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
lipid oxidation; glutathione; ascorbic acid; epithelial lining
fluid; lung injury; reactive absorption
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 NO2induced 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 (NO2
2 ), 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 aqueousphase conditions. The results from these initial studies
1040-0605/97 $5.00 Copyright r 1997 the American Physiological Society
L1265
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
Velsor, Leonard W., and Edward M. Postlethwait.
NO2-induced generation of extracellular reactive oxygen is
mediated by epithelial lining layer antioxidants. Am. J.
Physiol. 273 (Lung Cell. Mol. Physiol. 17): L1265–L1275,
1997.—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 gasphase NO2 and aqueous-phase GSH and/or AH2. Membrane
oxidation was antioxidant concentration and exposure duration dependent. Furthermore, studies indicated that GSHand AH2-mediated NO2 absorption lead to the production of
the reactive oxygen species (ROS) O2
2 z and H2O2 but not
to z OH and that Fe-O2 complexes likely served as the initiating oxidant. Similar results were also observed in combined
systems (GSH 1 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.
L1266
NITROGEN DIOXIDE-INDUCED REACTIVE OXYGEN SPECIES
demonstrate that, during NO2 exposure, the aqueoussoluble 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
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
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-a-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 5
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 5,58-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 2,6dichlorophenolindophenol (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 3 15 mm) were cleaned in boiling 10% (vol/vol)
nitric acid and rinsed profusely with 18 MV deionized water
(18 MV 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 MV 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 MV 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 gener-
L1267
NITROGEN DIOXIDE-INDUCED REACTIVE OXYGEN SPECIES
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
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 steadystate 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.
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 gasphase 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 rela-
Table 1. Disposition of RCM lipids and proteins during NO2 exposure in the 3-compartment model
Protein, mg/dish
Lipid, mg/dish
Compartment
Tested
Preexposure
Postexposure
Preexposure
Postexposure
Aqueous phase
Petri dish
ND
0.045 6 0.002
ND
0.049 6 0.005
ND
0.038 6 0.005
0.02 6 0.001
0.036 6 0.004
Results are expressed as means 6 SD for n $ 6 experiments. ND, not detected. Lipid and protein contents of both the aqueous-phases and
petri dishes (adhered biomolecules) were evaluated before and after NO2 exposure [10 µM glutathione (GSH)]. Data suggest that proteins and
lipids of red blood cell membranes (RCM) immobilized to the bottom of glass petri dishes are not released into the overlying aqueous-phase
during NO2 exposures. Small amount of lipids detected in the aqueous-phase is likely attributable to lipid oxidation products formed during
exposures.
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
ated 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.
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 5 2.00 ml) for 30 min. Under these conditions, the
accumulation of 824 6 62 nM (n 5 5) TBARS was observed.
The combination of iron complexes with reductants such as
GSH and especially AH2 is often utilized as an oxidantgenerating 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
6.2 and 84.3 6 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 AH2associated 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 6 18.5 and 70.1 6 16.3 nM TBARS at 0 and 30 min,
respectively).
Data analysis. All experimental measurements are expressed as means 6 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
L1268
NITROGEN DIOXIDE-INDUCED REACTIVE OXYGEN SPECIES
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, airexposure 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]) 5 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 6 SD for n 5 6. Significantly
different [analysis of variance (ANOVA); P # 0.05] TBARS (*) and
sulfhydryls (1) from the respective mean ([GSH] 5 10–250 µM)
air-exposed controls. [TBARS], TBARS concentration.
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 aqueousphase 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 6 SD for n 5 6. * Significance
(ANOVA) from equivalent air-exposed control at P # 0.05.
tionship 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.
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
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
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 [<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 6 SD for n $ 3 experiments. PB, phosphate buffer.
L1269
NITROGEN DIOXIDE-INDUCED REACTIVE OXYGEN SPECIES
exposure resulted in the accumulation of 217 6 21 nM
TBARS (n 5 4). There was no difference in TBARS
production between 15-min NO2 (117 6 8 nM; n 5 4)
and 15-min NO2 plus 15-min air (131 6 18 nM; n 5 4),
establishing that membrane oxidation was dependent
on the continuous generation of exposure-derived secondary oxidants rather than exposure-initiated autoxidative processes.
