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 |
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 |
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.
 |
METHODS |
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).
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)
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 |
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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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 ( )}.
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 ( 0.05 mM)
and Trolox at 0.05 and 0.1 mM. GSH, glutathione.
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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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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 ( 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.
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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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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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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 |
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 (
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.
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 |
1.
Alpert, S. E.,
R. W. Walenga,
G. L. Squadrito,
M. G. Salgo,
and
W. A. Pryor.
Lipid ozonation products (LOP) cause release of PGE2 and subsequent loss of cyclooxygenase activity in human tracheal epithelial (HTE) cells similar to in vitro ozone exposure (Abstract).
Am. J. Respir. Crit. Care Med.
155:
A196,
1997.
2.
Asplund, P. T.,
K. A. Ben-Jebria,
M. L. Rigas,
and
J. S. Ultman.
Longitudinal distribution of ozone absorption in the lung: effect of continuous inhalation exposure.
Arch. Environ. Health
51:
431-438,
1996[Medline].
3.
Banerjee, S. K,
and
J. B. Mudd.
Reaction of ozone with glycophorin in solution and in lipid vesicles.
Arch. Biochem. Biophys.
295:
84-89,
1992[Medline].
4.
Bastacky, J.,
C. Y. C. Lee,
J. Goerke,
H. Koushafar,
D. Yager,
L. Kenaga,
T. P. Speed,
Y. Chen,
and
J. A. Clements.
Alveolar lining layer is thin and continuous: low-temperature scanning electron microscopy of rat lung.
J. Appl. Physiol.
79:
1615-1628,
1995[Abstract/Free Full Text].
5.
Bergshoeff, G.,
and
R. W. Lanting.
Improved neutral buffered potassium iodide method for ozone in air.
Anal. Chem.
52:
541-546,
1980.
6.
Bidani, A.,
and
E. M. Postlethwait.
Kinetic determinants of reactive gas uptake.
In: Complexity in Structure and Function of the Lung, edited by M. P. Hlastala,
and H. T. Robertson. New York: Dekker, 1998, p. 243-295. (Lung Biol. Health Dis. Ser.)
7.
Cheek, J. M.,
G. L. Squadrito,
M. G. Salgo,
W. A. Pryor,
and
C. G. Plopper.
Lipid ozonation products (LOP) act as ozonation signal transduction molecules in alveolar epithelium in vitro (Abstract).
Am. J. Respir. Crit. Care Med.
153:
A810,
1996.
8.
Cross, C. E.,
P. A. Motchnik,
B. A. Bruener,
D. A. Jones,
H. Kaur,
B. N. Ames,
and
B. Halliwell.
Oxidative damage to plasma constituents by ozone.
FEBS Lett.
298:
269-272,
1992[Medline].
9.
Cueto, R.,
G. L. Squadrito,
E. Bermudez,
and
W. A. Pryor.
Identification of heptanal and nonanal in bronchoalveolar lavage from rats exposed to low levels of ozone.
Biochem. Biophys. Res. Commun.
188:
129-134,
1992[Medline].
10.
Cueto, R.,
G. L. Squadrito,
and
W. A. Pryor.
Quantifying aldehydes and distinguishing aldehydic product profiles from autoxidation and ozonation of unsaturated fatty acids.
Methods Enzymol.
233:
174-182,
1994[Medline].
11.
Dahl, A. R.
Contemporary issues in toxicology: dose concepts for inhaled vapors and gases.
Toxicol. Appl. Pharmacol.
103:
185-197,
1990[Medline].
12.
Freeman, B. A.,
and
J. P. Mudd.
Reaction of ozone with sulfhydryls of human erythrocytes.
Arch. Biochem. Biophys.
208:
212-220,
1981[Medline].
13.
Goerke, J.,
and
J. A. Clements.
Alveolar surface tension and lung surfactant.
