NO2-induced generation of extracellular reactive oxygen is mediated by epithelial lining layer antioxidants

Leonard W. Velsor and Edward M. Postlethwait

Departments of Internal Medicine and Experimental Pathology, Pulmonary Research Laboratories, University of Texas Medical Branch, Galveston, Texas 77555-0876

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Nitrogen dioxide (NO2) is an environmental oxidant that causes acute lung injury. Absorption of this aqueous insoluble gas into the epithelial lining fluid (ELF) that covers air space surfaces is, in part, governed by reactions with ELF constituents. Consequently, NO2 absorption is coupled to its chemical elimination and the formation of ELF-derived products. To investigate mechanisms of acute epithelial injury, we developed a model encompassing the spatial arrangements of the lung surface wherein oxidation of cell membranes immobilized below a chemically defined aqueous compartment was assessed after NO2 exposures. Because aqueous-phase unsaturated fatty acids displayed minimal NO2 absorptive activity, these studies focused on glutathione (GSH) and ascorbic acid (AH2) as the primary NO2 absorption substrates. Results demonstrated that membrane oxidation required both gas-phase NO2 and aqueous-phase GSH and/or AH2. Membrane oxidation was antioxidant concentration and exposure duration dependent. Furthermore, studies indicated that GSH- and AH2-mediated NO2 absorption lead to the production of the reactive oxygen species (ROS) O<SUP>−</SUP><SUB>2</SUB>⋅ and H2O2 but not to · OH and that Fe-O2 complexes likely served as the initiating oxidant. Similar results were also observed in combined systems (GSH + AH2) and in isolated rat ELF. These results suggest that the exposure-induced prooxidant activities of ELF antioxidants generate extracellular ROS that likely contribute to NO2-induced cellular injury.

lipid oxidation; glutathione; ascorbic acid; epithelial lining fluid; lung injury; reactive absorption

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

NITROGEN DIOXIDE (NO2) is a ubiquitous oxidant gas that generates a variety of exposure-induced pathophysiological alterations within the lung. Acute inhalation exposure causes a dose-dependent injury characterized by epithelial damage, altered air space permeability, and induction of an inflammatory response (12, 44). Long-term exposure leads to biochemical and microanatomical changes in airways that may contribute to the pathogenesis of chronic lung diseases (44). Although the pulmonary responses consequent to exposure are well characterized, the mechanisms underlying NO2-induced lung injury remain equivocal.

Undoubtedly, the detrimental effects of NO2 are directly related to its oxidizing potential as evidenced by the production of a broad spectrum of biomolecular oxidation products during exposure (30, 53). It is unlikely, however, that the initial stages of the cytotoxic events result from direct interactions between NO2 and the epithelium. Direct contact between gas-phase NO2 and epithelial cells is precluded by the aqueous epithelial lining fluid (ELF) that covers the entire pulmonary air space surface (1). Consequently, the oxidant burden must be relayed from the gas-phase through the ELF to the underlying cells. Rapid and irreversible reactions between NO2 and reduced constituents maintain the driving force for the continued net flux of this relatively aqueous-insoluble gas into the ELF, a process designated as "reactive absorption" (36). Thus the deleterious effects of inhaled NO2 are likely mediated by products of these initial NO2-ELF reactions rather than NO2 per se.

Under physiological conditions, the predominant pathway for NO2-induced oxidation of biomolecules occurs via hydrogen abstraction or electron transfer to produce nitrous acid or nitrite (NO<SUP>−</SUP><SUB>2</SUB>), respectively, and biomolecule-derived free radicals (35). Consequently, reactive absorption couples reduction of the primary oxidant, NO2, with production of initial reaction products that may or may not function as secondary oxidants. With the use of the rate of NO2 gas-phase disappearance as a measure of aqueous-phase reaction, recent studies have demonstrated that reduced glutathione (GSH) and ascorbic acid (AH2) are the preferential absorption substrates in rat ELF (38). These conclusions were based on the combined evidence that aqueous-phase GSH and AH2 displayed rapid kinetics for NO2 gas-phase disappearance, removal of low-molecular-weight ELF constituents notably reduced NO2 absorption rates, and treatment of rat bronchoalveolar lavage fluid (BALF) to specifically diminish GSH and AH2 concentrations eliminated most NO2 uptake. Although minimal, residual absorption activity was potentially attributable to ELF unsaturated fatty acids (UFA).

Delineating the reaction mechanisms that govern NO2 toxicity requires an approach that not only encompasses the spatial arrangements of the lung surface compartments (i.e., air space, ELF, and epithelium) but also allows control of experimental conditions within each compartment. In vivo and isolated lung approaches would be ideal except that the lack of control over the biochemical makeup and initial conditions of the ELF present profound methodological constraints. Furthermore, alteration of the extracellular milieu due to cell injury-induced release of intracellular elements during intact lung and/or in vitro cell exposures limits these approaches for delineating the initial events associated with NO2 toxicity. Consequently, we have developed an in vitro model of the lung surface wherein model epithelia, red blood cell membranes (RCM) immobilized to the bottom of petri dishes, were covered by defined aqueous layers and exposed to NO2 atmospheres. Oxidation of biomolecules in the model epithelia were measured as a function of gas- and aqueous-phase conditions. The results from these initial studies demonstrate that, during NO2 exposure, the aqueous- soluble antioxidants GSH and AH2 function as prooxidants by leading to the production of extracellular reactive oxygen species (ROS) that in turn produce oxidation of membrane constituents.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. Packed human red blood cells were obtained from the University of Texas Medical Branch Blood Bank. NO2 (548 ppm in N2) and nitric oxide (NO; 10 ppm in N2) were purchased from Liquid Carbonic (Houston, TX). L-alpha -Lecithin [egg phosphatidylcholine (EggPC)] for liposomes was purchased from Avanti Polar Lipids (Alabaster, AL). All other reagents were purchased from Sigma Chemical (St. Louis, MO).

Liposomes. As a model of ELF unsaturated lipids, liposomes prepared from EggPC containing ~20% (wt/wt) UFA were utilized. Liposomes were generated by drying the desired volume of EggPC in a large glass test tube using N2. Cold phosphate buffer (PB) was added, and the contents were vortexed and sonicated three times for 30 s at 65 W (42). Precautions to minimize exposure to light and air were taken to limit autoxidation of UFA.

Bronchoalveolar lavage. Male Sprague-Dawley rats (225-250 g; Harlan Sprague Dawley, Houston, TX) were used as donor animals for all lavage procedures. Rats were allowed free access to food and water before induction of anesthesia. Animals were anesthetized with intraperitoneal pentobarbital sodium (60 mg/kg), and the depth of anesthesia was verified via foot pinch. After tracheal cannulation, the heart and lungs were removed en bloc to a petri dish containing warm saline and lavaged with a single 10-ml aliquot of isotonic phosphate-buffered saline (50 mM PO4-0.6% NaCl, pH 7.0). Fluid was gently but rapidly instilled and withdrawn three times (36). Cells were removed via centrifugation (2,000 g for 10 min), and the resulting cell-free BALF was used immediately unless treated to modulate antioxidant concentrations. AH2 and GSH were depleted via the addition of ascorbate oxidase (AO) or N-ethylmaleimide (NEM), respectively (38). For AH2, 0.5 U AO/ml of BALF was added, whereas, for GSH, a small volume of concentrated NEM was added to the BALF to produce a final concentration of 100 µM. BALF was incubated for 15 min after each addition and used immediately thereafter. Treatment with AO and NEM reduced antioxidant concentrations below detectable limits.

Steady-state exposures. To characterize aqueous substrate reaction preferentiality, a limited number of studies were conducted using a previously described steady-state exposure protocol (22, 36). Briefly, solutions were exposed (25°C) under well-mixed conditions in small glass vessels to a constant inflow of NO2 in air. NO2 absorption was computed by determining gas-phase NO2 mass balance across the exposure vessel.

