Analysis of aromatic nitration, chlorination, and hydroxylation by gas chromatography-mass spectrometry.
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Animals↗
Biomedical subjects
C E Cross
Publications and source records attributed to C E Cross.
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 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.
Adolescent↗
Reactive nitrogen species and tyrosine nitration in the respiratory tract: epiphenomena or a pathobiologic mechanism of disease?
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Animals↗
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 (approximately 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 alpha-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-gamma1, 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.
Carbodiimides↗
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.
Ascorbic Acid↗
Macromolecular carbonyls in human stratum corneum: a biomarker for environmental oxidant exposure?
The presence of stratum corneum carbonyls may serve as an intrinsic dosimeter for environmental oxidative damage to skin. To investigate the accumulation of carbonyls in human stratum corneum, skin was tape-stripped, then tapes were sequentially incubated with 2,4-dinitrophenyl hydrazine (DNPH), rat anti-DNP, mouse anti-rat IgG conjugated with alkaline phosphatase, p-nitrophenyl phosphate and absorbance (405 nm) measured and carbonyls estimated. Stratum corneum exposed in vitro to oxidants: hypochlorous acid (1, 10, 100 mM), ozone (0, 1, 5, 10 ppm for 2 h) or UV light (280-400 nm; 0, 4, 88, or 24 J/cm2) contained increased carbonyls. Furthermore, stratum corneum carbonyls were elevated in tanned compared with untanned sites: dorsal hand (0.43 +/- 0.06 nmol/cm2) vs. lower arm (0.32 +/- 0.04, mean +/- S.E.M., n = 11; P < 0.003) and lower back (0.26 +/- 0.02) vs. buttock (0.21 +/- 0.02; n = 6, P < 0.01) indicating in vivo oxidative damage.
Aldehydes↗
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 (O2.-), 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.
Animals↗
Plasmodium chabaudi chabaudi (AS): inflammatory cytokines and pathology in an erythrocytic-stage infection in mice.
We have sought to characterize Plasmodium chabaudi chabaudi infection in mice for use as a model for malaria pathology. Different mouse strains vary in their susceptibility to the erythrocytic stages of this parasite and this is manifested not only in the outcome of infection (survival versus death) but also by differences in the numbers of circulating parasites at the peak of infection. We have shown that regardless of final outcome, both resistant and susceptible mice exhibit other parameters of disease such as loss in body weight and anemia. By contrast, other parameters such as hypothermia appear more severe in susceptible mice. The severe symptoms coincide with high levels of inflammatory cytokines in the circulation of susceptible mice, not seen in H-2-matched resistant mice. However, levels of mRNA for the same cytokines, measured in the spleen of the same mice was not significantly different between the two strains. Neutralization of IFN-gamma in vivo led to an increase in parasitemia, in both susceptible and resistant mice, but did not affect the final outcome of disease. Indeed, symptoms were exacerbated in the absence of IFN-gamma, presumably because of larger numbers of circulating parasites. These data suggest that IFN-gamma does not directly contribute to the lethal outcome of infection in susceptible strains of mice.
Anemia↗
Redox regulation of wound healing? NF-kappaB activation in cultured human keratinocytes upon wounding and the effect of low energy HeNe irradiation.
The complex process of wound healing as well as the signaling systems orchestrating this intricate process remain incompletely defined. Using human keratinocytes in primary culture, we sought to characterize their NF-kappaB responses to wounding alone or in combination with other treatments. We initially characterized these cultured human keratinocytes responses to known NF-kappaB activators (PMA, TNF-alpha and IL-1) using two different assays, immunohistochemistry and electrophoretic mobility shift (EMSA). After eliciting the expected NF-kappaB responses, we applied these same assays to assess responses to either wounding or HeNe irradiation alone. The results obtained indicated that only a modest/sporadic activation of NF-kappaB was elicited by these which was only detectable using immunohistochemistry. When the combination of wounding and HeNe irradiation on NF-kappaB status was assessed, a marked, localized activation of NF-kappaB in keratinocytes along the wound edge was found. Treatment induced NF-kappaB activation (e.g., wounding, HeNe irradiation and combined wounding and HeNe irradiation) was abrogated by pyrrolidine dithiocarbamate (PDTC) which inhibits NF-kappaB activation through an as yet incompletely understood (antioxidant?) mechanism. These data therefore suggest that NF-kappaB and oxidation mediated changes in its activation state likely play important roles in normal cutaneous wound healing.
DNA-Binding Proteins↗
Cigarette smokers and oxidant stress: a continuing mystery.
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Diet↗
Oxidative stress and antioxidants at biosurfaces: plants, skin, and respiratory tract surfaces.
Atmospheric pollutants represent an important source of oxidative and nitrosative stress to both terrestrial plants and to animals. The exposed biosurfaces of plants and animals are directly exposed to these pollutant stresses. Not surprisingly, living organisms have developed complex integrated extracellular and intracellular defense systems against stresses related to reactive oxygen and nitrogen species (ROS, RNS), including O3 and NO2. Plant and animal epithelial surfaces and respiratory tract surfaces contain antioxidants that would be expected to provide defense against environmental stress caused by ambient ROS and RNS, thus ameliorating their injurious effects on more delicate underlying cellular constituents. Parallelisms among these surfaces with regard to their antioxidant constituents and environmental oxidants are presented. The reactive substances at these biosurfaces not only represent an important protective system against oxidizing environments, but products of their reactions with ROS/RNS may also serve as biomarkers of environmental oxidative stress. Moreover, the reaction products may also induce injury to underlying cells or cause cell activation, resulting in production of proinflammatory substances including cytokines. In this review we discuss antioxidant defense systems against environmental toxins in plant cell wall/apoplastic fluids, dead keratinized cells/interstitial fluids of stratum corneum (the outermost skin layer), and mucus/respiratory tract lining fluids.