In: Handbook of Physiology. The Respiratory System. Mechanics of Breathing. Bethesda, MD: Am. Physiol. Soc., 1986, sect. 3, vol. III, pt. 1, chapt. 16, p. 247-262.
14.
Hamilton, R. F.,
M. E. Hazbun,
C. A. Jumper,
W. L. Eschenbacher,
and
A. Holian.
4-Hydroxynonenal mimics ozone-induced modulation of macrophage function ex vivo.
Am. J. Respir. Cell Mol. Biol.
15:
275-282,
1996[Abstract].
15.
Hatch, G. E.
Comparative biochemistry of the airway lining fluid.
In: Treatise on Pulmonary Toxicology: Comparative Biology of the Normal Lung, edited by R. A. Parent. Boca Raton, FL: CRC, 1992, p. 617-632.
16.
Hildebran, J. N.,
J. Goerke,
and
J. A. Clements.
Surfactant release in excised rat lung is stimulated by air inflation.
J. Appl. Physiol.
51:
905-910,
1981[Abstract/Free Full Text].
17.
Hu, S.-C.,
A. Ben-Jebria,
and
J. S. Ultman.
Longitudinal distribution of ozone absorption in the lung: quiet respiration in healthy subjects.
J. Appl. Physiol.
73:
1655-1661,
1992[Abstract/Free Full Text].
18.
Hu, S.-C.,
A. Ben-Jebria,
and
J. S. Ultman.
Longitudinal distribution of ozone absorption in the lung: effects of respiratory flow.
J. Appl. Physiol.
77:
574-583,
1994[Abstract/Free Full Text].
19.
Intersociety Committee for Air Sampling and Analysis.
Tentative method for the manual analysis of oxidizing substances in the atmosphere.
In: Methods of Air Sampling and Analysis. Washington, DC: American Public Health Association, 1977, p. 556-560.
20.
Jacobson, L. M.,
A. Bidani,
and
E. M. Postlethwait.
Influence of interfacial surface-active phospholipids on NO2 and O3 reactive absorption (Abstract).
Am. J. Respir. Crit. Care Med.
151:
A503,
1995.
21.
Kafoury, R. M.,
W. A. Pryor,
G. L. Squadrito,
M. G. Salgo,
X. Zou,
and
M. Friedman.
Lipid ozonation products (LOP) activate phospholipases A2 (PLA2), C (PLC), and D (PLD) in human bronchial epithelial cells (BEAS-2B) (Abstract).
Am. J. Respir. Crit. Care Med.
155:
A729,
1997.
22.
Kanofsky, J. R.,
and
P. D. Sima.
Singlet-oxygen generation at gas-liquid interfaces: a significant artifact in the measurement of singlet-oxygen yields from ozone-biomolecule reactions.
Photochem. Photobiol.
58:
335-340,
1993[Medline].
23.
Kanofsky, J. R.,
and
P. D. Sima.
Reactive absorption of ozone by aqueous biomolecule solutions: implications for the role of sulfhydryl compounds as targets for ozone.
Arch. Biochem. Biophys.
318:
52-62,
1995.
24.
Kirichenko, A.,
L. Li,
M. T. Morandi,
and
A. Holian.
4-Hydroxy-2-nonenal-protein adducts and apoptosis in murine lung cells after acute ozone exposure.
Toxicol. Appl. Pharmacol.
141:
416-424,
1996[Medline].
25.
Langford, S. D.,
A. Bidani,
and
E. M. Postlethwait.
Ozone-reactive absorption by pulmonary epithelial lining fluid constituents.
Toxicol. Appl. Pharmacol.
132:
122-130,
1995[Medline].
26.
Miller, F. J.,
J. H. Overton,
J. S. Kimbell,
and
M. L. Russel.
Regional respiratory tract absorption of inhaled reactive gases.
In: Toxicology of the Lung (2nd ed.), edited by D. E. Gardner,
J. D. Crapo,
and R. O. McClellan. New York: Raven, 1993, p. 485-525.