Biochemical determinations. RCM lipid oxidation was assessed via determination of thiobarbituric acid reactive substances (TBARS) appearing in the model ELF (32, 51). After exposure, butylated hydroxytoluene was added directly to the dishes to prevent further oxidation (final concentration = 0.087 mM), and aliquots of the model ELF were removed for TBARS determination. Tetraethoxypropane was utilized as a standard for the TBARS assay. In some experiments, RCM protein oxidation was evaluated by the loss of protein sulfhydryls using the reduction of 5,5'-dithio-bis-2(nitrobenzoic acid) (Ellman's reagent) added directly to the petri dishes (46). Before addition of Ellman's reagent, the model ELF was removed for TBARS analysis, and the dishes were carefully rinsed with PB (100 mosmol, pH 7.0) to remove residual antioxidants contributed by the model ELF. GSH concentrations were also determined with Ellman's reagent. AH2 concentrations were determined by the reduction of 2,6-dichlorophenolindophenol (47).

Overview of the three-compartment model. A model system to appropriately investigate the capacity of the ELF to modulate acute toxicity of inhaled oxidants must satisfy the following criteria. 1) It must contain the three compartments that comprise the lung surface. Most importantly, the model must distinctly separate the gas-phase and the epithelial compartment with an intervening aqueous layer. 2) Components of the model epithelium must not be released into the ELF where they may act as absorption substrates or influence subsequent oxidative events. 3) Initial conditions within the ELF must be controllable and known. For these studies, we modeled the epithelium by use of a monolayer of RCM immobilized within silanized glass petri dishes. An aqueous layer consisting of absorption substrates in PB was added over the RCM to mimic the ELF, and the prepared petri dishes were exposed to air or NO2 atmospheres in a small glass chamber.

Immobilization of red blood cell membranes. Pyrex petri dishes (60 × 15 mm) were cleaned in boiling 10% (vol/vol) nitric acid and rinsed profusely with 18 MOmega deionized water (18 MOmega dH2O). A small volume of 5% (vol/vol) 3-aminopropyltriethoxysilane (titrated to pH <= 4 with 6 N HCl) was added into each dish to completely cover the bottom surface. Dishes were heated at 70°C for 8 h, rinsed with 18 MOmega dH2O, and dried at 110°C overnight. This process coated the glass surface with positively charged 3-aminopropyl moieties that were covalently attached to the glass via silanol linkages (54). Dishes were subsequently incubated with 2.5% (vol/vol) glutaraldehyde in 50 mM PB (pH 5.0) to form imino linkages with the amino groups and generate a surface of reactive aldehydes with a high affinity for the NH2 termini of RCM proteins. Dishes were thoroughly rinsed with 18 MOmega dH2O to remove unreacted glutaraldehyde before cells were added.

Packed human red blood cells were suspended in ice-cold isotonic PB (310 mosmol, pH 7.4) and centrifuged at 4,000 g to remove plasma components (11). This was repeated until the supernatant was visibly clear. The washed red blood cells were resuspended (10% vol/vol) in N2-saturated buffer, and aliquots that contained >25-fold excess red blood cells needed for surface covering were added to the treated petri dishes. Dishes were incubated in the dark at room temperature with gentle swirling. This produced a monolayer of red blood cells covalently bound to the glass surface. Unadhered red blood cells were aspirated, and the dishes were rinsed with isotonic PB. To lyse the red blood cells, a small volume of hypotonic PB (100 mosmol, pH 7.0) was added to the dishes (11). Dishes were repeatedly rinsed with hypotonic PB to remove residual hemoglobin and other intracellular debris.

Exposure protocol. Just before exposure, 2.00 ml of hypotonic PB (pH 7.0) containing dissolved reagents were gently added over the RCM to serve as the model ELF. Reagents included the absorption substrates (i.e., GSH and AH2), the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), the iron chelators desferrioxamine (DFX) or diethylenetriaminepentaacetic acid (DETAPAC), or the hydroxyl radical scavenger mannitol (MAN). GSH and AH2 solutions were prepared immediately before use. The petri dishes were cyclically tilted (one time every 2 min) during NO2 exposures to generate an intermittent aqueous film over the upper one-half of the RCM monolayer. Model constructs were exposed to NO2 atmospheres in a minimal-volume (1,500 ml) glass chamber. The NO2 atmospheres were generated by blending (countercurrent injection) NO2/N2 into a constant flow of humidified air (20% O2 and 80% N2) via mass flow controllers (Scott Specialty Gases, Houston, TX) to obtain desired concentrations. NO concentrations in the exposure chamber were <0.02%. A total flow of 550 ml/min provided a chamber turnover every 3 min. Exposures were conducted under well-mixed, NO2 first-order (<= 10 ppm), steady-state conditions at 25°C. Control air exposures (no NO2) were conducted similarly. To minimize background NO2 losses by reaction with chamber walls, tubing, etc., all exposure system components were thoroughly conditioned with the NO2 atmosphere before use. NO2 concentrations were continuously monitored with a chemiluminescent-based NOx analyzer (model 42; Thermo Environmental, Franklin, MA). The instrument was calibrated by graded addition of ozone to an NO primary standard (10 ppm) to generate NO2. NO2 mass balance was calculated from the difference between inlet and exit concentrations multiplied by the gas flow rate and time.

Validation of the three-compartment model. Validation that RCM biomolecules were not released into the aqueous-phase was confirmed by subjecting immobilized RCM to the exposure/tilting protocol and extracting lipids from both the ELF and dishes using 2:1 methanol-chloroform. Extracts were dried, and total lipid content was evaluated gravimetrically (4). The method of Lowry et al. (24) was utilized to measure protein concentrations in the model ELF and directly in the dishes. Table 1 demonstrates that the exposure protocol led to negligible RCM-derived lipids and proteins appearing in the aqueous-phase, i.e., RCM remained adhered to the petri dish bottoms.

                              
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Table 1.   Disposition of RCM lipids and proteins during NO2 exposure in the 3-compartment model

Maximal lipid oxidation rates were determined by exposing RCM monolayers to an ROS-generating system containing 5 mU/ml xanthine oxidase and 0.20 mM xanthine (total volume = 2.00 ml) for 30 min. Under these conditions, the accumulation of 824 ± 62 nM (n = 5) TBARS was observed. The combination of iron complexes with reductants such as GSH and especially AH2 is often utilized as an oxidant-generating system (28, 43). In initial studies, however, addition of AH2 over the RCM led to minimal increases in aqueous-phase TBARS during a 30-min air exposure (62.9 ± 6.2 and 84.3 ± 7.6 nM TBARS at 0 and 30 min, respectively). Moreover, AH2 oxidation, when added over the RCM, assessed via the loss in absorbance at 265 nm, was <0.5% over 30 min, further indicating that spontaneous iron plus AH2-associated oxidation was minimal (5). In the absence of antioxidants, background TBARS formation in dishes containing buffer increased slightly during a 30-min air exposure (55.8 ± 18.5 and 70.1 ± 16.3 nM TBARS at 0 and 30 min, respectively).

Data analysis. All experimental measurements are expressed as means ± SD. Differences in red blood cells obtained from varied donors led to relative variations in the baseline values for RCM lipid oxidation. To normalize for these variations, results for most experiments are reported as a percent change from the respective air control group. Significant differences between experimental groups were assessed by a one-way analysis of variance and Dunnett's test post hoc (49). Significance was defined as P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

NO2 reactivity with ELF substrates. Although previous studies had demonstrated that ELF AH2 and GSH served as the predominant NO2 absorption substrates, some direct reaction with lipids could also have occurred (38). Because the ELF contains an abundance of UFA (41) and because NO2 exposure generates ELF lipid oxidation products (53), we determined the absorption potential of lipids to ascertain whether they represented a significant source of primary reaction products. NO2 gas-phase disappearance rates during steady-state exposures in small exposure vessels were utilized as an indicator of reaction preferentiality. EggPC liposomes were employed as a model of ELF lipids. Equimolar aqueous solutions of antioxidants (0.10 mM) with or without 0.50 mM EggPC liposomes (approx 0.10 mM UFA) were exposed to NO2 under steady-state conditions, and NO2 absorption was assessed (Fig. 1). Liposomes displayed little absorption over background, and the addition of liposomes to either GSH or AH2 did not increase absorption over the antioxidants alone. Exposure of GSH plus AH2 mixtures demonstrated that both substrates react when combined and strongly suggested that NO2 absorption is primarily mediated by the antioxidants and not by the UFA. Thus, despite the presence of UFA in the ELF, GSH and AH2 produce the bulk of the initial reaction products formed as a consequence of absorption. Consequently, the following studies focused on the potential of these antioxidants to generate exposure-induced secondary oxidation of cellular components in the model system.


