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1 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-0876; and 2 Biodynamics Institute, Louisiana State University, Baton Rouge, Louisiana 70803-1800
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Recent evidence suggests that inhaled ozone (O3) does not induce toxicity via direct epithelial interactions. Reactions with epithelial lining fluid (ELF) constituents limit cellular contact and generate products, including lipid ozonation products, postulated to initiate pathophysiological cascades. To delineate specific aspects of lipid ozonation product formation and to estimate in situ surface concentrations, we studied the O3 absorption characteristics of ELF constituent mixtures and measured hexanal, heptanal, and nonanal yields as a function of ascorbic acid (AH2) concentration. Exposures of isolated rat lungs, bronchoalveolar lavage fluid (BALF) and egg phosphatidylcholine (PC) liposomes were conducted. 1) O3 absorption by AH2, uric acid, and albumin exceeded that by egg PC and glutathione. O3 reaction with egg PC occurred when AH2 concentrations were reduced. 2) Aldehydes were produced in low yield during lung and BALF exposures in a time- and O3 concentration-dependent manner. 3) Diminishing BALF AH2 content lowered O3 uptake but increased aldehyde yields. Conversely, AH2 addition to egg PC increased O3 uptake but reduced aldehyde yields. Estimations of bioactive ozonation and autoxidation product accumulation within the ELF suggested possible nanomolar to low micromolar concentrations. The use of reaction products as metrics of O3 exposure may have intrinsic sensitivity and specificity limitations. Moreover, due to the heterogenous nature of O3 reactions within the ELF, dose-response relationships may not be linear with respect to O3 absorption.
ozone; pulmonary oxidant stress; epithelial lining fluid; lipid oxidation; aldehydes; ozonation products; ascorbic acid
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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A GROWING BODY of both theoretical and experimental evidence supports the concept that inhaled ozone (O3) likely does not exert its toxic effects via direct interactions with the pulmonary epithelium (17, 25, 39, 53, 54). Contact with the epithelium is limited by the process of "reactive absorption" wherein inspired O3 undergoes a chemical reaction at or near the air space gas-liquid interface with constituents of the pulmonary epithelial lining fluid (ELF) (6, 22, 25, 36). By chemically eliminating O3, this process not only maintains the driving force for the net flux of O3 from the gas phase but also limits the diffusion of dissolved O3. Reactive absorption (or uptake), first described within the lung for inhaled NO2 (34), demonstrates both aqueous substrate dependence and mass transfer limitations (6, 25, 35). Because the reaction of inhaled O3 appears to be predominantly localized to within the ELF compartment, its absorption is implicitly coupled to the production of ELF-derived reaction products. It is these products that are thought to initiate the cascade(s) that ultimately leads to the cellular pathophysiologies resulting from exposure (43, 44).
Current evidence suggests that most, if not all, of the pulmonary epithelial surface is covered by a continuous film of ELF (4), which has characteristics unique to the conducting airways and alveolar spaces (15, 47). However, both airway and alveolar surface fluids contain a multitude of constituents that can react with O3. On the basis of studies of both O3 reaction kinetics and the exposure-mediated loss of constituents from biological fluids, it can be reasonably predicted that the predominant absorption substrates are the water-soluble antioxidants ascorbic acid (AH2), glutathione (GSH), and uric acid (UA); proteins; and unsaturated lipids (3, 8, 12, 23, 25, 27, 29, 43, 45, 46, 54, 56). Macrophages, located on the air space surface of conducting airways, may protrude into the gas phase, allowing their membranes to directly contact inhaled O3. Under these circumstances, the initial molecular targets that react with O3 may differ appreciably from those within the ELF milieu.
Although ~20% of the lipids harvested by bronchoalveolar lavage are
unsaturated, the interfacial monolayer of surface active lipids is
generally considered to be highly enriched with saturated moieties
(13). The physicochemical status of the unsaturated lipids that lie
below the gas-liquid interface is unclear. Nonetheless, despite the
presence of proteins, antioxidants, and other solutes within this
compartment, during in vivo O3
exposure, lipid ozonation products (LOPs) are unquestionably produced
(42, 43, 46). Under physiological conditions, the reaction between
O3 and the double bonds of
phospholipid unsaturated fatty acids (UFAs) leads to bond cleavage and
the formation of aldehyde and hydroxyhydroperoxide end products. The
source fatty acids that form specific products, the reaction
mechanisms, and the product yields occurring from direct ozonation have
been previously well characterized (42, 43, 48, 51). During the Criegee
ozonation reaction, the shortened fatty acyl chain with either an
aldehydic or hydroperoxide function at the terminus can remain attached
to the lipid backbone. Recent observations (1, 7, 21) suggest that
these phospholipid products exhibit biological activity that could, in
part, account for the cytotoxic effects of
O3. Furthermore,
O3 exposure initiates autoxidation
of UFAs, which also leads to the formation of bioactive species such as
the 
-unsaturated alkenals (e.g., 4-hydroxynonenal) (14, 24).