27.
Mudd, J. B.,
P. J. Dawson,
and
J. Santrock.
Ozone does not react with human erythrocyte membrane lipids.
Arch. Biochem. Biophys.
341:
251-258,
1997[Medline].
28.
Mudway, I. S.,
D. Housley,
R. Eccles,
R. J. Richards,
A. K. Datta,
T. D. Tetley,
and
F. J. Kelly.
Differential depletion of human respiratory tract antioxidants in response to ozone challenge.
Free Radic. Res.
25:
499-513,
1996[Medline].
29.
Mudway, I. S.,
and
F. J. Kelly.
Modeling the interactions of ozone with pulmonary epithelial lining fluid antioxidants.
Toxicol. Appl. Pharmacol.
148:
91-100,
1998[Medline].
30.
Ohkawa, H.,
N. Ohishi,
and
K. Yagi.
Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.
Anal. Biochem.
95:
351-358,
1979[Medline].
31.
Overton, J. H.,
and
R. C. Graham.
Simulation of the uptake of a reactive gas in a rat respiratory tract model with an asymmetric tracheobronchial region patterned on completed conducting airway cast data.
Comput. Biomed. Res.
28:
171-190,
1995[Medline].
32.
Peden, D. B.,
M. Swiersz,
K. Ohkubo,
B. Hahn,
B. Emery,
and
M. A. Kaliner.
Nasal secretion of the ozone scavenger uric acid.
Am. Rev. Respir. Dis.
148:
455-461,
1993[Medline].
33.
Plopper, C.,
A. Wier,
J. Bric,
L. Putney,
E. Postlethwait,
V. Wong,
B. Tarkington,
J. Joad,
and
D. Hyde.
Mapping ozone-induced acute cytotoxicity in intrapulmonary airways of the rat using differential permeability dyes and laser scanning confocal microscropy (Abstract).
Am. J. Respir. Crit. Care Med.
155:
A746,
1997.
34.
Postlethwait, E. M.,
and
A. Bidani.
Reactive uptake governs the pulmonary airspace removal of inhaled nitrogen dioxide.
J. Appl. Physiol.
68:
594-603,
1990[Abstract/Free Full Text].
35.
Postlethwait, E. M.,
S. D. Langford,
and
A. Bidani.
Interfacial transfer kinetics of NO2 into pulmonary epithelial lining fluid.
J. Appl. Physiol.
71:
1502-1510,
1991[Abstract/Free Full Text].
36.
Postlethwait, E. M.,
S. D. Langford,
and
A. Bidani.
Determinants of inhaled ozone absorption in isolated rat lungs.
Toxicol. Appl. Pharmacol.
125:
77-89,
1994[Medline].
37.
Postlethwait, E. M.,
S. D. Langford,
L. M. Jacobson,
and
A. Bidani.
NO2 reactive absorption substrates in rat pulmonary surface lining fluids.
Free Radic. Biol. Med.
19:
553-563,
1995[Medline].
38.
Postlethwait, E. M.,
L. W. Velsor,
J. M. Cheek,
and
C. G. Plopper.
O3-induced acute pulmonary epithelial injury: analysis via fluorescent probes versus BAL protein and LDH (Abstract).
Am. J. Respir. Crit. Care Med.
151:
A499,
1995.
39.
Pryor, W. A.
How far does ozone penetrate into the pulmonary air/tissue boundary before it reacts?
Free Radic. Biol. Med.
12:
83-88,
1992[Medline].
40.
Pryor, W. A.
Ozone in all its reactive splendor.
J. Lab. Clin. Med.
122:
483-486,
1993[Medline].
41.
Pryor, W. A.
Mechanisms of radical formation from reactions of ozone with target molecules in the lung.
Free Radic. Biol. Med.
17:
451-465,
1994[Medline].
42.
Pryor, W. A.,
E. Bermudez,
R. Cueto,
and
G. L. Squadrito.
Detection of aldehydes in bronchoalveolar lavage of rats exposed to ozone.
