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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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

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. circle  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. circle  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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Table 3.   Particle characteristics with AA and GSH depletion rates and rates corrected for available particle SA


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 277(4):L719-L726
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