Modeling the interactions of particulates with epithelial
lining fluid antioxidants
Henryk
Zielinski1,
Ian S.
Mudway2,
Kelly A.
Bérubé3,
Samantha
Murphy3,
Roy
Richards3, and
Frank J.
Kelly2
1 Institute of Animal
Reproduction and Food Research, Polish Academy of Sciences, Olsztyn
10-747, Poland; 2 The Rayne
Institute, St. Thomas' Hospital, London SE1 7EH; and
3 Cardiff School of Biosciences,
Cardiff CF1 1ST, United Kingdom
 |
ABSTRACT |
Oxidative stress may be a fundamental mode of
injury associated with inspired particles. To examine this, we
determined the ability of three carbon black particles (CBPs; M120,
M880, and R250) and two forms of silicon dioxide, amorphous (Cabosil)
and crystalline (DQ12) quartz, to deplete epithelium lining fluid antioxidant defenses. Single and composite antioxidant solutions of
uric acid, ascorbic acid (AA), and reduced glutathione (GSH) were
examined in the presence of particle concentrations of 150 µg/ml.
Uric acid was not depleted by any particle considered. AA was depleted
in a near-linear fashion with time by the three different CBPs;
however, AA depletion rates varied markedly with CBP type and decreased
in the presence of metal chelators. An initially high GSH depletion
rate was noted with all CBPs, and this was always accompanied by the
appearance of oxidized glutathione. Exposure to Cabosil or DQ12 did not
result in the loss of GSH. Together, these data demonstrate that
particle type, size, and surface area are all important factors when
considering particle-antioxidant interactions in the airways.
oxidative stress; particles; air pollution; ascorbic acid; reduced
glutathione
 |
INTRODUCTION |
A NUMBER OF STUDIES (6, 29) have demonstrated a
correlation between daily variations in ambient particulate pollution, with fluctuations in mortality from cardiorespiratory disease, and
hospital admissions for respiratory and cardiovascular illness. These
associations persist after adjustment for potential confounders and are
thought to be causal. The mechanisms underlying these responses are not
yet established, but it has been suggested that particle toxicity
arises through oxidative challenge to the lung (8, 17).
Carbonaceous material is a principal component of airborne particulate
matter. It exists as either organic carbon or particulate elemental
carbon, also known as carbon black particle (CBP). In London, UK,
carbonaceous particles contribute up to 20% of the mass of particulate
matter < 10 µm in diameter
(PM10), whereas 87% of the elemental carbon is formed from diesel combustion. Diesel
exhaust therefore represents an important source of urban air
pollution, consisting of a complex of both oxidant gases and particulate matter. A wide variety of transition metals and
hydrocarbons derived from the combustion process are absorbed onto the
surface of these particles (7, 26). Although many of these gaseous components and surface-absorbed materials are recognized oxidants (14,
23, 25) capable of causing oxidative stress in and damage to the lung,
little is presently understood about the toxicity of CBPs formed during
diesel combustion. Although CBPs are generally considered to be
relatively inert, they could be toxic through their action as delivery
vectors for materials absorbed on their surfaces (24).
As an initial step toward establishing the mechanistic basis of
particle toxicity associated with diesel exhaust, we examined the
impact of three different CBPs of varying diameter, surface area, and
chemical properties on the antioxidant defenses in lung epithelial
lining fluid (ELF). For comparison, we also examined the effects of two
silicon dioxide particles known to have considerable toxicity in vivo.
We hypothesized that because ELF is the first physical interface
encountered by inspired particulate matter, interactions between
particles and ELF antioxidants may result in antioxidant depletion from
this compartment.
 |
MATERIALS AND METHODS |
Unless otherwise stated, chemicals used in the following protocols were
obtained from Sigma (Poole, UK) or British Drug House (Poole, UK).
Exposure system. To conduct these
experiments, we modified the exposure apparatus previously used to
examine the impact of gaseous oxidants on ELF antioxidants (19). In the
model system, ELF was represented in two ways: first as individual
antioxidant solutions of ascorbic acid (AA; 200 µmol/l), uric acid
(UA; 200 µmol/l), and reduced glutathione (GSH; 400 µmol/l) and
second as a composite mixture of these three antioxidants. These
concentrations were used because they reflect those present in ELF from
normal, nonsmoking individuals (4, 10, 27). Antioxidant solutions were
prepared in 0.9% (wt/vol) saline (Baxter Healthcare, Thetford, UK) and were adjusted to pH 7.4 to reflect normal human airway secretions. In some instances, antioxidant solutions were prepared in
the presence of EDTA and desferoxamine mesylate (0.1 mM final concentration). Details of the pH buffering capacity of these solutions
have been described previously (19), determined with standard protocols
(11).
