O3-induced formation of bioactive lipids: estimated surface concentrations and lining layer effects

Edward M. Postlethwait1, Rafael Cueto2, Leonard W. Velsor1, and William A. Pryor2

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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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 beta -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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 (approx 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 (approx 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 (Q; in ml/min) and time (t; in min) to yield the mass of O3 uptake (&Udot;) {i.e., &Udot; = ([O3]i - [O3]e) · Q · t}. Data are presented as either uptake rate (in nmol/min) or total uptake (in nmol).

Use of uptake rates as indexes of relative reactivity. The reactive absorption of O3 can be conceptualized as a two-step process. In this simplified scheme, O3 undergoes mass transfer across the gas-liquid interface to dissolve, forming a solute gas [(O3)s], followed by a chemical reaction with an available biomolecule. In the absence of a reaction, the aqueous interfacial thin film rapidly saturates with (O3)s. Under these conditions, net flux usually declines because only diffusive movement of (O3)s into the bulk phase reduces back pressure (aqueous to gas phase). Because the reaction eliminates the (O3)s, back pressure is minimized so that the driving force for the net transfer of O3 into the aqueous phase is maintained. The uptake rate is aqueous substrate dependent such that substrate concentration, physicochemical status (e.g., reactive site protonation), and intrinsic reactivity all influence the absorption rate (6, 23, 25, 35, 37). When the substrate is in sufficient excess relative to O3, the interfacial flux of O3 reaches saturation, and further increases in aqueous-substrate concentration produce no further increments in absorption (6, 25). An overall coefficient describing gas-phase disappearance (kO3) can be expressed as a conductance that is related to both a mass transfer coefficient (km) and a reaction coefficient (kr) (35)
<FR><NU>1</NU><DE><IT>k</IT><SUB>o<SUB>3</SUB></SUB></DE></FR> = <FR><NU>1</NU><DE><FR><NU>1</NU><DE><IT>k</IT><SUB>m</SUB></DE></FR> + <FR><NU>1</NU><DE><IT>k</IT><SUB>r</SUB></DE></FR></DE></FR>
Under the well-stirred conditions employed during the in vitro exposures, interfacial mass transfer resistance is minimized, although not entirely eliminated. Consequently, under nonsaturated conditions, the rate at which various substrates drive O3-reactive absorption can be used as a measure of their relative reaction with O3. In combined substrate systems, absorption rates that exceed those displayed by the most reactive individual component suggest that multiple substrates are reacting. Previous analyses of NO2 uptake, which is absorbed in an analogous manner, has revealed mass transfer limitations (6, 35), and similar conclusions also have been formed from human studies with bolus O3 exposure methodologies (2, 18). Thus, under conditions where the aqueous-phase interfacial thin film is rapidly depleted of either O3 or solute reactants, rate-limiting situations may occur wherein 1) if the reaction exceeds the diffusion of aqueous substrate into the reaction plane, (O3)s concentration will rise, resulting in back pressure and limitations in the uptake rate, and 2) under conditions where aqueous substrates react very rapidly, mass transfer limitations may constrain the rate of O3 dissolution [(O3)s formation] and preclude distinguishing differences in reactivity among the substrates.

Determination of aldehydes. As previously described (9, 10, 42), aldehydes were analyzed as oximes of pentafluorobenzylhydroxylamine with gas chromatography and electron capture detection. Briefly, test solutions were analyzed for the aldehydes hexanal (C-6; from linoleic and arachidonic acid autoxidation and/or ozonation), heptanal (C-7; from ozonation of palmitoleic acid), and nonanal (C-9; from ozonation of oleic acid). Solutions (1-2 ml) were allowed to react with 0.5 ml of pentafluorobenzylhydroxylamine (1.0 mg/ml) for 2 h, 3 drops of 18 N H2SO4 were added, and the oximes formed were extracted with 1 ml of hexane containing decafluorobiphenyl (50 µg/l) as the internal standard. The hexane layer was decanted, washed with 5 ml of 0.1 N H2SO4, and dried over anhydrous sodium sulfate. A Hewlett-Packard gas chromatograph model 5890 equipped with 63Ni electron capture detection, autosampler, cool on-column injector with electronic pressure control, and an HP-5 25-m × 0.2-mm × 0.33-µm column with a 5-m × 0.53-mm retention gap was used for analysis. Helium (2.9 ml/min) and argon-methane were used as the carrier and makeup gases, respectively. Chromatographic conditions included a detector temperature of 280°C and temperature programming starting at 50°C for 1 min. with a 5°C/min ramp to a final temperature of 220°C. Two-microliter sample volumes were injected, and the measured values were computed as micrograms per liter. In addition, thiobarbituric acid-reactive substances (TBARS) were determined in BALF before and after O3 exposure and with and without AO treatment. Butylated hydroxytoluene was added immediately on sample removal, and the TBARS analysis was performed with tetraethoxypropane as the standard (30, 52). On the basis of the specifics of the individual in vitro exposure or isolated lung BALF volumes, aldehydes were calculated to reflect total yields in picomoles or nanomoles.