Although many of these LOPs are unstable or reactive, saturated
aldehydes that are liberated during either ozonation [i.e.,
heptanal (C-7), nonanal (C-9)] or autoxidation [i.e.,
hexanal (C-6)] have sufficiently long biological half-lives (>2
h) to allow for their quantification after an exposure (10, 42). Because bond cleavage does not favor liberation of either the aldehydic
or hydroperoxide products, approximately equivalent amounts should be
produced. Consequently, the saturated aldehydes can be used as
surrogate measures for the production of the pool of potentially
bioactive LOP. However, uncertainties remain as to the predominance of
specific reaction pathways within the ELF, the amounts of bioactive
products that are formed, and how ELF composition may influence
specific product formation.
Accordingly, we examined the reactive absorption characteristics of ELF constituent mixtures and, utilizing biologically relevant investigational models (intact lung, isolated ELF, and liposome suspensions), explored the exposure-induced yields of both nonspecific autoxidation and O3-specific aldehydes relative to the dose of absorbed O3. Aldehyde yields also were determined as a function of the AH2 concentration. These data were used to estimate the concentrations of lipid-derived bioactive products that may occur within the ELF. The results suggest that within the ELF milieu, direct O3 reaction with UFAs occurs and produces bioactive LOPs in a relatively small yield (nanomolar to possibly low micromolar concentrations) that are inversely related to AH2 availability. Moreover, because the aqueous-phase concentration of AH2 in large part influences the rate of O3 absorption, the relationships between the amount of O3 absorbed (dose) and LOP production may be sufficiently complex to suggest that extrapolating the O3 dose based on a measured product may be difficult.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Reagents. All reagents were purchased from Sigma (St. Louis, MO) unless otherwise noted. Egg phosphatidylcholine (PC) and linolenic acid (18:3) were obtained from Avanti Polar Lipids (Alabaster, AL).
Animals. Male, viral antigen-free, Sprague-Dawley rats (250-275 g; Harlan Sprague Dawley, Houston, TX) served as donor animals for all isolated lung procedures and harvesting of ELF. Animal procedures, which met University of Texas Medical Branch (Galveston) Animal Care and Use Committee standards, were allowed free access to food and water until just before induction of anesthesia. For experimental procedures, the animals were anesthetized with 70 mg/kg of pentobarbital sodium intraperitoneally, with the depth of anesthesia verified via foot pinch.
Isolated lung exposures. Details of the isolated lung exposure protocol have been described (34, 36). Briefly, after tracheal cannulation and a midline thoracotomy, the pulmonary artery was cannulated and the left atrium was resected. Continuous positive-pressure support ventilation was initiated at the time of pneumothorax. The pulmonary vascular bed was perfused free of erythrocytes with 50 ml of Krebs bicarbonate buffer containing 8.3 mM glucose and 5 g/100 ml of BSA. During perfusion, the remaining heart tissue was trimmed free, the lower respiratory tract was resected en bloc, and the pulmonary arterial cannula was cut. The lungs were transferred to within an artificial thorax equipped to provide subatmospheric ventilation (50 breaths/min). The lungs were not perfused during exposure to limit the dilution volume of the aldehydes and maximize sensitivity and accuracy of quantification. Postlethwait et al. (36) have previously shown that acute O3 uptake is independent of vascular perfusion. Under the time (30-60 min), temperature (37°C), and nonperfused conditions, epithelial integrity is maintained, ELF GSH and AH2 levels are stable (air exposure; Postlethwait, unpublished observations), and the lungs continue to synthesize and secrete pulmonary surfactant (16). A range of exposure concentrations (0.25-1.0 ppm of O3) was selected that was previously shown not to induce overt epithelial damage as assessed by lactate dehydrogenase activity in postexposure bronchoalveolar lavage fluid (BALF) (36).
