nitrotyrosine as the most important products.