Statistics. All experimental measurements are presented as means ± SD of three to five observations. Significant differences between experimental groups were assessed by one-way ANOVA and Dunnett's test post hoc (50). Significance was defined as P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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 (approx 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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Fig. 1.   Relationship between aqueous-substrate concentration ([Substrate]) and ozone (O3) absorption for various epithelial lining fluid (ELF) reactants. Aqueous solutions (37.5 mM phosphate and 10 µM desferrioxamine, pH 7.0, 25°C) were exposed under steady-state conditions to O3 in air with constant stirring. O3 uptake was computed by determining rate of gas-phase disappearance during transit through exposure vessel {[inflow O3 concentration ([O3]i- exit O3 concentration] × flow rate (Q)}. Egg phosphatidylcholine (EggPC) liposomes were prepared as described in METHODS, and molar concentrations were computed based on manufacturer's reported mean molecular weight (760.2). Fatty acid-free BSA was tested only at 3.5 mg/ml (approx 0.05 mM) and Trolox at 0.05 and 0.1 mM. GSH, glutathione.

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 approx 1 mM; GSH concentration approx 0.5 mM), with the lipid concentration (7 mg/ml; approx  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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Fig. 2.   O3 uptake rates by equimolar mixtures of ELF substrates (each final concentration 0.5 mM). Solutions (37.5 mM phosphate buffer, pH 7.0) containing GSH, ascorbic acid (AH2), or EggPC liposomes alone or in designated combinations were exposed under steady-state conditions (25°C), and O3 uptake was evaluated. Addition of EggPC to GSH moderately elevated uptake over either substrate alone (* P < 0.05). Although AH2 displayed significantly greater absorption rates than either GSH or EggPC (** P < 0.05), addition of GSH and/or EggPC to AH2 produced no change in uptake rate over AH2 alone.


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Fig. 3.   O3 uptake rates by biologically relevant mixtures of ELF substrates. Solutions (37.5 mM phosphate buffer, pH 7.0) containing final concentrations of 0.5 mM GSH, 1.0 mM AH2, or 7 mg/ml (approx 1.9 mM) of EggPC were exposed alone or in combination to O3 under well-mixed, steady-state conditions (25°C), and O3 uptake was determined. Although addition of a fourfold excess of EggPC to GSH produced a significant increase in O3 absorption rate (* P < 0.05), substrate addition to AH2 produced no alteration in absorption over AH2 alone. When initial AH2 concentration was reduced to 0.1 mM, combination with 1.9 mM EggPC resulted in a signficant increase in absorption rate over 0.1 mM AH2 alone (** P < 0.05), suggesting that under these conditions both substrates were reacting with O3.

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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Table 1.   O3 exposure parameters and absorption rates in isolated rat lungs


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Fig. 4.   Yields of lipid ozonation products (LOPs) occurring in bronchoalveolar lavage fluid (BALF) from isolated rat lungs relative to absorbed dose of inhaled O3. Isolated rat lungs were exposed (37°C) to O3 (0.27-0.98 ppm) under steady-state conditions for 30 or 60 min, lungs were lavaged immediately postexposure, and resulting BALF was analyzed for O3-specific aldehydes heptanal and nonanal. Data are plotted based on total absorbed dose rather than on exposure rate or time. Specifics of exposure parameters can be found in Table 1. Data suggest a positive correlation (r2 = 0.98) between extent of O3 uptake and yield of LOPs. Note that absorption is expressed in nanomoles, whereas aldehyde yield is in picomoles.