To standardize the system, a model CBP (M120) was employed at three
concentrations (50, 150, and 500 µg/ml). These values were chosen because they represent those that would be present in ELF
following 1) the maximum UK hourly
average value, 2) the mean UK 8-h
running average and simultaneously the UK
PM10 upper limit, and
3) the maximum UK daily average,
respectively (22). These CBP doses were arrived at with the assumption
that only 65% of the available CPB dose is deposited in the lung and
that the "morphological volume" of the mucous and alveolar lining
layers in humans is 12.6 ml (10, 28). For comparison, we subsequently examined the impact of two different types of CBP (M880 and R250) and
amorphous (Cabosil) and crystalline (DQ12) silicon dioxides on ELF antioxidants.
Exposures were carried out in a 5.6-liter Perspex chamber. The chamber
was maintained throughout at 37 ± 2.8°C, and the whole apparatus was mounted on an orbital shaker to facilitate mixing of the
aqueous phase. Antioxidant solutions were incubated with particles as
1-ml aliquots in multiwell plates (Becton Dickinson UK, Oxford, UK)
fixed within the exposure chamber. Each well had a diameter of 1.5 cm,
giving an exposed surface area of 1.78 cm2. Before addition of the
particles, the solutions were allowed to equilibrate to 37°C.
Concentrated particle solutions were then added to specific wells to
give the desired final particle concentration. The chamber was closed,
and the orbital shaker was switched on. At set intervals during the
exposure (30, 60, 120, 240, and 360 min), the shaker was switched off,
the chamber was opened, and three samples were removed. These samples
were centrifuged at 10,000 g for 5 min
(4°C) to separate the particles from the aqueous components, and
the supernatant was decanted to a fresh tube and snap-frozen in liquid
nitrogen. At the end of the experiment, all samples were transferred to
80°C for longer-term storage.
Characteristics of particles. The
three different types of CBP used in this study were donated by Cabot
(Billerica, MA). Before the particles were utilized, their morphology
and surface chemistry were established. The diameter of single
particles of carbon black type M120 was 50 nm, of type M880 20 nm, and
of R250 40 nm. The surface areas were 32, 220, and 62 m2/g for M120, M880, and R250,
respectively. The diameter of a single particle of amorphous silicon
dioxide (Cabosil) was 7 nm and of crystalline silicon dioxide quartz
(DQ12) 440 nm. All particles tended to aggregate to form bunches or
chains in solution (18). Size distribution (<300-nm equivalent
spherical diameter) of these aggregates was 21 (M120), 49 (R250), 41 (M880), 39 (DQ12), and 74% (Cabosil).
Electron-probe X-ray microanalysis studies confirmed that M120 was a
pure carbon black, which contained only chloride and sulfur (20).
Sulfur was presumably derived from the fuel source during particle
generation. M880 was found to contain sodium, silicon, sulfur,
chloride, calcium, and potassium, whereas R250 has silicon, sulfur, and
iron. Cabosil and DQ12 were shown to be pure silicon dioxide, with
surfaces that contain no contaminating elements.
Preparation of M120 stock solution.
Three stock solutions of M120 were prepared as follows. Ten, thirty, or
one hundred milligrams of dry M120 were suspended in 10 ml of 0.9%
(wt/vol) saline adjusted to pH 7.4 at room temperature. These
solutions were continuously mixed for 2 h, and then 50 µl of each
were added to 1-ml aliquots of the antioxidant solution in multiwell
plates, giving final CBP concentrations of 50, 150, and 500 µg/ml,
respectively. Control samples, which did not contain M120, were run in
parallel throughout the exposure. In a similar manner, stock solutions
of M880, R250, and Cabosil were prepared. The DQ12 sample was first
cleaned of amorphous contaminants by being washed over a 24-h period
with four changes of 4 M HCl. The dust was then extensively washed with
HPLC-grade water and dried before use.