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Fig. 1.   NO2 reactive absorption by equimolar antioxidants and unsaturated lipids. Solutions (pH 7.0) of glutathione (GSH, 0.10 mM), ascorbic acid (AH2, 0.10 mM), or both (0.050 mM each) were exposed in the absence or presence of 0.50 mM egg phosphatidylcholine (EggPC) liposomes [approx 0.1 mM unsaturated fatty acids (UFA)] under well-mixed, steady-state conditions to 10 ppm NO2 for 30 min (25°C). Absorption was determined by computing NO2 mass balance across the exposure vessel. Liposomes displayed negligible absorption either alone or in the presence of antioxidants. When combined, both GSH and AH2 react to drive absorption. Results are expressed as means ± SD for n >=  3 experiments. PB, phosphate buffer.

Antioxidant-mediated oxidation of RCM membranes. NO2 exposure (10 ppm; 20 min) of PB-covered RCM produced negligible membrane oxidation over air controls (Figs. 2 and 3), suggesting that sufficient gas-phase NO2 did not penetrate the aqueous film to induce demonstrable oxidative events. Under identical exposure conditions, oxidation of RCM lipids and protein sulfhydryls was evaluated using GSH concentrations from 0 to 250 µM (Fig. 2). The addition of low GSH concentrations produced maximal exposure-related RCM biomolecule oxidations that declined at more elevated concentrations. A similar dose-response relationship was observed for both lipid and protein sulfhydryl oxidations. In all subsequent experiments, we chose to assess RCM oxidation by the appearance of aqueous-phase TBARS because RCM lipid oxidation resulted in product accumulation that enhanced detection sensitivity, whereas oxidative loss of protein sulfhydryls reduced measurement accuracy. For AH2, a similar dose-response relationship was observed but with maximal RCM lipid oxidation occurring at 25 µM (Fig. 3). For both antioxidants, air exposures produced little RCM oxidation, indicating that membrane oxidation was initiated by the exposure-related production of antioxidant-derived products.


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Fig. 2.   GSH-mediated red blood cell membrane (RCM) oxidation during NO2 exposure. RCM were covalently bound to the bottom of petri dishes, covered with GSH in PB (0-250 µM; pH 7.0), and exposed under steady-state conditions (25°C) to 10 ppm NO2 for 20 min with cyclic tilting. Membrane oxidation was evaluated by aqueous-phase thiobarbituric acid reactive substance (TBARS) formation and loss of protein sulfhydryls. To simplify the figure, air-exposure results for TBARS are only shown for 10 µM GSH, since lipid oxidation in air-exposed dishes did not vary over the range of GSH concentrations tested. Similarly, membrane sulfhydryl content did not change over GSH concentration during air exposures or from NO2 exposure with GSH concentration ([GSH]) = 0 (reference bar shown). Both TBARS production and loss of membrane sulfhydryls displayed [GSH] dependence during NO2 exposure. RCM oxidation was also dependent on a minimal aqueous covering because lipid oxidation in untilted dishes (10 µM GSH) was only slightly increased. Results are expressed as means ± SD for n = 6. Significantly different [analysis of variance (ANOVA); P <=  0.05] TBARS (*) and sulfhydryls (+) from the respective mean ([GSH] = 10-250 µM) air-exposed controls. [TBARS], TBARS concentration.


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Fig. 3.   AH2-mediated RCM oxidation during NO2 exposure. RCM covered by increasing concentrations of AH2 were exposed under steady-state conditions to 10 ppm NO2 or air for 20 min (25°C) with cyclic tilting and were evaluated for lipid oxidation via aqueous-phase TBARS formation. RCM oxidation did not occur during air exposure, regardless of AH2 concentration ([AH2]), or in the absence of NO2 but was AH2 concentration dependent during NO2 exposure. Results are expressed as means ± SD for n = 6. * Significance (ANOVA) from equivalent air-exposed control at P <=  0.05.

RCM oxidation was also dependent on aqueous layer thickness. When petri dishes containing 10 µM GSH were not tilted during exposure, only minimal TBARS were formed (Fig. 2), demonstrating that a relatively thin aqueous layer was necessary for translocation of secondary oxidants to the RCM. The prevention of RCM oxidation in the presence of a thick overlying layer was likely due to quenching of the secondary oxidants.

These results suggested that, if the initial absorption substrate concentrations were elevated, a lag period should occur during which absorption substrates are consumed. With the use of GSH, the time course of substrate loss versus RCM oxidation was determined (Fig. 4). During NO2 exposure, the concentration of absorption substrate decreased to a critical threshold before the onset of lipid oxidation. At physiologically relevant initial GSH concentrations (e.g., 500 µM in rat ELF), exposure-induced membrane oxidation only began when GSH concentrations fell below ~100 µM. In addition, the exposure dependence on TBARS accumulation was evaluated. RCM covered with 50 µM AH2 were exposed to either air, NO2 for 15 or 30 min, or NO2 for 15 min followed by 15 min of air. A 30-min NO2 exposure resulted in the accumulation of 217 ± 21 nM TBARS (n = 4). There was no difference in TBARS production between 15-min NO2 (117 ± 8 nM; n = 4) and 15-min NO2 plus 15-min air (131 ± 18 nM; n = 4), establishing that membrane oxidation was dependent on the continuous generation of exposure-derived secondary oxidants rather than exposure-initiated autoxidative processes.


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Fig. 4.   Temporal relationship between GSH disappearance and RCM lipid oxidation during NO2 exposure. RCM were covered with 500 µM GSH and exposed to 10 ppm NO2 for up to 105 min during which aqueous-phase TBARS formation and GSH concentrations were monitored. Only when GSH concentrations were decreased to a critical threshold (<100 µM) did TBARS accumulation become apparent. GSH and TBARS results are expressed as means ± SD for n >=  3.

Effect of pH and O2 concentration on the association between GSH loss and NO2 uptake. The potential for NO2 exposure-induced antioxidant redox cycling and the dependence on O2 was studied by exposing solutions to NO2 under steady-state conditions in an exposure flask at varying pH and O2 content. GSH was utilized due to its lower rate of spontaneous autoxidation. The effects of pH and O2 on NO2 uptake, GSH consumption, and the ratio of consumption to uptake were determined (Table 2). Despite the profound pH-induced alteration in the absorption rate, the GSH-to-NO2 ratio from air atmospheres reflected a threefold excess in GSH loss relative to NO2 uptake, which was unchanged across pH. When exposures were conducted under N2 atmospheres with N2-saturated PB (PO2 decreased >= 75%), a notable decline in the GSH-to-NO2 ratio occurred with increasing pH. This decline was due, in part, to the combined effects of decreased GSH consumption and increased NO2 uptake. In additional studies, the potential for O<SUP>−</SUP><SUB>2</SUB>⋅ and H2O2 to react directly with NO2 was evaluated (data not shown). H2O2 (1 mM) displayed little or no absorption activity. However, O<SUP>−</SUP><SUB>2</SUB>⋅, generated via xanthine plus xanthine oxidase, produced significant rates of NO2 uptake. O<SUP>−</SUP><SUB>2</SUB>⋅, estimated in the near-interfacial reaction plane as <= 1 µM, produced equivalent absorption rates as 100 µM GSH, suggesting that a back reaction between GSH-derived O<SUP>−</SUP><SUB>2</SUB>⋅ (6) and NO2 could also contribute to absorption.