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 pHinduced alteration in the absorption rate, the GSH-toNO2 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
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 Fe21 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 6 SD for n $ 7 in all groups except MAN
and FeCl2 in which n 5 3. Significance (ANOVA; P # 0.05) from the
air control (*) or GSH (1).
consumption and increased NO2 uptake. In additional
studies, the potential for O2
2 z and H2O2 to react directly
with NO2 was evaluated (data not shown). H2O2 (1 mM)
displayed little or no absorption activity. However, O2
2 z,
generated via xanthine plus xanthine oxidase, produced significant rates of NO2 uptake. O2
2 z, 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 O2
2 z (6)
and NO2 could also contribute to 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,
Table 2. Effect of pH and O2 concentrations on the ratio between GSH consumption and NO2 absorption
Air
Nitrogen
pH
GSH loss, nmol
NO2 uptake, nmol
GSH/NO2
GSH loss, nmol
NO2 uptake, nmol
GSH/NO2
5.0
7.0
8.0
101 6 14
331 6 22
719 6 48
32 6 2
112 6 10
222 6 10
3.1:1
3.0:1
3.0:1
133 6 16
137 6 11
112 6 4
43 6 3
131 6 3
291 6 32
3.1:1
1.0:1
0.4:1
Exposures were conducted under steady-state conditions in a flow-through system with constant aqueous-phase mixing (25°C). GSH [200
µM in phosphate buffer (PB)] was exposed to 7.5 ppm NO2 in either air or N2 for 20 min, and samples were analyzed for total reduced
sulfhydryls immediately postexposure. PB was equilibrated with N2 , and pH was adjusted immediately before exposure. NO2 uptake was
determined by computing NO2 mass balance across the exposure vessel. PO2 : air 5 165 6 6 mmHg; N2 5 42 6 9 mmHg. Results are expressed
as means 6 SD for n 5 4.
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
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 6 SD for
n $ 3.
L1270
NITROGEN DIOXIDE-INDUCED REACTIVE OXYGEN SPECIES
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 (z OH) in either GSH- or AH2-mediated lipid
oxidation was tested by inclusion of the z OH scavenger
MAN (10 mM). MAN failed to prevent RCM lipid
oxidation by either GSH or AH2 during NO2 exposures
(Figs. 5 and 6).
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 Fe31 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 (ONOO2 ), the DFX-mediated protection of membrane lipids was potentially
attributable to its ONOO2 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 ONOO2. The prevention of
GSH-mediated TBARS formation by DETAPAC during
NO2 exposures (110.3 6 13.5% relative to air controls)
confirmed that the observed DFX-mediated protection
was due to iron sequestration. The addition of excess
Fe21 (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-
Fig. 7. Modulation of GSH- 1 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 6 SD with n $ 8 in all groups
except for FeCl2 and MAN in which n 5 4. Significance (ANOVA: P #
0.05) from air control (*) and AH2 1 GSH (1).
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
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 Fe21 did not enhance lipid oxidation. Results were
normalized to the air control and are presented as means 6 SD for
n $ 7 in all groups except MAN and FeCl2 in which n 5 3.
Significance (ANOVA: P # 0.05) from the air control (*) and AH2 (1).
induced RCM oxidation indicated that insufficient ironAH2 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).
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 6 0.6 and 34.4 6 4.7 µM, respectively. Because rat BALF also contains surfactant and
airway lipids (1.8 6 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 Fe12 ) substantially enhance production of
TBARS.
NITROGEN DIOXIDE-INDUCED REACTIVE OXYGEN SPECIES
DISCUSSION
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 aqueousinsoluble 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 phasederived 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 ROSgenerating 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
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
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 1 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 6 SD for n $ 7 except for
FeCl2 in which n 5 4. Significance (ANOVA; P # 0.05) from air control
(*) or NO2-exposed (1) groups.
L1271
L1272
NITROGEN DIOXIDE-INDUCED REACTIVE OXYGEN SPECIES
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 z (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 O2
2 z 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 1 AH2 ), scavenging
either O2
2 z 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 z OH
involvement.
In all three systems, Fe31 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 O2
2 z and H2O2 generation occurred in the
presence of iron, Fe-O2 complexes likely served as the
initiating oxidant rather than z 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 z and A2z
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
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
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 a-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 z) and the ascorbyl
(A2z) 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 NO2
2
via electron transfer from AH2 and thiolate (GS2 ).
