Macular pigment and macular volume in eyes of patients with cystic fibrosis.

BACKGROUND: Because patients with cystic fibrosis (CF) are living longer, chronic malabsorption of carotenoids associated with CF resulting in decreased macular pigment (MP) may affect macular long-term health in later-life pathology. This study compared the macular pigment optical density (MPOD) and corresponding central macular volume (MV) of adult CF subjects and age-matched normal controls subjects to determine whether chronic malabsorption associated with CF could adversely affect macular photoreceptor anatomy. OBJECTIVE: Our aim was to compare MPOD with measurements of central MV in CF patients with age-matched controls. Design. In nine adult CF patients (ages: 29-46) without a history of carotenoid supplementation or known retinal or optic nerve disease MPOD and MV were measured by heterochromatic flicker photometry (HFP) and optical coherence tomography (OCT), respectively, and compared to results obtained from 14 age-matched controls. RESULTS: MPOD was significantly reduced at 15' and 30' eccentricities in CF subjects compared to normal subjects (mean difference -0.21 at 15', -0.25 at 30', p < 0.005). No significant difference, in MV noted at any of the eccentricities tested between CF and normal subjects (CF: normal MV ratios ranged from 0.94 to 1.1 for all eccentricities with p > 0.1 at all eccentricities). Best corrected vision acuity and fundus examination were normal in all subjects. CONCLUSIONS: Unsupplemented CF patients have markedly lower levels of macular carotenoids (e.g., lutein and zeaxanthin), but well-maintained visual function and no significant reductions in central MV primarily composed of macular photoreceptors. Future studies are needed to determine whether the lifelong decrease in protective central retinal carotenoids predisposes CF patients to later-life retinal pathology.

Nitroxide radical TEMPO reduces ozone-induced chemokine IL-8 production in lung epithelial cells.

Exposure to high levels of ozone (O(3)) damages respiratory tract epithelial cells. This research evaluated the ability of TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a stable nitroxide free radical, to decrease O(3)-mediated injury to a respiratory tract-derived cell line (A549 cells) by monitoring in this cell system the interleukine-8 (IL-8) production. TEMPO reduced O(3)-induced IL-8 production in A549 cells, as evidenced by PCR analysis, Western blot and ELISA assays. This behaviour is explainable on the basis of the reactivity between TEMPO with O(3) and/or O(3)-derived free radicals in biological systems. The study provides evidence that TEMPO reacts with O(3) and/or its cytotoxic products and may provide protections against O(3)-induced biotoxicities.

Tocopherol transfer protein deficiency modifies nuclear receptor transcriptional networks in lungs: modulation by cigarette smoke in vivo.

Dietary factors and environmental pollutants initiate signaling cascades that converge on AhR:Nrf2:NF-kappaB transcription factor (TF) networks and, in turn, affect the health of the organism through its effects on the expression of numerous genes. Reactive oxygen metabolites (ROMs) have been hypothesized to be common mediators in these pathways. alpha-Tocopherol (AT) is a potent, lipophilic, scavenger of ROMs in vitro and has been hypothesized to be a major chain-breaking anti-oxidant in lipoproteins and biological membranes in vivo. The lung offers a vital organ to test the various postulated actions of AT in vivo. Lung AT concentrations can be manipulated by several methods that include dietary and genetic techniques. In this study we have used mice with severe AT deficiency inflicted at birth by the deletion of AT transfer protein (ATTP) which is abundantly expressed in the liver and regulates systemic concentrations of AT. Mice and humans deficient in ATTP are AT deficient. Female ATTP-deficient (ATTP-KO) mice and their congenic ATTP normal (WT) mice fed a diet containing 35 IU AT/kg diet were used to test our hypothesis. The mice (n=5/group) were exposed to either air or cigarette smoke (CS, total suspended particles 60 mg/m(3), 6h/day), a source of ROM, for 3 or 10 days. Post-exposure lung tissue was dissected, RNA extracted from each lung and it was pooled group-wise and processed for GeneChip analysis (Affymetrix 430A 2.0). Differential analysis of the transcriptomes ( approximately 16,000 mRNAs) identified CS sensitive genes that were modulated by lung AT-concentration. CS activated AhR driven genes such as cyp1b1 whose induction was augmented in CS-exposed, AT-deficient lungs. However, CS-induced expression of some of the Nrf2 driven genes was not potentiated in the AT-deficient lungs. Largest clusters of CS-AT sensitive genes were lymphocyte and leukocyte specific genes. These gene-clusters included those encoding cytokines and immunoglobulins, which were repressed by CS and were modulated by lung AT concentrations. Our genome-wide analysis suggests reciprocal regulation of xenobiotic and immune response genes by CS and a modulatory role of lung AT concentration on the expression of these clusters of genes. These data suggest that in vivo network of AT, AT-metabolites and ATTP affects the transcription of genes driven by AhR, Nrf2 and NF-kappaB, transcription factor networks that transduce cellular metabolic signals and orchestrate adaptive responses of lungs to inhaled environmental pollutants.