                              
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Table 2.   Effect of pH and O2 concentrations on the ratio between GSH consumption and NO2 absorption

Initial assessment of ROS generation. Based on the above suggestion of O2 dependence, SOD (100 U/ml) and/or CAT (250 U/ml) was used to determine whether NO2-induced, antioxidant-mediated RCM oxidation involved ROS. Neither enzyme was inactivated by the NO2 exposure conditions employed. Inclusion of SOD significantly inhibited GSH-mediated oxidation of RCM lipids (Fig. 5). Inactivated SOD did not prevent the GSH-mediated lipid oxidation (data not shown). Addition of CAT, however, had little effect on TBARS production (Fig. 5), suggesting a limited role for H2O2 in the GSH-mediated RCM oxidation. In contrast to GSH, AH2-mediated membrane oxidation was somewhat prevented by CAT but not by SOD, indicating a partial dependence on H2O2 (Fig. 6). A role for the hydroxyl radical (· OH) in either GSH- or AH2-mediated lipid oxidation was tested by inclusion of the · OH scavenger MAN (10 mM). MAN failed to prevent RCM lipid oxidation by either GSH or AH2 during NO2 exposures (Figs. 5 and 6).


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Fig. 5.   Modulation of GSH-mediated RCM lipid oxidation during NO2 exposure. RCM were covered with 10 µM GSH (pH 7.0) with or without reactive oxygen species (ROS) scavengers, iron chelators, or supplemental Fe2+ and exposed to 10 ppm NO2 for 30 min (25°C) with cyclic tilting. Superoxide dismutase (SOD, 100 U/ml), catalase (CAT, 250 U/ml), mannitol (MAN, 10 mM), desferrioxamine (DFX, 50 µM), or FeCl2 (50 µM) was added just before exposure. SOD and DFX blocked NO2 exposure-induced TBARS production, whereas CAT and MAN had no effect. Addition of FeCl2 did not amplify membrane oxidation over GSH alone. Results are normalized to the air control and are presented as means ± SD for n >=  7 in all groups except MAN and FeCl2 in which n = 3. Significance (ANOVA; P <=  0.05) from the air control (*) or GSH (+).


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Fig. 6.   Modulation of AH2-mediated RCM lipid oxidation during NO2 exposure. RCM exposures and ROS modulator addition studies analogous to those presented in Fig. 5 were performed for AH2 (25 µM). In contrast to GSH, CAT, to a moderate extent, and DFX inhibited TBARS production, whereas SOD and MAN had no effect. Addition of excess Fe2+ did not enhance lipid oxidation. Results were normalized to the air control and are presented as means ± SD for n >=  7 in all groups except MAN and FeCl2 in which n = 3. Significance (ANOVA: P <=  0.05) from the air control (*) and AH2 (+).

Iron dependence and antioxidant combinations. The use of red blood cells as a source of cellular membranes clearly predisposes this model system to iron contamination. To characterize the importance of iron in the antioxidant-mediated RCM oxidation during NO2 exposure, iron addition and chelation studies were conducted. Chelation of Fe3+ using aqueous-phase DFX (50 µM) completely blocked both GSH- and AH2-mediated lipid oxidation during exposure. Because previous studies (9) suggested that reactions between NO2 and GSH may produce peroxynitrite (ONOO-), the DFX-mediated protection of membrane lipids was potentially attributable to its ONOO- scavenging properties rather than chelation of iron. To clarify the function of DFX in the model system, exposures with GSH were conducted in the presence 400 µM DETAPAC, which chelates iron but does not scavenge ONOO-. The prevention of GSH-mediated TBARS formation by DETAPAC during NO2 exposures (110.3 ± 13.5% relative to air controls) confirmed that the observed DFX-mediated protection was due to iron sequestration. The addition of excess Fe2+ (50 µM FeCl2) to either antioxidant system resulted in only minor enhancement of RCM lipid oxidation (Figs. 5 and 6), demonstrating that, although iron served a pivotal role, membrane oxidation was not rate limited by ambient iron concentrations. Importantly, due to the inherent presence of iron, spontaneous oxidation of RCM could have occurred upon AH2 addition (28, 41). However, the lack of significant AH2-induced RCM oxidation indicated that insufficient iron-AH2 redox cycling occurred in the absence of NO2.

Exposure-induced RCM oxidation was also determined in combined GSH plus AH2 systems. Under these conditions, substantial TBARS accumulated during NO2 exposures (Fig. 7). The addition of either SOD or CAT to the aqueous-phase reduced lipid oxidation by one-half, whereas combined SOD plus CAT treatment was completely protective. Similar to their effects in single substrate systems, inclusion of DFX completely prevented RCM lipid oxidation, whereas MAN had no effect (Fig. 7).


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Fig. 7.   Modulation of GSH- + AH2-mediated RCM lipid oxidation during NO2 exposure. RCM were covered by model ELF containing both GSH (10 µM) and AH2 (25 µM) and exposed to 10 ppm NO2 under steady-state conditions with cyclic tilting. Antioxidant enzymes were tested alone or in combination, in addition to DFX and FeCl2. Both SOD and CAT produced a significant decrease in TBARS production, whereas their combination and DFX both reduced lipid oxidation to near air control levels. Results were normalized to the air controls and are presented as means ± SD with n >=  8 in all groups except for FeCl2 and MAN in which n = 4. Significance (ANOVA: P <=  0.05) from air control (*) and AH2 + GSH (+).

Rat lung ELF. To test these observations in the biochemically complex milieu of the ELF, studies were conducted using rat BALF as the aqueous-phase. Initially, rat BALF GSH and AH2 concentrations were measured to be 20.5 ± 0.6 and 34.4 ± 4.7 µM, respectively. Because rat BALF also contains surfactant and airway lipids (1.8 ± 0.2 mg of total BALF lipids/lung), it was necessary to evaluate exposure-induced lipid oxidation within the BALF in the absence of RCM. NO2 exposure of BALF alone resulted in moderate TBARS production, most likely due to the combined presence of the antioxidants and iron (Fig. 8). In the presence of RCM, lipid oxidation increased over BALF alone, demonstrating secondary oxidation of the underlying membranes. Depletion of GSH and AH2 by NEM and AO, respectively, inhibited NO2-induced lipid oxidation in both BALF alone (data not shown) and the RCM composite. Inclusion of SOD in BALF moderately inhibited TBARS formation, whereas inclusion of SOD plus CAT or DFX completely blocked lipid oxidation. Similar to the model ELF systems, addition of MAN did not diminish lipid oxidation nor did iron supplementation (50 µM Fe+2) substantially enhance production of TBARS.