Antioxidant determinations. UA, AA,
GSH, and oxidized glutathione (GSSG) were determined as previously
described (12). Briefly, UA and AA were measured simultaneously
according to the method of Iriyama et al. (13) with reverse
chromatography with a 25-cm Apex II ODS column eluted with a mobile
phase of 0.2 M
KH2PO4 and 0.25 mM octanesulfonic acid (pH 2.1). Electrochemical detection was
employed (current sensitivity 0.1 nA), and the sample concentration was
determined with reference to standard curves of 0-6.3 and 0-12.5 µM for AA and UA, respectively. Detection limits were 10 (AA) and 5 (UA) nM. GSH and GSSG were measured with the enzyme recycling assay (1). Sample concentrations were determined with
reference to standard curves for GSH and GSSG of 0-6.6 and 0-3.3 µM, respectively. Detection limits were 50 (GSH) and 10 (GSSG) nM. Analyses were performed within 7 days postexposure.
Correction for evaporative loss. Due
to evaporation, sample volume decreased ~15% over 6 h. To correct
for this change in concentration, the concentration of
Na+ in control and CBP samples was
determined with a blood gas analyzer.
Determination of antioxidant depletion rates.
The rate of antioxidant loss or GSSG formation in the present study
does not reflect a conventional reaction rate. Rather, the change in
concentration of these moieties with time, expressed as time per
microgram of particle, represents a composite of 1) direct
oxidative consumption by the particle, 2) that arising from
secondary oxidative reactions, and 3) that lost through
adsorption onto the particle surface. The rates are therefore expressed
either as the change in concentration with time (in M/s) or as the
change in concentration with time per particle concentration (in
M · s
1 · µg
1).
The former rate was determined from the gradient of a linear regression
passed through a plot of antioxidant-to-GSSG concentrations against
exposure time at each particle concentration used. In the majority of
cases, this relationship was linear with time. The "goodness of
fit" of the regression through each set of data points is indicted
by the regression coefficient
(r2) and the
probability that the regression line does not deviate significantly
from the linear model (P). Gradients
derived from linear regressions were only used when
P < 0.05. Where data could not be
fitted by linear regression, the "best fit" polynomial regression
was performed, and the rate of consumption was determined over the
linear portion. The latter rate expression
(M · s
1 · µg
1)
was similarly derived by plotting the consumption rates observed under
each particle concentration, derived as described above, against
particle concentration.
Statistical analysis. Overall
variations in data sets were determined with a two-way,
repeated-measures analysis of variance. The factors employed for each
analysis were particle concentration (0-, 50-, 150-, and 500-µg
particles) and antioxidant (AA, UA, and GSH) for the rate of
consumption (in M/s) with respect to the pure antioxidant and composite
antioxidant data sets and antioxidant (AA, UA, and GSH) and exposure
model (pure antioxidant and composite antioxidant solution) for the
overall consumption rate (in
M · s
1 · µg
1)
data set for each model system employed. Pairwise comparisons of group
means within each data set were conducted with the Student-Newman-Keuls test, with corrections for multiple comparisons. Linearity of the
response was determined by linear regression analysis with the
least-squares method. Significance for all comparisons of means and
linear regression analysis was accepted at
P < 0.05.
 |
RESULTS |
M120-antioxidant interactions in pure and composite
solutions. Antioxidant depletion kinetics achieved by
M120 in pure and composite antioxidant solutions are illustrated in
Fig. 1. Of note, different rates of loss
from supernatant fluids were observed for AA, UA, and GSH. Although UA
was not depleted at any concentration of M120 considered, increasing AA
loss was seen with time under each M120 concentration employed. Similar
AA depletion kinetics were seen in both the pure and composite
antioxidant models. In comparison to the near-linear depletion rates
seen for AA, more complex depletion kinetics were observed for GSH.
After an initial rapid loss of GSH with the 150 and 500 µg/ml M120
concentrations, new steady-state GSH levels were established at ~80
and 50% of time 0 concentrations,
respectively. Importantly, no appreciable loss of AA or GSH was
observed in the absence of M120 (Fig. 1).

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Fig. 1.
Consumption of ascorbic acid (AA;
A), uric acid (UA;
B), and reduced glutathione (GSH;
C) by carbon black particles (CBPs;
M120) in pure and composite antioxidant solutions. Solutions were
incubated in indicated final concentrations of CBPs. Results are
expressed as percent change in concentration, with assumption that
concentration at time 0 (before CBP
incubation) was 100%. Each point represents mean (±SD) converted
concentration of antioxidant at a specific time within exposure
protocol; n = 3 experiments. Details
of linear regression analysis are given in Table 1. Over a
360-min exposure to 500 µg/ml of M120, fluctuations in pH of pure AA,
UA, and GSH solutions and of composite antioxidant solutions prepared
and exposed as outlined above were negligible.