Acrolein-induced cytotoxicity in cultured human bronchial epithelial cells. Modulation by alpha-tocopherol and ascorbic acid.

Acrolein is a highly reactive unsaturated hazardous air pollutant of human health concern, particularly as a component of cigarette smoke. In this study, the mechanisms of acrolein-induced cytotoxicity in human bronchial epithelial cells (HBE1) and the modulating effects of antioxidants were examined. Our results show that acrolein induces a cell death pathway in human bronchial epithelial cells, which retain key features of apoptosis, as indicated by phosphatidylserine (PS) externalization and DNA fragmentation. Acrolein-induced apoptosis was associated with depletion of cellular GSH and intracellular generation of oxidants. Supplementation of cells with either alpha-tocopherol or ascorbic acid was found to strongly inhibit acrolein-induced apoptosis and to prevent the increase in the generation of intracellular oxidants, although GSH depletion was unaffected. Moreover, recovery of cellular GSH levels after acrolein exposure was enhanced following either alpha-tocopherol or ascorbic acid supplementation. The intracellular generation of oxidants following acrolein exposure seems to be an important event triggering the apoptotic response in this model system.

Phagocyte oxidants and nitric oxide in cystic fibrosis: new therapeutic targets?

Chronic persistent respiratory tract (RT) infection and overly exuberant activation of host inflammatory-immune processes represent the paramount pathobiologic consequences of cystic fibrosis (CF). The host inflammatory-immune system activation includes the production of reactive oxygen species (ROS) and nitric oxide (NO*), which are helpful in RT antimicrobial defenses but under conditions of excess are believed to be harmful to host tissues. However, the genetic defect underlying CF is recognized to uniquely affect inflammatory-immune (oxidative) processes, including alterations in cytokine release, phagocyte activation, antioxidant mechanisms, and NO* synthesis and metabolism, and these appear to contribute importantly to the persistent inflammation and infection in patients with CF. The dysregulation of inflammatory-immune responses has also been observed in CF epithelial cells and in some mouse models of CF, and marked advances in our understanding of these processes can be expected from future studies in such model systems. The emerging ideas of how ROS may influence molecular events that control inflammatory protease/antiprotease homeostasis and RT epithelial cell signaling and gene expression, and the role that augmentation of local antioxidant micronutrients (aerosolized and/or systemically administrated) might play in these effects, still remain to be further established and clarified. Further understanding of these processes can be expected to play an important role in future treatment directed toward the devastating effects of CF on the RT of patients with this disorder.

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.

Ultraviolet B-mediated phosphorylation of the small heat shock protein HSP27 in human keratinocytes.

Exposure of human keratinocytes to environmental stress is known to induce changes in the expression, phosphorylation, and subcellular relocalization of the 27 kDa heat shock protein. This study demonstrates that ultraviolet B (280-320 nM) irradiation with physiologic doses induces a dose-dependent phosphorylation of 27 kDa heat shock protein, generating the more acidic 27 kDa heat shock protein B, C, and D isoforms. Ultraviolet B also induces perinuclear cytoplasmic relocation and nuclear translocation of 27 kDa heat shock protein and caused aggregation of cytoplasmic actin filaments into a broad perinuclear distribution. The ultraviolet B-induced phosphorylation is reversible, returning to baseline levels 4 h after exposure, and this coincides with the reversal of ultraviolet B-induced actin reorganization. The ultraviolet B-induced phosphorylation is not affected by the protein kinase C inhibitor, GF 109203X, is partially inhibited by epidermal growth factor receptor tyrosine kinase inhibitor, PD 153035, and is substantially inhibited by the specific p38 mitogen-activated protein kinase inhibitor, SB 203580. In addition, pretreatment of cells with the anti-oxidant N-acetyl cysteine partially inhibits ultraviolet B-and oxidant-induced 27 kDa heat shock protein phosphorylation. The p38 mitogen-activated protein kinase cascade is thus the major transduction pathway for ultraviolet B-induced 27 kDa heat shock protein phosphorylation, and reactive oxygen species generated in response to ultraviolet B also contribute to this phosphorylation. As 27 kDa heat shock protein phosphorylation and relocalization has been associated with increased cell survival after environmental insult, our data suggest that ultraviolet B, in addition to initiating recognized cytotoxic events in keratinocytes, also initiates a signaling pathway that may provide cellular protection against this ubiquitous environmental insult.

Myeloperoxidase and protein oxidation in cystic fibrosis.