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Fig. 8.   Rat lung bronchoalveolar lavage fluid (BALF)-mediated RCM lipid oxidation during NO2 exposure. RCM were covered with BALF and exposed to 10 ppm NO2 for 30 min, and TBARS accumulation in the aqueous-phase was evaluated relative to air-exposed dishes. SOD, SOD + CAT, MAN, DFX, and FeCl2 were added as noted in Figs. 5-7. BALF was also treated with ascorbate oxidase (AO, 0.5 U/ml) and N-ethylmaleimide (NEM, 0.1 mM) to deplete AH2 and GSH, respectively. All treatments except MAN significantly inhibited exposure-induced membrane oxidation. Results were normalized to the air controls and are expressed as means ± SD for n >=  7 except for FeCl2 in which n = 4. Significance (ANOVA; P <=  0.05) from air control (*) or NO2-exposed (+) groups.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

NO2 exposure produces cellular injury and oxidation of both ELF and epithelial components before the onset of an inflammatory response. Because the ELF covers the epithelial surfaces, acute cell injury likely occurs due to transduction of oxidant species through this intervening layer to the underlying cells. The mechanisms that account for how this aqueous-insoluble but reactive gas penetrates a chemically reactive aqueous layer to initiate oxidative damage have not been fully delineated. Because solubility is low, but reactivity with substrates present in the ELF is high, direct interaction between inhaled NO2 per se and the pulmonary epithelium is most likely limited. The extent to which the inhaled toxicant may directly contact the epithelium is governed by the balance between diffusion and reaction of the dissolved (solute) gas within the ELF compartment. NO2 and the similarly aqueous- insoluble but reactive gas O3 undergo reactive absorption wherein the rate of gas-phase removal by aqueous solutions is greatly enhanced in the presence of reactive substrates (13, 18, 36, 37). The overall flux of NO2 into the aqueous-phase is gas-phase mass transfer limited (2) so that, within the ELF milieu, reaction likely exceeds formation of solute NO2. Thus the absorption of NO2 is coupled to its chemical elimination within the near-interfacial reaction plane. This serves to maintain the driving force for net flux across the gas-liquid interface, limit diffusion of solute NO2, and produce ELF-derived reaction products. However, it should be noted that, if NO2 concentrations are very high and/or reactions with either solutes or the solvent are sufficiently limited, diffusion of NO2 may exceed reaction, thereby permitting direct interaction with cellular membranes.

Previous kinetic analysis of NO2 gas-phase disappearance by ELF demonstrated that GSH and AH2 were the predominant rat ELF absorption substrates and that, although uric acid is highly reactive, its contribution to absorption within the distal rat (Sprague-Dawley) lung is negligible (16, 38). Because those studies did not definitively quantify the potential for UFA participation, studies to determine NO2 absorption rates by UFA alone or in combination with GSH or AH2 were performed (Fig. 1). Although an appreciable proportion of secreted surfactant lipids contain UFA (41), it is generally thought that the interfacial phospholipid film is highly enriched in saturated moieties (14, 23). Despite their presence in the ELF subphase, the exact physicochemical status of the UFA remains largely undefined. Because the UFA reside in an aqueous environment and dissolution with detergents limits reactivity with gas-phase NO2 (38), EggPC liposomes were used as a model of ELF UFA. This model likely facilitates some interfacial cycling (adsorption) of UFA so that limited contact with gas-phase NO2 could have occurred. Despite this, results from the steady-state exposure studies suggest that UFA are not primary targets for gas-phase NO2 even when present at the interface. Similar to studies using human plasma, NO2 absorption by solutions containing both antioxidants was additive, indicating that both substrates react with NO2 in multisubstrate systems (16). Because EggPC liposomes did not effectively compete with GSH and/or AH2 for removal of gas-phase NO2, their direct interaction with inhaled NO2 is likely limited. However, if NO2 is generated in close proximity to membrane UFA, such as from NO oxidation or HONOO reaction (8, 40), then reaction with UFA may occur. Moreover, if exposure decreases ELF concentrations of GSH and AH2 to very low levels, UFA may begin to react due to the lack of more preferential substrates, although the data in Fig. 1 imply that only limited reaction with gas phase-derived NO2 would occur.

The technique of covalently bonding RCM to a glass substratum prevented release of membrane lipid and protein into the aqueous phase (Table 1). Under conditions of direct contact, lipids and proteins undergo rapid oxidation by NO2 (13, 21, 39), which could be mimicked within the RCM by addition of an ROS-generating system to the model ELF. The lack of detectable exposure-induced membrane oxidation in RCM covered only by PB (Fig. 2) clearly demonstrated that the diffusion/reaction limitations within the aqueous-phase restricted direct interactions between NO2 and RCM even though the overlying film was relatively thin.

Based on the above, our initial studies focused on the ELF absorption substrates AH2 and GSH. Addition of low concentrations of either GSH or AH2 produced substantial membrane oxidation during NO2 exposure (Figs. 2 and 3), which agrees with previous studies that demonstrated that plasma lipid oxidation during NO2 exposure was initiated only when antioxidants were nearly depleted (16). Our results also coincide with a previous in vitro study in which DNA strand breaks and liposome oxidation under relatively severe NO2 exposure conditions (60-80 ppm) were enhanced in the presence of cysteine and GSH (20). Because the rate of NO2 absorption is directly related to the aqueous substrate concentration (36), the divergence between absorbed dose and effect seems contradictory. High antioxidant concentrations lead to enhanced NO2 absorption rates but little secondary oxidation of the underlying RCM, suggesting that the reactive species generated during absorption were quenched. At low antioxidant concentrations, where the rate of NO2 uptake is substantially less, diminished quenching reactions enhanced secondary oxidant interactions with the membranes. Consequently, a bell-shaped curve would appropriately describe these observed relationships between the magnitude of the oxidant dose (NO2 uptake) and the extent of membrane oxidation. However, it cannot be definitively ruled out that high antioxidant concentrations may have reduced oxidized lipids and proteins and thus diminished the marker end points (TBARS and RCM sulfhydryl) rather than prevented the oxidation of the membrane biomolecules.

The prolonged preparation times and chemical procedures to covalently bind the RCM produced a modest background of TBARS at the time of exposure commencement. Nonetheless, exposure to air, air plus antioxidant, or NO2 only further increased the pool of TBARS ~30%, whereas combined NO2 plus antioxidant exposure resulted in >300% increases. Variation in background oxidation may have also reflected the red blood cell source and the presence of the lipophilic antioxidant alpha -tocopherol (AT). Clearly, AT in the RCM could serve to quench oxidants and/or chain terminate lipid radicals. Differences in RCM AT content could affect both baseline levels of RCM oxidation and the response to NO2 exposure-induced oxidations, although normalization of results to preparation-matched controls should have accounted for potential variations. The presence of AT in cellular membranes, however, is representative of the lung surface where AT is localized to the epithelial cells. Although pure chemical studies demonstrated a high rate of reaction between NO2 and AT (38), nearly all of the AT found in BALF is associated with the cells and not with the extracellular milieu (48).

The initial products of GSH and AH2-mediated NO2 reactive absorption are the thiyl (GS ·) and the ascorbyl (A-·) radicals, respectively. The strong pH dependence of NO2 uptake correlates with the acidic dissociation constant for each substrate (AH2 approx  4.2; GSH approx  8.6) and likely reflects direct univalent reduction of NO2 to NO<SUP>−</SUP><SUB>2</SUB> via electron transfer from AH- and thiolate (GS-). Accordingly, the range of most pronounced pH dependence (pH 5-8) was utilized to investigate whether GS- and O2 availability influenced NO2 uptake and GSH loss (Table 2). The relationships between NO2 uptake and GSH loss were notably variable across pH and O2 conditions and suggested that, depending on the aqueous-phase, pH, and PO2, reactions subsequent to absorption led to the production of secondary oxidants and/or reductants with associated effects on 1) GSH oxidation, 2) the net loss of GSH due to secondary reduction of oxidized species, and 3) NO2 uptake. Previous publications have characterized the formation of GSH- and O2-derived oxidant and reductant species by GS · (6, 45, 55). Consequently, studies were initiated to determine whether NO2 exposure-dependent oxidations were mediated by ROS.

ROS production in systems containing one or both antioxidants was evaluated via the addition of specific scavengers and iron chelators to the model ELF (Figs. 5-7). Solutions containing SOD, CAT, MAN, DFX, or DETAPAC produced no demonstrable NO2 uptake at their respective concentrations so that any inhibitory effects could not be attributed to direct quenching of the NO2. Differential ROS production was observed between GSH and AH2, with GSH-mediated membrane oxidation being O<SUP>−</SUP><SUB>2</SUB>⋅ but not H2O2 dependent (Fig. 5). On the other hand, AH2-mediated oxidation was not SOD inhibitable but was partially ameliorated by CAT (Fig. 6). In combined systems (GSH + AH2), scavenging either O<SUP>−</SUP><SUB>2</SUB>⋅ or H2O2 decreased the extent of membrane oxidation, suggesting that both antioxidant-specific ROS production pathways were operative (Fig. 7). Combined treatment with SOD and CAT reduced TBARS production to near air control levels. The addition of MAN did not suppress exposure-induced membrane oxidation, indicating a probable lack of · OH involvement.