For exposure, a continuous stream of
O3 in 5%
CO2-95% air flowed
past the tracheal cannula in excess of peak inspiratory flow. O3 was generated by passing 100%
O2 through a silent arc electrode, and via a system of mass flow controllers, an appropriate amount bled
into the flow of previously warmed and humidified ventilation gas to
achieve the desired exposure concentration. A pneumotachograph, located
downstream from the tracheal cannula, permitted breath-by-breath assessment of tidal volume (
2.5 ml). End-expiratory transpulmonary pressures were adjusted to produce functional residual capacities of
~4 ml. Immediately postexposure, the lungs were removed and bronchoalveolar lavage was performed.
Lung lavage and BALF treatment. The
lungs were lavaged via a tracheal cannula with 8 ml of warmed
(37°C) PBS (pH 7.0; 310 mosmol) that was gently instilled and
withdrawn three times to yield BALF. The total time for lavage was
<20-25 s, with >90% recovery of instilled fluid. For in vitro
exposures, the concentration of ELF constituents within the BALF was
increased by lavaging a second lung with the lavage fluid recovered
from the first lung (BALF2) (25,
37). The BALF was centrifuged (4,000 g
for 10 min at 4°C) to remove cells and either frozen
(
70°C) immediately for later aldehyde analysis or used
without delay for in vitro exposure. In some cases, cell-free
BALF2 from donor lungs was incubated with 1.0 U/ml of ascorbate oxidase (AO) for 10 min before in
vitro exposure. This treatment reduced
AH2 concentrations below detection.
In vitro exposures. Substrates were individually dissolved in 37.5 mM phosphate buffer containing 10 µM desferrioxamine to limit iron-catalyzed autoxidation. To model the UFAs in ELF, we utilized egg PC, which contains ~20% UFAs and 80% saturated fatty acids, as liposome suspensions. Unilamellar egg PC liposomes were prepared by drying chloroform solutions of egg PC in a large test tube under N2 followed by the addition of 37.5 mM phosphate buffer and sonication in an ice bath. The aqueous solution was sonicated (Heat Systems Ultrasonic Processor) under full power at a 50% duty cycle for 1 min for a total of three treatments (51). In some cases, to increase the extent of liposome unsaturation, 10% linolenic acid (18:3) by dry weight was added during the initial drying process, and the liposomes were prepared as above. All substrates were kept on ice under N2 in the dark and used within 90 min. Solutions (8- or 10-ml final volume) of model or BALF systems were placed in a glass 50-ml Erlenmeyer flask and exposed under steady-state conditions (constant O3 inflow rate), with continuous gas- and aqueous-phase stirring achieved by a stir bar that protruded through the gas-liquid interface (25, 35). Empty flasks were conditioned until inflow and exit concentrations of gas-phase O3 were equal, after which the test solution was injected through a long stainless steel needle into the bottom of the flask. Test solutions were brought to room temperature just before exposure. The O3 was generated and delivered to the exposure flasks in a manner analogous to that used for the isolated lung exposures. Exposure concentrations (0.5-1 ppm) and gas flow rates (135-415 ml/min) were optimized for the various protocols. For uptake studies, more elevated dose rates limited initial uptake efficiency but allowed for detection of enhanced uptake rates by substrate mixtures. For the aldehyde production studies, dose rates were adjusted to enhance production without excessive substrate depletion.
Gas-phase O3 analysis and computation of
uptake.
For isolated lung exposures, samples for gas-phase
O3 analysis were collected by
diverting a constant stream of ventilation gas (100 ml/min), withdrawn
downstream from the tracheal cannula, through a KI bubbler for 5 min
(5, 19, 36). Sampling flow rates were kept to a minimum to maximize the
effect of lung absorption on the downstream
O3 concentration
([O3]). To prevent
rebreathing of expired gases but permit sensitive detection of
downstream changes in
[O3], the stream of
ventilation gas across the tracheal cannula required a flow rate that
just exceeded peak inspiratory flow plus the sampling withdrawal rate.
Because peak inspiratory flow occurs for only a brief period,
throughout the respiratory cycle the gas phase downstream from the
tracheal cannula is a variable admixture of both expired and noninhaled
gases. [O3] was
quantified based on daily standard curves. For the in vitro exposures,
a model 49 Thermo Environmental UV Photometric
O3 Analyzer (Franklin, MA) was
used for continuous assessment of
O3 exit (downstream) concentration
([O3]e).