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With these data, individual supernatant depletion rates (in M/s) for AA
and GSH were calculated (Table 1).
Significantly increased rates of AA loss were seen at 50, 150, and 500 µg/ml of M120 from both single and composite antioxidant solutions. GSH (initial rate) loss was significantly greater at 150 and 500 µg/ml of M120 than under control (0 µg/ml) conditions in both pure
and composite antioxidant solutions (Table 1). To clarify the
relationship between the antioxidant depletion rates and M120 concentration, plots of antioxidant depletion rate against M120 concentration were performed (plots not illustrated). In the case of AA
and GSH, a near-linear, positive relationship was observed: r2 = 0.98 and
P < 0.05 for AA and
r2 = 0.98 and
P < 0.05 for GSH. The gradients of
the individual regression lines were then calculated and are expressed
as molarity per second per microgram. Comparison of these rates, which
are broadly indicative of the substrate reactivity toward CBPs, are summarized in Table 2. These data suggest a
significant difference in the overall depletion rates between GSH and
AA versus UA and GSH versus AA, suggesting an overall reactivity
hierarchy within the pure antioxidant model of GSH > AA
UA.
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Table 1.
Antioxidant consumption rates in pure and composite antioxidant
solutions with linear regression analysis
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Table 2.
Overall antioxidant depletion rates in pure antioxidant and composite
antioxidant solutions with linear regression analysis
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GSSG formation. As with GSH loss,
complex formation kinetics were observed for GSSG formation. A rapid
initial increase in GSSG at 150 and 500 µg/ml of M120 was followed by
the establishment of a new steady-state concentration after 2-4 h
of exposure (data not shown). Because of these kinetics, GSSG formation
rates were calculated only over the initial linear portion of the best
fit polynomial regression. In all experiments, the rate of GSSG
formation was significantly enhanced in comparison with the 0 µg/ml
control exposure. Overall GSSG formation rates per unit of M120
calculated from the gradients of linear regressions were 14.3 ± 3.2 × 10
12
M · s
1 · µg
1
in the pure GSH model
(r2 = 0.98;
P < 0.05) and 25.3 ± 0.9 × 10
12
M · s
1 · µg
1
in the composite model
(r2 = 0.99;
P < 0.05). These rates,
approximately one-half of the corresponding GSH overall consumption
rates, support a 2:1 GSH-to-GSSG reaction stochiometry.
CBP- and SiO2 particle-antioxidant
interactions in composite solutions.
All these experiments were carried out at particle concentrations of
150 µg/ml and with composite antioxidant solutions containg UA, AA,
and GSH. None of the particles studied (M120, M880, R250, Cabosil, or
DQ12) had any impact on UA. In contrast, both GSH (Fig.
2) and AA (Fig.
3) were depleted by exposure to M880,
M120, and R250, although no consumption of either antioxidant by
Cabosil or DQ12 was seen. The particle reactivities were M880 > R250 > M120
DQ12 = Cabosil for AA, whereas those for GSH were M880
R250 > = M120
DQ12 = Cabosil. However, when these reactivities were indexed to particle surface area, the hierarchies quoted above
altered to R250 > M120 > M880 for AA and M120 > R250 = M880 for
GSH (Table 3). Inclusion of
chelators significantly lowered the rate of AA depletion (Fig. 3) but
not GSH loss or GSSG formation (Fig. 2).

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Fig. 2.
GSH depletion (A) and GSSG formation
(B) rates observed in unchelated
( ) and chelated (+) composite antioxidant solutions exposed to
CBPs (M120, R250, and M880) and
SiO2-based particles (Cabosil and
DQ12) at 150 µg/ml. DES, desferoxamine mesylate. Values are means ± SD; n = 3 experiments.
Comparison of rates between particles and of particles with and without
chelator systems was performed with 2-way ANOVA, with pairwise
comparisons performed with Student-Newman-Keuls test. A significant
difference in rates was assumed when P < 0.05. Significant
difference between depletion or formation rates between particles in
unchelated systems.
Significant difference
between depletion or formation rates between particles in chelated
systems. * Rates observed for Cabosil and DQ12 were all
significantly less than those observed with CBPs. Bottom:
GSH-to-GSSG ratios after incubation with each type of particle.