In all three systems, Fe3+ chelation was notably effective. Because membranes were derived from red blood cells, catalytic iron that could initiate oxidative reactions in the presence of added GSH or AH2 was recognized as a possible limitation of the model. In our initial studies, the lack of both AH2 oxidation and substantial TBARS formation in air-exposed dishes indicated that the contaminating iron displayed only minor potential to spontaneously catalyze RCM lipid oxidation. Taken together, these results suggest that, although O<SUP>−</SUP><SUB>2</SUB>⋅ and H2O2 generation occurred in the presence of iron, Fe-O2 complexes likely served as the initiating oxidant rather than · OH produced via Fenton or Haber-Weiss reactions. The fact that neither spontaneous redox reactions (antioxidant addition without exposure) nor continued autoxidation reactions after exposure cessation (NO2 exposure stopped but tilting continued) were sufficient to produce or amplify RCM oxidation suggests that these oxidative events were not sustainable without continued formation of GS · and A-· from NO2 reactive absorption.

Rat lung BALF was used as a model of the biologically complex ELF. Techniques to specifically harvest air space cells or lipids generally rely on repetitive washings. In these studies, we utilized a relatively mild lavage procedure to limit contamination of the BALF (22, 38). Although this approach may have undersampled the surface lipid pools, it was necessary to minimize contamination by cellular constituents (enzymes, antioxidants, etc.) due to diffusion of cytosolic elements or overt disruption of cells. Because BALF contains absorption substrates and UFA, NO2 exposure in the absence of RCM produced BALF-derived TBARS formation (Fig. 8). When RCM were covered with BALF, TBARS formation exceeded the BALF alone exposures, suggesting that membrane oxidation was also occurring. As in the pure chemical systems, addition of SOD, SOD plus CAT, or DFX reduced exposure-induced TBARS formation to near air control levels, whereas MAN had no effect. Depletion of GSH and AH2 in BALF before exposure also reduced the extent of lipid oxidation. It is interesting to note that, despite the presence of aqueous-phase UFA, the depletion of antioxidants, addition of ROS scavengers, or iron chelation all reduced TBARS formation even in the presence of RCM, suggesting that direct NO2-induced oxidation of UFA in the ELF is governed by the initial reactions between NO2 and antioxidants. Lavage of a single rat lung produces an ~100-fold dilution of the ELF constituents. However, based on the time- and concentration- dependent studies (Fig. 4), under in situ conditions, exposure-induced antioxidant consumption should ultimately lead to the prooxidant activities of GSH and AH2 observed in both the pure chemical and BALF studies.

Previous publications have detailed the specific pathways by which GS · and A-· are able to produce ROS (3, 6, 25, 28, 43). For GS ·, reactions with GS- produce the reductants GSSG-· and O<SUP>−</SUP><SUB>2</SUB>⋅
NO<SUB>2</SUB> + GS(H) → GS⋅ + H<SUP>+</SUP> + NO<SUP>−</SUP><SUB>2</SUB>
GS⋅ + GS<SUP>−</SUP> → GSSG<SUP>−</SUP>⋅ + O<SUB>2</SUB> → GSSG  + O<SUP>−</SUP><SUB>2</SUB>⋅
Subsequent interactions between O<SUP>−</SUP><SUB>2</SUB>⋅ and iron lead to Fe-O2 complex formation (15). GS · itself may serve as an oxidant or react with O2 to produce the additional oxidants GSOO · and GSO2OO · (6, 45). When GS- is limiting (e.g., acidic pH), these peroxyl-generating pathways may be more predominant. The consumption of GSH in excess of NO2 uptake may stem from GSSG-· formation and reaction with oxidant products (e.g., GSOO ·) such that ELF depletion potentially occurs more rapidly than the NO2 uptake rate. GSSG-· or O<SUP>−</SUP><SUB>2</SUB>⋅ may undergo direct electron transfer to NO2. In steady-state exposures, we observed that xanthine oxidase-derived O<SUP>−</SUP><SUB>2</SUB>⋅ produced notable NO2 uptake despite being in very low concentration, indicating that this direct quenching reaction (NO2 + O<SUP>−</SUP><SUB>2</SUB>⋅ right-arrow NO<SUP>−</SUP><SUB>2</SUB> + O2) may proceed at near-diffusion-limited rates (17). Furthermore, the addition of SOD to GSH solutions diminished NO2 uptake by approx 10% (unpublished observations). Presumably, this reaction could occur either directly from GSH-mediated sequelae or during O<SUP>−</SUP><SUB>2</SUB>⋅ release from activated air space surface cells. In addition, O<SUP>−</SUP><SUB>2</SUB>⋅ and GSSG-· may also reduce GS ·, which would result in no net GSH loss even though NO2 would be absorbed. These multiple interactions potentially lead to complex alterations in the relationship between NO2 uptake and GSH loss such that measurement of ELF substrate disappearance may not yield a straightforward indication of the extent of GSH reaction with NO2 or of the oxidant burden.

Interactions between A-· and iron lead to both H2O2 and Fe-O2 complex formation (3, 25, 28, 43)
AH<SUP>−</SUP> + NO<SUB>2</SUB> → A<SUP>−</SUP>⋅ + NO<SUP>−</SUP><SUB>2</SUB> + H<SUP>+</SUP>
AH<SUP>−</SUP> + Fe<SUP>3+</SUP> → A<SUP>−</SUP>⋅ + Fe<SUP>2+</SUP>; Fe<SUP>2+</SUP> + O<SUB>2</SUB> → Fe-O<SUB>2</SUB>
Fe-O<SUB>2</SUB> + A<SUP>−</SUP>⋅ + H<SUP>+</SUP> → Fe<SUP>3+</SUP> + A + H<SUB>2</SUB>O<SUB>2</SUB>
Because CAT only marginally reduced, but DFX completely blocked, AH2-induced membrane oxidation, it is likely that CAT served to limit Fe-O2 complex formation rather than direct membrane oxidation. Collectively, the results are consistent with the prooxidant activities of both antioxidants being mediated through interactions with iron wherein NO2 exposure produces ROS leading to Fe-O2 complex formation, which serves to initiate membrane oxidation. In both the pure chemical and BALF systems, there was sufficient iron such that further Fe2+ addition did not markedly increase membrane oxidation (Figs. 5-8). Based on preliminary evaluations of BALF, we have calculated that rat lung ELF contains ~50 µM chelatable iron. Human BALF has also been reported to contain appreciable iron concentrations (7, 31). Consequently, iron-mediated membrane oxidation should occur in situ during NO2 exposure. A previous study with rats demonstrated that histological evidence of lung epithelial injury after an NO2 exposure was substantially decreased by intravenous administration of DFX, although distribution of DFX to the lung surface was not reported (27).

The data characterizing the complex relationships among antioxidant concentration, NO2 absorption, catalytic iron, and membrane oxidation in this model can be theorized to infer the following. 1) Under basal air conditions, membrane-associated iron was not particularly available for redox cycling so that, when antioxidants were added, little spontaneous membrane oxidation occurred. 2) At initially elevated antioxidant concentrations, because of the high rates of NO2 uptake, product formation was rapid within the reaction plane, which promoted self quenching (e.g., A-· disproportionation, GS · dimerization, or reduction by O<SUP>−</SUP><SUB>2</SUB>⋅ or GSSG-·) of the reaction products, which diminished potential iron interactions. Consequently, little membrane oxidation occurred despite the large dose of absorbed oxidant. In addition, "thick" aqueous layers introduced longer diffusional distances for the products, which increased the potential for self quenching and thwarted membrane oxidation. 3) At lower antioxidant concentrations, self quenching was less likely due to the coupled decrease in product formation rates. Under these conditions, the formation of species with greater reducing potential than the parent antioxidants (e.g., A-·, O<SUP>−</SUP><SUB>2</SUB>⋅, and GSSG-·; see Ref. 6) facilitated formation of the iron-based oxidant species that initiated membrane oxidation.