Under these conditions, the total flask flow was mixed with filtered
air to provide the necessary sampling flow required by the analyzer
(1 l/min). All flows were measured with a soap bubble meter so that
dilution factors could be accurately computed. The disappearance
(uptake) of O3 was determined by
computing the O3 mass balance
across either the isolated lungs or exposure flask.
[O3]e
(in ng/ml) was subtracted from inflow (inspired) concentration
([O3]i),
and the difference was multiplied by the flow rate
(
; in ml/min) and time
(t; in min) to yield the mass of
O3 uptake (
)
{i.e., = ([O3]i [O3]e) · · t}.
Data are presented as either uptake rate (in nmol/min) or total uptake (in nmol).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Reactive absorption by ELF substrate
mixtures. The
O3-reactive absorption
characteristics of ELF substrates were initially investigated by
exposing pure chemical solutions under steady-state, well-mixed
conditions at room temperature. Figure 1
displays the aqueous-substrate concentration dependence for
AH2, GSH, and egg PC exposed to a
fixed O3 dose rate. For
comparative purposes, fatty acid-free BSA, two concentrations of the
water-soluble vitamin E analog Trolox, dimethylthiourea (DMTU), and a
range of UA concentrations were also studied. Under the employed
exposure conditions, AH2, UA, and
Trolox displayed essentially analogous absorption activity that was
significantly greater than either GSH or egg PC at all concentrations
tested. DMTU induced ~80% of the
O3 uptake exhibited by
AH2 (data not shown). BSA, at the
single, low concentration tested, was intermediate in its ability to
drive O3 uptake. Treatment of BSA
with N-ethylmaleimide (100 µM final
concentration) to diminish sulfhydryl groups significantly
decreased the BSA-mediated rate of
O3 uptake (32%; data not
shown), suggesting that reaction with protein sulfhydryls
appreciably contributes to its overall rate of
O3-reactive absorption. As
previously demonstrated with GSH (25), increasing concentrations of
AH2, UA, and egg PC above substrate-specific critical thresholds were associated with saturation of the O3 uptake rate.
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We subsequently determined the relative propensity for
substrates to drive O3 uptake in
mixed substrate systems. Both equimolar and more biologically relevant
conditions were studied with exposure criteria similar to those
employed above. Under equimolar conditions (Fig.
2), combinations of egg PC and GSH produced
a moderate but significant increase in
O3 uptake over their individual
reactivity. Addition of GSH, egg PC, or both to
AH2 produced no discernable alteration in the O3 uptake rate
attributable to AH2 alone. When more biologically relevant initial concentrations of substrate mixtures
were investigated, a similar pattern was evident (Fig. 3). In general, the antioxidant
concentrations selected approximated those that occur in rat lung ELF
(AH2 concentration 1 mM; GSH concentration 0.5 mM), with the lipid concentration (7 mg/ml; 1.9 mM) only ~50% of that projected for in situ ELF.
When the concentration of egg PC exceeded GSH by approximately
fourfold, the significant elevation in the rate of
O3 gas-phase disappearance suggested that both substrates were reacting to drive
O3 absorption. Analogous to the
equimolar studies, the presence of 1 mM
AH2 governed the overall
absorption rate despite addition of the other substrates. However, when
the initial AH2 concentration was
reduced, as would happen during exposure-induced consumption, the
augmented absorption rates of mixed
AH2 plus egg PC systems suggested
that lipid reaction was, in part, contributing to the overall uptake of
O3.
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Aldehyde yields in the isolated lung. Isolated rat lungs were exposed to a range of O3 concentrations (0.25-1.0 ppm) for either 30 or 60 min. Comparable to previous observations (36), O3 uptake for all exposures was constant over exposure time, with mean fractional uptake rates for all exposure groups equaling 0.92 ± 0.06 (Table 1). Figure 4 is a plot relating total O3 uptake to the measured yield of the O3-specific aldehydes heptanal and nonanal measured in BALF from postexposure lungs. A positive correlation between the total absorbed dose of O3 and the measured accumulation of aldehydes is apparent. Table 2 presents the relative aldehyde yields as detected in the BALF, the ratio between each total aldehyde and O3 uptake, and the proportion that each total yield represents of the absorbed O3 dose. As is evident, although the ratios between each aldehyde and O3 uptake are relatively consistent regardless of the varying exposure conditions, the measured aldehyde yields are quite low. Only picomoles of aldehyde were detected as a result of nanomoles of O3 uptake. In general, the combined accumulation of the heptanal plus nonanal only accounted for ~0.2% of the O3 absorbed dose regardless of [O3]i. Hexanal is derived from both ozonation and autoxidation (10). However, because the precise contribution of each pathway was not determined, we chose to consider hexanal yield as a marker of autoxidation, recognizing the inherent overestimation. The measured hexanal yield was slightly less (nonsignificant) than the total LOP yield, suggesting that within the lung surface compartment, the rate of UFA autoxidation did not exceed the rate of direct ozonation.