Air Pollutants↗
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 microM) 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.
Catalysis↗
Ozone depletes tocopherols and tocotrienols topically applied to murine skin.
To evaluate ozone damage to hairless mouse skin, two parameters of oxidative damage, vitamin E depletion and malondialdehyde (MDA) production, were measured in vitamin E-enriched and in control skin from mice exposed to ozone (10 ppm). A 5% vitamin E solution (tocotrienol-rich fraction, TRF) in polyethylene glycol (PEG) was applied to 2 sites on the back of hairless mice, PEG to 2 sites. After 2 h, the sites were washed, one of each pair of sites covered and the mice exposed ozone for 2 h. Ozone exposure (compared with covered sites) increased epidermal MDA in PEG-treated sites, while vitamin E was unchanged. In contrast, ozone exposure significantly depleted vitamin E in TRF-treated sites, while significant MDA accumulation was prevented. This is the first demonstration that ozone exposure causes damage to cutaneous lipids, an effect which can be attenuated by vitamin E application.
Administration, Topical↗
Marijuana. Respiratory tract effects.
Daily marijuana smoking has been clearly shown to have adverse effects on pulmonary function and produce respiratory symptomatology (cough, wheeze, and sputum production) similar to that of tobacco smokers. Based on the tobacco experience, decrements in pulmonary function may be predictive of the future development of chronic obstructive pulmonary disease (COPD). However, in the absence of alpha-1-antitrypsin deficiency, the habitual marijuana-only smoker would likely have to smoke 4-5 joints per day for a span of at least 30 yr in order to develop overt manifestations of COPD. The mutagenic/carcinogenic properties of marijuana smoke are also well-established. The potential for induction of laryngeal, oropharyngeal, and possibly bronchogenic carcinoma from marijuana has been documented by several case reports and observational series. Despite this, a relative risk ratio for the development of these tumors has not yet been quantified. Based on a higher frequency of case reports for upper airway cancer compared to bronchogenic carcinoma, marijuana smoking may have a more deleterious effect on the upper respiratory tract. However, this hypothesis remains speculative at best, pending confirmation by longitudinal studies.
Cannabis↗
In vivo exposure to ozone depletes vitamins C and E and induces lipid peroxidation in epidermal layers of murine skin.
To evaluate skin susceptibility to ozone (O3) and to localize possible oxidative damage within the skin layers, hairless mice were exposed to 10 ppm O3 or air (0 ppm O3) for 2 h. The mice were euthanized, the skin removed and frozen. Three skin layers (upper epidermis, lower epidermis/papillary dermis, and dermis) were separated, antioxidant concentrations (alpha-tocopherol and ascorbic acid) and the lipid peroxidation product malondialdehyde (MDA) measured. In the upper epidermis, O3 significantly depleted alpha-tocopherol (22%; p < .05) and ascorbic acid (55%; p < .01). These antioxidants were unchanged by O3 in the lower skin layers. More remarkably, MDA increased ten-fold in the upper epidermis (p < .001) and two-fold in the lower epidermis/papillary epidermis (p < .05); it was unchanged in the dermis. Thus, exposure to O3 in vivo depletes ascorbic acid and alpha-tocopherol and strongly induces lipid peroxidation in skin. High MDA concentrations measured in the upper epidermis suggest that O3 reacts directly with fatty acids on the skin surface layers. These results further suggest that chronic exposure to lower O3 concentrations found in urban smog could potentially have implications for skin health.
Animals↗
Adult respiratory distress syndrome: a radical perspective.
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Adult↗
Oxidative stress in cystic fibrosis: does it occur and does it matter?
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Administration, Inhalation↗
CR3-dependent phagocytosis by murine macrophages: different cytokines regulate ingestion of a defined CR3 ligand and complement-opsonized Cryptococcus neoformans.
Phagocytosis is a fundamental process in innate resistance to infection. We have used the pathogenic yeast Cryptococcus neoformans to study the interaction of this encapsulated organism with murine macrophages in vitro. In the absence of exogenous opsonins the encapsulated yeast is almost totally resistant to ingestion by murine macrophages. Owing to its ability to activate the alternative complement pathway, the anti-phagocytic properties of the polysaccharide capsule can be partially overcome following opsonization in vitro with non-immune mouse serum and subsequent phagocytosis via complement receptors. Here, we demonstrate the importance of the complement receptor type 3 (CR3) in in vitro phagocytosis of the yeast and in in vivo resistance to infection. In vitro, 70% of a population of resident murine macrophages are able to ingest C. neoformans and then only inefficiently (1-2 organisms per cell). Previously we have shown that tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) efficiently enhance ingestion of serum-opsonized encapsulated C. neoformans, and we now show that the cytokines convert a population of resident macrophages to a state where all the cells are competent for ingestion of large numbers of yeasts (6-8 per cell). We also show that these cytokines have a direct effect on CR3, as enhanced levels of complement-opsonized sheep red blood cells (EIgMC) bind to macrophages activated in this way. However, cytokines that have previously been shown to enhance phagocytosis of EIgMC have no effect on ingestion of encapsulated C. neoformans. These results demonstrate that the cytokines regulating CR3-dependent ingestion of C. neoformans are different to those regulating ingestion of EIgMC and reinforce the importance of studying pathogens rather than inert ligands in understanding the regulation of phagocytosis.
Animals↗