The regional deposition of inhaled NO2 is heterogenous (29, 34, 52) so that specific ELF microenvironments may experience more rapid exposure-induced antioxidant consumption than others. In preliminary studies using an isolated perfused rat lung model (unpublished observations), we have observed declines in BALF GSH and AH2 concentrations after relatively brief NO2 exposures. Alterations in BALF antioxidant concentrations have also been observed in humans after short-term, low-concentration NO2 exposures (19). Whole lung BALF represents a lumped sample of all airway surfaces, including the far distal air spaces where NO2 is not likely to penetrate (29). Therefore, a decline in the overall BALF antioxidant concentrations implies that localized regions may undergo substantial antioxidant depletion due to consumption exceeding resupply. Thus, as illustrated in Fig. 4, the early phases of in vivo exposure may be characterized by the rapid consumption of antioxidants down to a critical threshold at which point quenching is lost and epithelial injury begins to occur.

Ultimately, it must be determined whether NO2-induced extracellular ROS-mediated secondary oxidations occur within the lung during in vivo exposures. Although lung tissue is rich in antioxidant enzymes, including extracellular SOD (10), the extent to which SOD and CAT are present on the lung surface has not been completely resolved. Although recent in situ hybridization studies show that type II cells and alveolar macrophages contain extracellular SOD mRNA (50), immunohistochemical localization studies do not clearly show high concentrations associated with the apical side of the epithelium (33). Analysis of BALF reveals both SOD and CAT activities, but whether these arise from lavage-induced artifacts is not clear (26). However, differential production and scavenging of extracellular ROS on the lung surface could, in part, account for the disparate toxicities between NO2 and O3 even though they display similar physicochemical characteristics.

In conclusion, we have utilized a new model system of the lung surface to demonstrate a potential pathway for NO2-induced lung injury wherein the oxidant burden of gas-phase NO2 is transduced through the ELF to underlying membranes via GSH- and AH2-dependent prooxidant activities. NO2 alone was not sufficient to initiate oxidation of membranes sequestered below a substrate-free aqueous film. Antioxidant-specific generation of extracellular ROS with coupled Fe-O2 complex formation provoked the secondary oxidative events. The data further suggest that, within the lung, dose-response relationships may be complex such that there are nonlinear proportionalities between oxidant uptake and the extent of cell membrane damage. Consequently, delineation of mechanisms of action and extrapolation of NO2-induced biological effects across and among in vitro and in vivo experimental systems should be viewed with appropriate caution.

    ACKNOWLEDGEMENTS

We recognize the constructive suggestions and valuable insights provided by Dr. Bruce A. Freeman in preparation of this work.

    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-54696, by funds from the Center for Indoor Air Research 90-23, by National Institute of Environmental Health Sciences Grant T32-ES-07254 (to L. W. Velsor), and by United States Army Grant 1796M0081.

Address for reprint requests: E. M. Postlethwait, Div. of Pulmonary and Critical Care Medicine, 0876, Dept. of Internal Medicine, Univ. of Texas Medical Branch, 301 Univ. Blvd., Galveston, TX 77555-0876.

Received 18 April 1997; accepted in final form 5 September 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Bastacky, J., C. Y. C. Lee, J. Goerke, H. Koushafar, D. Yager, L. Kenaga, T. P. Speed, Y. Chen, and J. A. Clements. Alveolar lining layer is thin and continuous: low-temperature scanning electron microscopy of rat lung. J. Appl. Physiol. 79: 1615-1628, 1995[Abstract/Free Full Text].

2.  Bidani, A., and E. M. Postlethwait. Kinetic determinants of reactive gas uptake. In: Complexities in Structure and Function of the Lung, edited by C. Lenfant. New York: Dekker. In press.

3.   Bielski, B. H. Chemistry of ascorbic acid radicals. In: Ascorbic Acid, Chemistry, Metabolism, and Uses, edited by P. A. Seib, and B. M. Tolbert. Washington, DC: Am. Chem. Soc., 1982, p. 81-100.

4.   Bligh, E. G., and W. J. Dyer. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911-917, 1959.

5.   Buettner, G. R. Use of ascorbate as test for catalytic metals in simple buffers. Methods Enzymol. 186: 125-127, 1990[Medline].

6.   Buettner, G. R. The pecking order of free radicals and antioxidants: lipid peroxidation, alpha -tocopherol and ascorbate. Arch. Biochem. Biophys. 300: 535-543, 1993[Medline].

7.   Corhay, J., T. Bury, J. Delavignette, F. Baharloo, M. Radermecker, P. Hereng, A. Fransolet, G. Weber, and I. Roelandts. Nonfibrous mineralogical analysis of bronchoalveolar lavage fluid from blast-furnace workers. Arch. Environ. Health 50: 312-319, 1995[Medline].

8.   Crow, J. P., C. Spruell, J. Chen, C. Gunn, H. Ischiropoulos, M. Tsai, C. D. Smith, R. Radi, W. H. Koppenol, and J. S. Beckman. On the pH-dependent yield of hydroxyl radical products from peroxynitrite. Free Radic. Biol. Med. 16: 331-338, 1994[Medline].

9.   Davidson, C. A., P. M. Kaminski, M. Wu, and M. S. Wolin. Nitrogen dioxide causes pulmonary arterial relaxation via thiol nitrosation and NO formation. Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H1038-H1043, 1996[Abstract/Free Full Text].

10.   Davis, W. B., and E. R. Pacht. Extracellular antioxidant defenses. In: The Lung, Scientific Foundations, edited by R. G. Crystal, and J. B. West. New York: Raven, 1991, p. 1821-1887.

11.   Dodge, J. T., C. Mitchell, and D. J. Hanahan. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch. Biochem. Biophys. 100: 119-130, 1963.

12.   Evans, M. J., L. J. Cabral, R. J. Stephens, and G. Freeman. Renewal of alveolar epithelium in the rat following exposure to NO2. Am. J. Pathol. 70: 175-198, 1973[Medline].

13.   Gallon, A. A., and W. A. Pryor. The reaction of low levels of nitrogen dioxide with methyl linoleate in the presence and absence of oxygen. Lipids 29: 171-176, 1994[Medline].

14.   Goerke, J., and J. A. Clements. Alveolar surface tension in lung surfactant. In: Handbook of Physiology. Respiration. Bethesda, MD: Am. Physiol. Soc., 1985, sect. 3, vol. III, pt. 1, chapt. 16, p. 247-261.

15.   Gutteridge, J. M. C. Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by superoxides. FEBS Lett. 201: 291-295, 1986[Medline].

16.   Halliwell, B., M. Hu, S. Louie, T. R. Duvall, B. K. Tarkington, P. Motchnik, and C. E. Cross. Interaction of nitrogen dioxide with human plasma: antioxidant depletion and oxidative damage. FEBS Lett. 313: 62-66, 1992[Medline].

17.   Huie, R. E. The reaction kinetics of NO2. Toxicology 89: 193-216, 1994[Medline].

18.   Kanofsky, J. R., and P. D. Sima. Singlet-oxygen generation at gas-liquid interfaces: a significant artifact in the measurement of singlet-oxygen yields from ozone-biomolecule reactions. Photochem. Photobiol. 58: 335-340, 1993[Medline].

19.   Kelly, F. J., A. Blomberg, A. Frew, S. T. Holgate, and T. Sandstrom. Antioxidant kinetics in lung lavage fluid following exposure of humans to nitrogen dioxide. Am. J. Respir. Crit. Care Med. 154: 1700-1705, 1996[Abstract].