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Aldehyde yields during in vitro exposure of BALF and model systems. BALF was harvested from rat lungs and exposed as BALF2 under well-mixed, steady-state conditions at room temperature. Figure 5 displays the relationship between the absorbed dose of O3 and the measured amounts of heptanal and nonanal when the O3 dose was varied by removing samples after 15 and 30 min of exposure. Similar to the isolated lung, a positive correlation between O3 dose and aldehyde accumulation was observed. Table 3 displays the results for the net accumulation of the hexanal, heptanal, and nonanal aldehydes across a variety of aqueous-phase conditions during a 30-min exposure in vitro. Under these exposure conditions, the combined accumulation of heptanal plus nonanal accounted for <1% of the absorbed O3 dose. When ELF AH2 was enzymatically oxidized before exposure, we observed a significant decrease in the rate of O3 uptake but a profound increase in the measured yield of all three aldehydes and in the percentage of absorbed O3 accounted for by the heptanal plus nonanal products. As measured, the measured yields of heptanal and nonanal relative to the O3 absorbed dose increased by ~350 and 560%, respectively. To determine whether aldehyde yields were being grossly underestimated due to volatilization into the flowing stream of exposure gas, we injected a known amount of O3 directly into a sealed vessel containing BALF2. The results suggest that the loss from volatility may have resulted in a twofold underestimation of aldehyde production. The yields of hexanal result, in part, from lipid peroxidation; consequently, for comparison, we measured TBARS in BALF2. The ratio of TBARS production to O3 uptake for BALF2 was 12.5 ± 3.5 pmol/nmol (1.3 ± 0.3% of total O3 uptake). AO treatment of BALF2 significantly increased the ratio to 25.6 ± 7.4 pmol TBARS/nmol O3, which represented 2.6 ± 0.7% of the total O3 uptake.
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Exposure of egg PC liposomes produced greater heptanal and nonanal yields than those from BALF, in accordance with the greater concentration of 16:1 and 18:1 fatty acids within the egg PC system. Because the UFA moieties represented the only available O3-reactive absorption substrates, heptanal plus nonanal accounted for a larger proportion of the absorbed O3 dose in this egg PC system than was observed for BALF. However, when increasing amounts of AH2 were added to the liposome suspension, measured aldehyde yields decreased in the presence of increasing O3 uptake. Interestingly, when 18:3 fatty acids were added to egg PC, we observed a significant increase in O3 uptake that exceeded the modest augmentation of unsaturated bonds contributed by the linolenic acid. The extent of fatty acid autoxidation, indicated by hexanal accumulation, increased concomitant with the elevated O3 uptake, as would be expected from the inclusion of the more autoxidizable UFAs. Addition of 200 µM AH2 to the egg PC plus 18:3 composite liposome suspension further elevated O3 uptake but resulted in a overall significant decline in the measured aldehyde yields, with heptanal and nonanal levels relatively comparable to the liposome suspension not containing linolenic acid.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Importance of O3 initial reactions and
assessment of absorption substrate preferentiality.
Despite the abundant information characterizing the pathophysiological
consequences of O3 exposure, the
specific mechanisms by which cellular injury is induced remain
equivocal. The efficient absorption of
O3 is maintained via reactive
absorption in which the rate of O3
gas-phase disappearance is dependent on a rapid chemical reaction of
dissolved O3 with aqueous-phase
substrates (6, 23, 25, 36). Although
O3 displays limited aqueous solubility [6.4 (mol · l
air
1)/(mol · l
water1) (26)] so
that only limited amounts of
(O3)s
are required to saturate the interfacial thin film, dissolution into
the aqueous phase occurs rapidly. Diffusion of
(O3)s
to the underlying epithelium is constrained due to the combined effects
of the low aqueous-phase concentration, mass transfer limitations, and
rapid reactions with diverse ELF biomolecules (6, 25, 39, 40, 53). Thus
it is reasonable to assume that the ELF-derived products, at least in
part, initiate the cellular perturbations resulting from exposure (25,
39, 44).