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Fig. 3.
AA depletion rates observed in unchelated and chelated composite
antioxidant solutions exposed to CBPs and
SiO2-based particles at 150 µg/ml. Values are means ± SD; n = 3 experiments. Comparison of rates between particles and of particles
with and without chelator systems was performed with 2-way ANOVA, with
pairwise comparisons performed with Student-Newman-Keuls test. A
significant difference in rates was assumed when
P < 0.05. Significant difference
between depletion or formation rates between particles in unchelated
systems. Significant
difference between depletion or formation rates between particles in
chelated systems.
+ Significant difference
between rate of consumption between chelated and unchelated systems.
* Rates observed for Cabosil and DQ12 were all significantly less
than those observed with CBPs. UA was not consumed by any of the
particles outlined above in either system.
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 |
DISCUSSION |
Concern about the health effects of particulate air pollution continues
to grow on an international scale. As a consequence, increased effort
is being made to understand the mechanisms underlying the biological
toxicity of particulate matter. Although the toxicity of pollutant
gases such as ozone and nitrogen dioxide is widely acknowledged to
involve oxidative stress, the mechanisms behind particulate toxicity
are less well understood. Because many of the surface-absorbed
materials on particles are recognized oxidants, oxidative stress may
also be an important component of particle-induced lung injury.
Moreover, because the surface of the lung is protected by a network of
soluble antioxidant defenses (4, 5, 14, 27), particles with oxidative
capacity may deplete this antioxidant screen. In this respect, particle
surface chemistry may play a major role in determining their oxidative capacity.
To examine this hypothesis, experiments were conducted to determine
whether M120, a simple form of carbon black, would deplete AA, UA, or
GSH from a model ELF system. In an initial dose-response study, M120
was found to preferentially deplete AA and GSH but not UA. Loss of AA
and GSH could be the result of 1)
oxidative depletion, 2) adsorption
of these antioxidants onto the surface of the particles, or
3) a combination of these two
events. The loss of GSH, accompanied by the appearance of its oxidation
product GSSG, and the time-related depletion of AA suggest that
oxidative depletion is probably the predominant reason for antioxidant
loss in this system. The specific loss of AA and GSH in the presence of
M120 is in marked contrast to findings with the pollutant gases ozone
(19) and nitrogen dioxide (16), which react avidly with and deplete AA
and UA but not GSH. Combining these observations leads us to the
conclusion that different respiratory pollutants may target different
antioxidants. Because pollution episodes rarely consist of a single
pollutant, this specificity may help explain why a network of
antioxidant defenses are required to protect the airways.
To investigate particle-antioxidant interactions further, we next
considered the antioxidant depletion capacity of two further forms of
CBPs and two forms of silica particles. The objective of this second
study was to determine the overall reactivity hierarchy of antioxidants
with particles and to determine whether particles of variable
morphology and surface properties have different reactivities toward
ELF antioxidants. The findings of this study demonstrate that particle
surface area and surface chemistry are both important determinants of
particle reactivity. Whereas none of the particles examined caused the
loss of UA, exposure to M880 or R250 caused greater depletion of AA and
GSH than of M120. However, when these rates of loss were corrected for
particle surface area, the hierarchy changed to R250 > M120 > M880
for AA and M120 > R250 = M880 for GSH. These data illustrate the
importance of available surface area of the particles for these
antioxidant interactions. Although the three types of CBPs examined
depleted AA and GSH but not UA, the depletion kinetics seen for each
antioxidant were quite different. For example, AA was depleted in a
near-linear fashion in a dose- and time-dependent manner, whereas
although the initial depletion kinetics of GSH were rapid, they
subsequently slowed down until a new, lower, steady-state GSH
concentration was established.
Surface area may not be the only factor responsible for particle
reactivity. Each CBP has a slightly different surface chemistry that
may influence its oxidative and/or antioxidant adsorption capacity.