                              
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Table 2.   ELF aldehyde yields and relationship to O3 absorption consequent to exposure of isolated rat lungs

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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Fig. 5.   Yields of LOPs resulting from in vitro O3 exposure of rat lung BALF. Sets of 2 rat lungs were sequentially lavaged to increase concentration of ELF constituents twofold to produce BALF2. BALF2 was exposed in a small glass vessel to O3 under steady-state conditions. At 15 and 30 min of exposure, samples were removed and frozen for later aldehyde analysis. Data are plotted as total aldehyde yields vs. total O3 absorbed dose. Similar to isolated lung, increases in O3 absorbed dose concomitantly increased LOP yield (r2 = 0.97). Note that O3 absorption is in nanomoles, whereas LOP yield is in picomoles.

                              
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Table 3.   Aldehyde yields and relationships to O3 absorbed dose during in vitro exposure of rat lung BALF and liposome model systems

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.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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 water-1) (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).

Brief O3 exposure, even at relatively low concentrations, produces acute epithelial injury (33, 38). This initial wave of cellular injury occurs within a sufficiently short time to exclude apoptosis, inflammation, or upregulation of cytokine-mediated pathways as the causative mechanism. As exposure continues, the epithelium undergoes further injury that may be related to either ensuing oxidative insult or alterations in metabolic and/or cellular processes. The composition of the ELF likely changes over the time course of exposure as a result of substrate consumption, cellular lysis, changes in air space permeability, and compensatory adaptation mechanisms. Because O3 doubtlessly reacts via a number of different pathways (e.g., reaction with AH2, protein, or UFAs), delineating the mechanisms of O3-induced toxicity requires not only identifying the bioactive products that are formed but also quantifying the extent and temporal sequence of their formation rates. For a specific product to be causally linked to O3-induced cellular perturbations, it must be demonstrated to produce appropriate cellular responses at concentrations equivalent to those generated in vivo.

Recent in vitro studies have suggested that both LOPs (e.g., 1-palmitoyl-2-oleoyl PC aldehydes and ozonides) and lipid autoxidation products (e.g., 4-hydroxynonenal) produce cellular effects qualitatively associated with O3 exposure (1, 7, 14, 21, 24). To date, information regarding the cytotoxic potential of non-lipid-derived O3 products is limited. As a first approach to characterizing the potential production rates of lipid-derived bioactive products, we investigated the relationship between O3 uptake and the measured yields of saturated aldehydes that were used as surrogate metrics for the pool of LOPs.

Our initial studies explored the propensity of ELF substrates to drive O3-reactive absorption. Although the generation of O3-specific aldehydes unequivocally occurs in vivo (9, 42), our objective was to investigate whether a direct O3 reaction with UFAs could be observed via gas-phase disappearance and what factors favored these reactions within the ELF milieu. Although rat ELF UA concentrations are low due to endogenous uricase activity, UA has been implicated as a major O3 scavenger in humans (28, 29, 32) and was therefore included for comparative purposes. Protein (as BSA), Trolox, and DMTU were included because protein has been suggested to be an important O3 target (3, 27, 45, 54) and Trolox and DMTU are used to quench intracellular free radicals. Egg PC concentrations were intentionally limited [<= 7 mg/ml (approx 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 approx  UA > BSA > GSH approx  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 beta -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.

In conclusion, despite the presence of more reactive substrates, O3 undergoes direct reaction with ELF UFAs to a relatively minor extent. Although antioxidants such as AH2 and UA detoxify much of the inhaled O3, in vivo exposure clearly induces acute injury, suggesting that only modest amounts of specific product(s) may be required to induce cytotoxicity. Whereas even minimal amounts of AH2 were observed to inhibit lipid ozonation in vitro, it is possible that O3 reactions with antioxidants may generate cytotoxic species (22, 40, 41), as has been demonstrated for NO2 (57). Because the specific nature of the bioactive products remains undefined, issues as to what appropriately represents the "effective" dose (11) of O3, as opposed to the inhaled or absorbed dose, persist. Furthermore, due to the influence of ELF constituent profiles on the proportionalities between O3 uptake and specific product formation, dose-response relationships may not be linear with respect to O3 absorption, potentially confounding extrapolations across species and exposure time.

    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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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