7 mg/ml (1.9 mM)] to ~50% of the ELF lipid concentration
because concentrated liposome suspensions result in appreciable lipid adsorption to the gas-liquid interface that constrains
O3 flux rates (20). BSA
concentrations were also limited to mimic in situ lipid-to-protein
ratios. Under our conditions, the rank order for
O3 absorption by the ELF
substrates was AH2 UA > BSA > GSH egg PC. For BSA, nonsulfhydryl sites governed the majority
of reaction because, despite the high molar ratio of cysteine residues per BSA molecule, N-ethylmaleimide
conjugation reduced uptake rates < 35%. Due to the high
O3 reactivity of both Trolox and DMTU, their use in O3 exposure
studies as intracellular antioxidants should be viewed with caution
because they may also react directly with
O3 if present in the extracellular
space.
These results compare favorably with a previous study (25) that
demonstrated that both AH2 and ELF
solutes 
10,000 molecular weight, but not GSH, substantially
contribute to O3 uptake by rat
lung BALF. Cross et al. (8) and Van der Vliet et al. (56), in studies
of human plasma, and Mudway and colleagues (28, 29), in studies of
human BALF and model systems, have shown that both UA and
AH2 undergo greater rates of
exposure-induced consumption than GSH, with UA disappearance likely
exceeding that of AH2. In
addition, we observed approximately the same differences in O3 uptake between
AH2 and GSH as those observed by
Kanofsky and Sima (23), even though they employed substantially greater
gas-phase O3 and aqueous-substrate
concentrations. Both our data illustrate the dependence of
O3 disappearance from the gas
phase on substrate characteristics, supporting the use of uptake rates
as a measure of relative reactivity. However, despite the well-mixed
conditions in our steady-state exposures, it is possible that some
compounds react rapidly enough to induce diffusion and/or mass
transfer limitations. Under such conditions, our methods would not
allow us to distinguish reactivity differences, if any, among, for
example, substrates such as AH2,
UA, and Trolox.
Under biologically relevant substrate concentrations,
AH2 reaction with
O3 predominated (Fig. 3). Thus, at
exposure onset, most O3 uptake
should be attributable to ELF AH2
(or other equivalent reactants, e.g., UA). However, interfacial
depletion of the most kinetically active substrates may allow for
reaction with other species. For example,
O3 uptake rates were augmented
when 7 mg/ml of egg PC were added to 0.1 mM
AH2, suggesting that
O3 reaction with other substrates
may begin to occur when AH2
concentrations within the reaction plane fall sufficiently. Kanofsky
and Sima (23) computed the substrate conditions required to prevent
surface depletion and reported values at pH 7.0 of 1.4 mM
AH2 and 0.5 mM GSH, concentrations
that may not meet basal ELF conditions within many respiratory systems
(49) or may not be maintained during exposure.
Aldehyde yields. Isolated rat lungs
exposed to a combination of inspired concentrations and exposure times
(Table 1) displayed absorption characteristics similar to those
observed during preceding studies (36). It should be noted that the
determination of both O3 uptake
and LOP accumulation represented lumped measures. Within the lung,
specific regions (e.g., proximal airways) and sites (e.g., downstream
of airway branch points) likely undergo proportionally greater rates of
O3 absorption. Consequently,
within these areas, depletion of surface
AH2 may occur more rapidly so that
the extent of lipid reaction and, therefore, product formation may
coincide. However, as AH2 is
consumed, the absorption efficiency also diminishes, which allows for
O3 distribution to more distal
airways. Our method to assess uptake does not permit analysis of the
intrapulmonary distribution of inhaled
O3. Moreover, because the LOPs
were quantified in samples of BALF recovered from whole lung lavage,
similar limitations exist for estimating regional aldehyde production.
Despite these confines, substantial information can still be derived
from the isolated lung studies. The accumulation of both heptanal and
nonanal exhibited linear correlations with the total dose of absorbed
O3 regardless of
[O3]i
or time (Fig. 4). Importantly, the overall measured yield of heptanal
plus nonanal accounted for small but consistent proportions of the
absorbed O3 (0.22 ± 0.01%).