CBPs have many organic and inorganic substances on their surface, which
are thought to be the principal toxic constituents of inhaled diesel
exhaust particles. In the present study, AA depletion by all CBPs was
reduced significantly by the addition of metal chelators (Fig. 3). This
is unlikely to reflect a general shielding effect by chelators
absorbing onto the particle surface because a similar effect was not
seen for GSH. Rather, it may reflect the release of iron from the
particle surface and an iron-catalyzed depletion of AA (9). Analysis of
the surface chemistry of the particles utilized in the present study
revealed that only R250 had iron on its surface and this was only a
minimal amount. The core elemental composition of CBPs can, however, be
very different from their surface chemistry (2), and it cannot be ruled
out that transition metals leach out from the particle core with time. Although the CBPs used in the present study were not studied in detail,
it is known that more complicated diesel exhaust particles show
differences in surface chemistry when collected as an aerial sample or
after treatment in solution (3). Sonication of particles in solution
makes them smoother in appearance and causes distribution of core and
surface elements, in effect making them less reactive. If similar
changes occur after sonication of CBPs in solution, then we may have
underestimated their true reactivity in the present study. Diesel
exhaust particles have been shown to produce superoxide anions in vitro
and to induce oxidative stress and oxidative DNA damage in vivo when
instilled intratracheally in mice (21, 25). Moreover, when these
particles were washed with methanol, they lost their oxidant capacity,
suggesting that surface, and perhaps core, chemistry play an important
role in this response. Based on these observations, it is feasible that
exposure to aerial particles would result in different findings from
those seen in the present study.
Despite the finding that CBPs were able to elicit a marked depletion of
AA and GSH, suggesting that they would pose a significant oxidative
stress, AA and GSH loss did not occur in the presence of amorphous or
crystalline quartz. These findings are contrary to what would be
predicted from a previous study in vivo (20), which established that
inhaled or instilled quartz has high biological reactivity. A low dose
(1 mg) of respirable crystalline quartz instilled in rats produced
significant increases in lung permeability, persistent surface
inflammation, progressive increases in pulmonary surfactant, and
activities of epithelial marker enzymes up to 12 wk after primary
exposure. Although ultrafine amorphous silica (Cabosil) did not induce
such progressive effects, it did promote early epithelial damage with
permeability changes, which subsequently regressed with time. By
contrast, M120 had little, if any, effect on lung permeability,
epithelial markers, or inflammation despite being given at a dose that
readily translocated the epithelium and that has been reported to
induce inflammation. It is always difficult to extrapolate in vitro
results to those from animal studies. However, it should be noted that
the doses employed in the present study were chosen to reflect actual
human exposures. As a consequence, the particle doses employed in the
rat study quoted above were ~20-fold higher than those used in the
present study. This may explain, in part, the differences noted in
particle toxicity.
Because lung ELF is the first interface encountered by inspired
particles, the reactions between particles and antioxidants as they
pass through this fluid layer will presumably modify the impact of
these particles on the pulmonary epithelium. Indeed, it has been
proposed that the antioxidants in this compartment help protect the
lung from oxidative injury arising from inspired gaseous and
particulate pollutants (5, 15). One interpretation of the present
results is that because CBPs readily react with AA and GSH, their
surface chemistry is changed as they pass through the ELF compartment,
and, as a consequence, they are less reactive when they reach the
pulmonary epithelium. Conversely, because quartz particles do not react
with antioxidants present in ELF, they reach the pulmonary epithelium
largely unchanged. Even so, it must follow that the mechanism of
toxicity of these different classes of particles must be fundamentally
different. Notwithstanding these differences, if the above assumption
is correct, the capacity of particles to react with ELF antioxidants
represents an important process in detoxifying certain particles and
hence limiting their subsequent biological activity.
Previously, we observed that exposure to diesel exhaust results in a
transient increase in nasal lavage fluid AA concentration (3a). In
light of the present findings, the increased secretion of AA into the
airways may represent a protective response against particle toxicity.
If ELF antioxidants are acting in this way, this possibility requires
further consideration because individual variations in the
concentrations of these moieties may explain, in part, the differential
susceptibility of subjects to
PM10.
In conclusion, the findings of this study contribute toward an improved
understanding of particle-mediated lung injury. The finding that
relatively chemically inert particles can result in a marked loss of
antioxidants suggests that particles in combination with oxidant gases
will synergistically reduce the level of endogenous airway protection,
thereby increasing the chances of subsequent epithelial cell injury.
 |
ACKNOWLEDGEMENTS |
We thank the British Council, the Wellcome Trust, and the Medical
Research Council (UK) for supporting these studies.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: F. Kelly,
Cardiovascular Research, The Rayne Institute, St. Thomas' Hospital,
London SE1 7EH, UK (E-mail: frank.kelly{at}kcl.ac.uk).
Received 16 February 1999; accepted in final form 18 May 1999.
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