Fundam. Appl. Toxicol.
34:
148-156,
1996[Medline].
43.
Pryor, W. A.,
and
D. F. Church.
Aldehydes, hydrogen peroxide, and organic radicals as mediators of ozone toxicity.
Free Radic. Biol. Med.
11:
41-46,
1991[Medline].
44.
Pryor, W. A.,
G. L. Squadrito,
and
M. Friedman.
The cascade mechanism to explain ozone toxicity: the role of lipid ozonation products.
Free Radic. Biol. Med.
19:
935-941,
1995[Medline].
45.
Pryor, W. A.,
and
R. M. Uppu.
A kinetic model for the competitive reactions of ozone with amino acid residues in proteins in reverse micelles.
J. Biol. Chem.
268:
3120-3126,
1993[Abstract/Free Full Text].
46.
Rabinowitz, J. L.,
and
D. J. P. Bassett.
Effect of 2 ppm ozone exposure on rat lung lipid fatty acids.
Exp. Lung Res.
14:
477-489,
1988[Medline].
47.
Rooney, S. A.
Phospholipid composition, biosynthesis, and secretion.
In: Treatise on Pulmonary Toxicology: Comparative Biology of the Normal Lung, edited by R. A. Parent. Boca Raton, FL: CRC, 1992, p. 511-544.
48.
Santrock, J.,
R. A. Gorski,
and
J. F. O'Gara.
Products and mechanisms of the reaction of ozone with phospholipids in unilamellar phospholipid vesicles.
Chem. Res. Toxicol.
5:
134-141,
1992[Medline].
49.
Slade, R.,
K. Crissman,
J. Norwood,
and
G. Hatch.
Comparison of antioxidant substances in bronchoalveolar lavage cells and fluid from humans, guinea pigs and rats.
Exp. Lung Res.
19:
469-484,
1993[Medline].
50.
Sokal, R. R.,
and
F. J. Rohlf.
Biometry. New York: Freeman, 1981.
51.
Squadrito, G. L.,
R. M. Uppu,
R. Cueto,
and
W. A. Pryor.
Production of the Criegee ozonide during the ozonation of 1-palmitoyl-2-oleoyl-sn-glcero-3-phosphocholine liposomes.
Lipids
27:
955-958,
1992[Medline].
52.
Uchiyama, M.,
and
M. Mihara.
Determination of malonaldehyde precursor in tissues by thiobarbituric acid test.
Anal. Biochem.
86:
271-278,
1978[Medline].
53.
Ultman, J. S.
Transport and uptake of inhaled gases.
In: Air Pollution, the Automobile, and Public Health, edited by A. Y. Watson,
R. R. Bates,
and D. Kennedy. Washington, DC: National Academy Press, 1988, p. 323-366.
54.
Uppu, R. M.,
R. Cueto,
G. L. Squadrito,
and
W. A. Pryor.
What does ozone react with at the air/lung interface? Model studies using human red blood cell membranes.
Arch. Biochem. Biophys.
319:
257-266,
1995[Medline].
55.
US Environmental Protection Agency.
Air Quality Criteria for Ozone and Related Photochemical Oxidants. Washington, DC: Environmental Protection Agency, 1996, vol. III (EPA/600/P-93/004cF)
56.
Van der Vliet, A.,
C. A. O'Neill,
J. P. Eiserich,
and
C. E. Cross.
Oxidative damage to extracellular fluids by ozone and possible protective effects of thiols.
Arch. Biochem. Biophys.
321:
43-50,
1995[Medline].
57.
Velsor, L. W.,
and
E. M. Postlethwait.
Nitrogen dioxide-induced generation of extracellular reactive oxygen is mediated by epithelial lining layer antioxidants.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L1265-L1275,
1997[Abstract/Free Full Text].
Am J Physiol Lung Cell Mol Physiol 274(6):L1006-L1016
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