Similar to in vivo studies, aldehyde yields generally reflected the ELF
content of their respective precursor fatty acids (9, 42). Accumulation
of hexanal, the designated autoxidation marker, also showed a good
positive correlation with O3
absorption (r2 = 0.98), suggesting that the extent of lipid autoxidation was dependent
on O3 flux into the ELF. However,
under our exposure conditions, we measured only 1.75 ± 0.31 pmol
hexanal/nmol O3 uptake, which was
marginally less than the measured LOP yield.
In vitro exposure of BALF produced results that were qualitatively both
similar and dissimilar from the isolated lung studies. Similar to the
isolated lung, heptanal and nonanal accumulation showed good
correlation with the O3 dose (Fig.
5) and accounted for slightly <1% of the
O3 uptake (Table 3). Notable was
the BALF yield of hexanal, which was some 16-fold greater per nanomole of O3 uptake than in the isolated
lung. However, both heptanal and nonanal also showed relative yields in
excess (3- to 5-fold) of lung values. This may be attributable to a
number of factors including 1)
diffusion of ELF aldehydes into the tissue; the mild lavage technique
and binding to tissue elements may have limited recovery of
intracellular materials; 2) loss of
aldehydes due to volatilization being facilitated by the large
surface-to-volume ratio in the ventilating lung;
3) destruction of aldehydes due to
further reaction with O3; and
4) repletion of
O3 scavengers into the BALF not
occurring in our model, but diffusion of
AH2, for example, from the tissue
into the ELF may constrain the extent of direct
O3 interactions with ELF lipids
and quench lipid autoxidation.
Exposure of egg PC liposomes resulted in substantially greater measured
yields of all three aldehydes than was observed for either the isolated
lung or BALF (Table 3). This was likely due to the lack of direct
O3 scavengers (i.e.,
AH2) and the concentration of
the precursor fatty acids within the liposome system. The preferential production of nonanal accords with the greater content of oleic acid in
egg PC relative to that in ELF. Oxidation of
AH2 with AO produces
H2O2,
which may have contributed to the augmented hexanal yield in the BALF
plus AO system. Contrary to the effects of
AH2 removal from BALF, even a
marginal AH2 addition to the egg
PC system increased O3 uptake but
diminished relative aldehyde yields, notably altering the ratios
between O3 uptake and the measured
aldehydes. Although addition of linolenic acid (18:3) elevated
O3 uptake over that of egg PC
alone, the distribution of aldehyde yields did not substantially
differ, indicating product formation reflects their UFA precursors.
Previous investigators, using plasma or erythrocyte ghost suspensions
as exposure models, have reported a lack of aldehyde production, both
O3 specific and autoxidative (8),
and have concluded that O3
preferentially reacts with membrane proteins rather than with UFA (27).
These differences from our results may be explained as follows. Plasma
protein levels far exceed ELF concentrations, and as illustrated in
Fig. 1, the combination of protein, UA,
AH2, and sulfhydryl groups may
outcompete lipids and protect them from direct ozonation. In situ,
exposure-induced consumption of ELF antioxidants within site-specific
anatomic regions and the modest ELF protein concentrations likely allow for the nominal direct O3
interactions with lipids we report herein. Moreover, under in vitro
conditions where substrate availability substantially differs from the
lung surface, making depletion measurements against a large background
pool of parent substrate may be potentially difficult. Furthermore,
spatial compartmentation may influence the rate of specific substrate
loss and/or product formation. As alluded to above,
O3 reactions with the ELF likely occur within a thin subinterfacial plane so that the extent of any
given reaction pathway is, in part, governed by the specific availability of reactants within the plane (6, 23, 53). Exposure
modalities wherein O3 is bubbled
through an aqueous phase increase the effective interfacial surface
area in an undefined fashion and, due to influences on diffusional
movement, likely disrupt this spatial arrangement and may lead to
reactant interactions that are normally limited under in situ
conditions.
Estimation of bioactive product
formation. As a first approximation of lipid-derived
bioactive product formation within rat lung ELF during
O3 exposure, we arbitrarily
utilized a number of simplifying assumptions.
1) The cumulative yield of heptanal plus nonanal is a surrogate measure for the LOPs remaining attached to
the phospholipid backbone that likely comprise the majority of
lipid-derived bioactive products resulting from direct
O3 reaction. Based on data in
Tables 2 and 3, the heptanal plus nonanal yield represents 0.5% of the
O3 absorbed dose. However, this
value underestimates actual yield by 50% due to loss from volatility
(Table 3). Thus an overall yield of 1% of absorbed
O3 is assumed.