Accordingly, the range of most pronounced pH dependence (pH 5–8) was utilized to investigate whether GS2
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
L1273
NITROGEN DIOXIDE-INDUCED REACTIVE OXYGEN SPECIES
NO2 1 GS(H) = GS z 1 H1 1 NO2
2
GSz 1 GS2 = GSSG2z 1 O2 = GSSG 1 O2
2z
Subsequent interactions between O2
2 z and iron lead to
Fe-O2 complex formation (15). GS z itself may serve as
an oxidant or react with O2 to produce the additional
oxidants GSOO z and GSO2OO z (6, 45). When GS2 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 GSSG2z
formation and reaction with oxidant products (e.g.,
GSOO z) such that ELF depletion potentially occurs
more rapidly than the NO2 uptake rate. GSSG2z or O2
2z
may undergo direct electron transfer to NO2. In steadystate exposures, we observed that xanthine oxidasederived O2
2 z produced notable NO2 uptake despite being
in very low concentration, indicating that this direct
2
quenching reaction (NO2 1 O2
2 z = NO2 1 O2 ) may
proceed at near-diffusion-limited rates (17). Furthermore, the addition of SOD to GSH solutions diminished
NO2 uptake by <10% (unpublished observations). Presumably, this reaction could occur either directly from
GSH-mediated sequelae or during O2
2 z release from
activated air space surface cells. In addition, O2
2 z and
GSSG2z may also reduce GS z, 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 A2z and iron lead to both H2O2
and Fe-O2 complex formation (3, 25, 28, 43)
1
AH2 1 NO2 = A2z 1 NO2
2 1 H
AH2 1 Fe31 = A2z 1 Fe21;
Fe21 1 O2 = Fe-O2
Fe-O2 1 A2z 1 H1 = Fe31 1 A 1 H2O2
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 Fe21 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., A2z disproportionation, GS z dimerization, or reduction by O2
2 z or
GSSG2z) 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 antioxi2
dants (e.g., A2z, O2
2 z, and GSSG z; 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
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
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 exposureinduced 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 concentrationdependent 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 z and A2z are able to produce ROS (3,
6, 25, 28, 43). For GS z, reactions with GS2 produce the
reductants GSSG2z and O2
2z
L1274
NITROGEN DIOXIDE-INDUCED REACTIVE OXYGEN SPECIES
We recognize the constructive suggestions and valuable insights
provided by Dr. Bruce A. Freeman in preparation of this work.
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
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.
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.
6. Buettner, G. R. The pecking order of free radicals and antioxidants: lipid peroxidation, a-tocopherol and ascorbate. Arch.
Biochem. Biophys. 300: 535–543, 1993.
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.
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.
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.
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.
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.
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.
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.
17. Huie, R. E. The reaction kinetics of NO2. Toxicology 89: 193–
216, 1994.
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.
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.
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.
21. Lai, C. C., and B. J. Finlayson-Pitts. Reactions of dinitrogen
pentoxide and nitrogen dioxide with 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine. Lipids 26: 306–314, 1991.
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
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 NO2induced 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, doseresponse 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.
NITROGEN DIOXIDE-INDUCED REACTIVE OXYGEN SPECIES
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
units in peptides and proteins. Arch. Biochem. Biophys. 243:
125–134, 1985.
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.
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.
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.
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.
Schlesinger, R. B. Nitrogen oxides. In: Environmental Toxicants, edited by M. Lippman. New York: Van Nostrand Reinhold, 1992, p. 412.
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.
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.
Skoza, L., A. Snyder, and Y. Kikkawa. Ascorbic acid in
bronchoalveolar wash. Lung 161: 99–109, 1983.
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.
Sokal, R. R., and F. J. Rohlf. Biometry. New York: Freeman,
1981.
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.
Uchiyama, M., and M. Mihara. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal.
Biochem. 86: 271–278, 1978.
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.
US Environmental Protection Agency. Air Quality Criteria
for Oxides of Nitrogen. Environmental Criteria/Assessment.
Washington, DC: US Environmental Protection Agency, 1995, p.
14–39.
Weetall, H. H. Preparation of immobilized proteins covalently
coupled through silane coupling agents to inorganic supports.
Appl. Biochem. Biotechnol. 41: 157–188, 1993.
Winterbourn, C. C. Superoxide as an intracellular radical sink.
Free Radic. Biol. Med. 14: 85–90, 1993.
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
22. Langford, S. D., A. Bidani, and E. M. Postlethwait. Ozonereactive absorption by pulmonary epithelial lining fluid constituents. Toxicol. Appl. Pharmacol. 132: 122–130, 1995.
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.
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.
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.
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.
28. Miller, D. M., and S. Aust. Studies of ascorbate-dependent,
iron-catalyzed lipid peroxidation. Arch. Biochem. Biophys. 271:
113–119, 1989.
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.
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.
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.
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.
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.
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.
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.
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
L1275