Cystic fibrosis (CF) is associated with chronic pulmonary inflammation and progressive lung dysfunction, possibly associated with the formation of neutrophil myeloperoxidase (MPO)-derived oxidants. Expectorated sputum specimens from adult CF patients were analyzed for MPO characteristic protein modifications and found to contain large amounts of active MPO as well as high levels of protein-associated 3-chlorotyrosine and 3,3'-dityrosine, products that result from MPO activity, compared with expectorated sputum from non-CF subjects. Sputum levels of nitrite (NO(2)(-)) and nitrate (NO(3)(-)), indicating local production of nitric oxide (NO. ), were not elevated but in fact were slightly reduced in CF. However, there was a slight increase in protein-associated 3-nitrotyrosine in CF sputum compared with controls, reflecting the formation of reactive nitrogen intermediates, possibly through MPO-catalyzed oxidation of NO(2)(-). CF sputum MPO was found to contribute to oxidant-mediated cytotoxicity toward cultured tracheobronchial epithelial cells; however, peroxidase-dependent protein oxidation occurred primarily within sputum proteins, suggesting scavenging of MPO-derived oxidants by CF mucus and perhaps formation of secondary cytotoxic products within CF sputum. Our findings demonstrate the formation of MPO-derived oxidizing and possibly nitrating species within the respiratory tract of subjects with CF, which collectively may contribute to bronchial injury and respiratory failure in CF.

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.

Ozone potentiates vitamin E depletion by ultraviolet radiation in the murine stratum corneum.

As the outermost layer of the skin, the stratum corneum is exposed to environmental oxidants. To investigate putative synergisms of environmental oxidative stressors in stratum corneum, hairless mice were exposed to ultraviolet radiation (UV) and ozone (O(3)) alone and in combination. Whereas a significant depletion of alpha-tocopherol was observed after individual exposure to either a 0.5 minimal erythemal dose of UV or 1 ppm O(3) for 2 h, the combination did not increase the effect of UV alone. However, a dose of 0.5 ppm O(3) x 2 h, which had no effect when used alone, significantly enhanced the UV-induced depletion of vitamin E. We conclude that concomitant exposure to low doses of UV and O(3) at levels near those that humans can be exposed to causes additive oxidative stress in the stratum corneum.

Vitamin C, uric acid, and glutathione gradients in murine stratum corneum and their susceptibility to ozone exposure.

The stratum corneum has been recognized as the main cutaneous oxidation target of atmospheric ozone (O3), a major part of photochemical smog. This study reports the presence and distribution of vitamin C, glutathione, and uric acid in murine stratum corneum, and evaluates their susceptibility to acute environmental exposure to O3. Based on tape stripping and a modified extraction method with high performance liquid chromatography electrochemical analysis, we detected vitamin C (208.0 +/- 82.5 pmol per 10 consecutive pooled tapes), glutathione (283.7 +/-96.3), and uric acid (286.4 +/-47.1) in murine stratum corneum as compared with only 16.5 +/- 1.4 pmol alpha-tocopherol. Vitamin C, glutathione (both p < 00.001), and urate (p < 0.01) were found to exhibit a gradient with the lowest concentrations in the outer layers and a steep increase in the deeper layers. To investigate the effect of O3 exposure on hydrophilic antioxidants, we exposed SKH-1 hairless mice to O3 concentrations of 0, 0.8, 1, and 10 p.p.m., and stratum corneum was analyzed before and after exposure. Whereas mock exposure with 0 p.p. m. for 2 h had no significant effect, O3 doses of 1 p.p.m. for 2 h and above showed depletion of all three antioxidants. Vitamin C was decreased to 80% +/- 15% of its pretreatment content (p < 0.05), GSH to 41% +/- 24% (p < 0.01), and uric acid to 44% +/- 28% (p < 0.01). This report demonstrates the previously unrecognized role of hydrophilic antioxidants in the stratum corneum and provides further evidence that O3 induces oxidative stress in this outer skin layer.

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.

Micronutrient antioxidants and smoking.

Cigarette smoking is a major risk factor in such human diseases as cardiovascular disease (especially atherosclerosis), lung cancer (the leading world-wide cancer killer), and chronic obstructive pulmonary disease (COPD). An avalanche of studies has suggested that a diet rich in fruit and vegetables is associated with decreased risk for atherosclerosis and cancer. However, the dietary intake of fruits and vegetables, as well as antioxidant micronutrients, is decreased in smokers. This, along with evidence of increased utilization of ascorbic acid and alpha-tocopherol, possibly on the basis of increased oxidative stress, contributes to the low plasma antioxidant concentrations seen in many smokers. This review addresses selected mechanistic considerations of this relationship.

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.

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.

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.

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.