Departments of Internal Medicine and Experimental Pathology, Pulmonary Research Laboratories, University of Texas Medical Branch, Galveston, Texas 77555-0876
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ABSTRACT |
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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)
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
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INTRODUCTION |
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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
(), 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.
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METHODS |
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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--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 M deionized water (18 M
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 M
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 M
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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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.
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RESULTS |
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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 (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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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
and
H2O2
to react directly with NO2 was
evaluated (data not shown).
H2O2
(1 mM) displayed little or no absorption activity. However,
, generated via xanthine plus
xanthine oxidase, produced significant rates of
NO2 uptake.
, 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
(6) and
NO2 could also contribute to
absorption.
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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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DISCUSSION |
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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 -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
4.2; GSH
8.6) and
likely reflects direct univalent reduction of
NO2 to
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 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
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
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
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Interactions between A· and iron lead to both
H2O2
and Fe-O2 complex formation (3,
25, 28, 43)
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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
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
·,
, 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.
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ACKNOWLEDGEMENTS |
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We recognize the constructive suggestions and valuable insights provided by Dr. Bruce A. Freeman in preparation of this work.
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FOOTNOTES |
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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.
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