2) Hexanal is a similar surrogate
measure for bioactive autoxidation products such as -unsaturated
aldehydes and malondialdehyde. The BALF studies suggest that although
qualitatively parallel results between hexanal and TBARS are observed,
hexanal is a more sensitive metric than TBARS. For the autoxidation
products, the isolated lung studies are likely most applicable because
diffusion of reducing species from the tissue into the ELF quenches
lipid peroxidation. Thus a 2% yield of absorbed
O3 is assumed.
3) A 0.25 ppm
[O3]i
is assumed, of which 50% is scrubbed in the upper respiratory tract.
The remaining O3 undergoes 100%
uptake uniformly distributed throughout the conducting airways and
proximal alveoli, the predominant sites of both uptake and acute cell
injury (17, 18, 26, 31, 33, 36, 38). This represents an approximate ELF
volume of 60 µl for a rat lung (15).
4) A minute volume of 150 ml over a
60-min exposure period is assumed. The absorbed dose
(0.125 ppm × 150 ml/min × 60 min) equates to 46 nmol
O3.
5) On the basis of the linear
accumulation of aldehyde over exposure concentration and time (Figs. 4
and 5), steady-state concentrations within the ELF are assumed.
One percent of the absorbed dose equals 0.46 nmol/h. If a volume of
distribution of 60 µl is assumed, 7.7 µM directly formed products
may be achieved after 60 min of exposure. For the autoxidation products, a 2% yield equates to ~15 µM concentrations within the ELF. We chose not to extend this projection past a 1-h period because
exposure-induced changes in ELF composition are unknown. Due to the
simplifying assumptions, these projections may be viewed as maximal
rates of accumulation. Presumably, only a few specific species of the
spectrum of LOPs produce significant biological effects. Thus it is
important to note that for any given product, the extent of formation
will be substantially less than the total value predicted by our
estimations.
Use of O3-derived products as predictors
of O3 dose.
Previous approaches to identify
O3 exposure biomarkers have
utilized varied techniques. Generic end points such as inflammatory cells or injury and/or permeability markers (e.g., BALF lactate dehydrogenase, protein) correlate with
O3 exposure but are also interdependent and are altered by a variety of inhaled agents. The use
of products formed only in ozonation reactions would be preferable.
However, several factors can introduce confounding effects. 1) Because
ambient O3 levels span a
relatively narrow range [0.25 ppm (55)], only limited
amounts of directly formed products are produced. Moreover, because
inhaled O3 reacts with a variety
of substrates, only a fraction of the absorbed
O3 results in measurable end
products, as evidenced by the low LOP yields observed in this study.
The isolated lung studies suggest that exposure-mediated autoxidation
may have similar limitations. Although oxidant release by inflammatory
cells further contributes to aldehyde production, this pathway is
independent of direct O3 reaction. Thus the use of specific reaction products to assess real-world exposures has substantial intrinsic sensitivity and specificity limitations. 2) The importance of
any given reaction pathway also must be considered within the context
of the lung surface compartment. Model systems that do not
appropriately address compartmentation or exposure modality may have
limited application to addressing mechanistic issues specific to
inhaled O3.
3) The ELF composition varies not
only as a function of anatomic location, strain, and species but also
likely with exposure. Although the data from both the isolated lung and
BALF studies suggest that LOP formation is proportional to the absorbed
dose of O3, the direct
proportionalities only held true within any given experimental system.
For example, the ratio between O3
uptake and LOP formation varied inversely with
AH2 or UFA content. Thus
substantial inhomogeneities in product formation relative to the
absorbed dose likely exist among differing respiratory systems or
investigational models. Although the detection of specific reaction
products, including O3-oxygen
addition, clearly demonstrates that exposure has occurred, using such
values to extrapolate back to the
O3 exposure dose may be
problematic.
| |
ACKNOWLEDGEMENTS |
|---|
We acknowledge the significant technical assistance of Brian K. Burleson.
| |
FOOTNOTES |
|---|
This research was supported by funding provided by National Heart, Lung, and Blood Institute Grant HL-54696 (to E. M. Postlethwait) and National Institute of Environmental Health Sciences Grants T32-ES-07254 (to L. W. Velsor) and ES-08663 (to W. A. Pryor).
Address for reprint requests: E. M. Postlethwait, Division of Pulmonary and Critical Care Medicine, 0876, Depts. of Internal Medicine, Pharmacology and Toxicology, and Pathology, Univ. of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0876.
Received 10 December 1997; accepted in final form 12 March 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
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