20.   Kikugawa, K., K. Hiramoto, Y. Okamoto, and Y. Hasegawa. Enhancement of nitrogen dioxide-induced lipid peroxidation and DNA strand breaking by cysteine and glutathione. Free Radic. Res. 21: 399-408, 1994[Medline].

21.   Lai, C. C., and B. J. Finlayson-Pitts. Reactions of dinitrogen pentoxide and nitrogen dioxide with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Lipids 26: 306-314, 1991[Medline].

22.   Langford, S. D., A. Bidani, and E. M. Postlethwait. Ozone-reactive absorption by pulmonary epithelial lining fluid constituents. Toxicol. Appl. Pharmacol. 132: 122-130, 1995[Medline].

23.   Longo, M. L., A. M. Bisagno, J. A. N. Zasadzinski, R. Bruni, and A. J. Waring. A function of lung surfactant protein SP-B. Science 261: 453-456, 1993[Medline].

24.   Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951[Free Full Text].

25.   Martell, A. E. Chelates of ascorbic acid. In: Ascorbic Acid, Chemistry, Metabolism, and Uses, edited by P. A. Seib, and B. M. Tolbert. Washington, DC: Am. Chem. Soc., 1982, p. 153-178.

26.   Matalon, S., B. A. Holm, R. R. Baker, M. K. Whitfield, and B. A. Freeman. Characterization of antioxidant activities of pulmonary surfactant mixtures. Biochim. Biophys. Acta 1035: 121-127, 1990[Medline].

27.   Meulenbelt, J., J. A. M. A. Dormans, L. van Bree, P. J. A. Rombout, and B. Sangster. Desferrioxamine treatment reduces histological evidence of lung damage in rats after acute nitrogen dioxide intoxication. Hum. Exp. Toxicol. 12: 389-395, 1993[Medline].

28.   Miller, D. M., and S. Aust. Studies of ascorbate-dependent, iron-catalyzed lipid peroxidation. Arch. Biochem. Biophys. 271: 113-119, 1989[Medline].

29.   Miller, F. J., J. H. Overton, E. T. Myers, and J. A. Graham. Pulmonary dosimetry of nitrogen dioxide in animals and man. In: Air Pollution by Nitrogen Oxides, edited by T. Schneider, and L. Grant. New York: Elsevier, 1982, p. 377-386.

30.   Mustafa, M. G., and D. F. Tierney. Biochemical and metabolic changes in the lung with oxygen, ozone and nitrogen dioxide toxicity. Am. Rev. Respir. Dis. 118: 1061-1090, 1978[Medline].

31.   Nelson, M. E., A. R. O'Brien, and L. J. Wesselius. Regional variation in iron and iron-binding proteins within the lungs of smokers. Am. J. Respir. Crit. Care Med. 153: 1353-1358, 1996[Abstract].

32.   Ohkawa, H., N. Ohishi, and K. Yagi. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95: 351-358, 1979[Medline].

33.   Oury, T. D., L. Chang, S. L. Marklund, and J. D. Crapo. Immunocytochemical localization of extracellular superoxide dismutase in human lung. Lab. Invest. 70: 889-898, 1994[Medline].

34.   Overton, J. H., and F. J. Miller. Absorption of inhaled reactive gases. In: Toxicology of the Lung, edited by D. E. Gardner, J. D. Crapo, and E. J. Massaro. New York: Raven, 1988, p. 477-507.

35.   Postlethwait, E. M., and A. Bidani. Mechanisms of pulmonary NO2 absorption. Toxicology 89: 217-237, 1994[Medline].

36.   Postlethwait, E. M., S. D. Langford, and A. Bidani. Reactive absorption of nitrogen dioxide by pulmonary epithelial lining fluid. J. Appl. Physiol. 69: 523-531, 1990[Abstract/Free Full Text].

37.   Postlethwait, E. M., S. D. Langford, and A. Bidani. Kinetics of NO2 air space absorption in isolated rat lungs. J. Appl. Physiol. 73: 1939-1945, 1992[Abstract/Free Full Text].

38.   Postlethwait, E. M., S. D. Langford, L. M. Jacobson, and A. Bidani. NO2 reactive absorption substrates in rat pulmonary surface lining fluids. Free Radic. Biol. Med. 19: 553-563, 1994.

39.   Prutz, W. A., H. Monig, J. Butler, and E. J. Land. Reactions of nitrogen dioxide in aqueous model systems: oxidation of tyrosine units in peptides and proteins. Arch. Biochem. Biophys. 243: 125-134, 1985[Medline].

40.   Pryor, W. A., and G. L. Squadrito. The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L699-L722, 1995[Abstract/Free Full Text].

41.   Rooney, S. A. Phospholipid composition, biosynthesis, and secretion. In: Treatise on Pulmonary Toxicology: Comparative Biology of the Normal Lung, edited by R. A. Parent. Boca Raton, FL: CRC, 1992, p. 511-544.

42.   Rubbo, H., R. Radi, M. Trujillo, R. Telleri, B. Kalyanaraman, S. Barnes, M. Kirk, and B. A. Freeman. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. J. Biol. Chem. 269: 26066-26075, 1994[Abstract/Free Full Text].

43.   Scarpa, M., R. Stevanto, P. Viglino, and A. Rigo. Superoxide anion as active intermediate in the autoxidation of ascorbate by molecular oxygen. J. Biol. Chem. 258: 6695-6697, 1983[Abstract/Free Full Text].

44.   Schlesinger, R. B. Nitrogen oxides. In: Environmental Toxicants, edited by M. Lippman. New York: Van Nostrand Reinhold, 1992, p. 412.

45.   Schoneich, C., U. Dillinger, F. von Bruchhausen, and K. Asmus. Oxidation of polyunsaturated fatty acids and lipids through thiyl and sulfonyl radicals: reaction kinetics, and influence of oxygen and structure of thiyl radicals. Arch. Biochem. Biophys. 292: 456-467, 1992[Medline].

46.   Sedlak, J., and R. Lindsay. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal. Biochem. 25: 192-205, 1968[Medline].

47.   Skoza, L., A. Snyder, and Y. Kikkawa. Ascorbic acid in bronchoalveolar wash. Lung 161: 99-109, 1983[Medline].

48.   Slade, R., K. Crissman, J. Norwood, and G. Hatch. Comparison of antioxidant substances in bronchoalveolar lavage cells and fluid from humans, guinea pigs and rats. Exp. Lung Res. 19: 469-484, 1993[Medline].

49.   Sokal, R. R., and F. J. Rohlf. Biometry. New York: Freeman, 1981.

50.   Su, W., R. Folz, J. Chen, J. D. Crapo, and L. Chang. Extracellular superoxide dismutase mRNA expressions in the human lung by in situ hybridization. Am. J. Respir. Cell Mol. Biol. 16: 162-170, 1997[Abstract].

51.   Uchiyama, M., and M. Mihara. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal. Biochem. 86: 271-278, 1978[Medline].

52.   Ultman, J. S. Transport and uptake of inhaled gases. In: Air Pollution, the Automobile, and Public Health, edited by A. Watson, R. R. Bates, and D. Kennedy. Washington, DC: Natl. Acad. Press, 1988, p. 323-366.

53.   US Environmental Protection Agency. Air Quality Criteria for Oxides of Nitrogen. Environmental Criteria/Assessment. Washington, DC: US Environmental Protection Agency, 1995, p. 14-39.

54.   Weetall, H. H. Preparation of immobilized proteins covalently coupled through silane coupling agents to inorganic supports. Appl. Biochem. Biotechnol. 41: 157-188, 1993[Medline].

55.   Winterbourn, C. C. Superoxide as an intracellular radical sink. Free Radic. Biol. Med. 14: 85-90, 1993[Medline].


AJP Lung Cell Mol Physiol 273(6):L1265-L1275
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