Role of soluble metals in oil fly ash-induced airway epithelial injury and cytokine gene expression

Janice A. Dye1, Kenneth B. Adler2, Judy H. Richards1, and Kevin L. Dreher1

1 National Health and Environmental Effects Research Laboratory, Experimental Toxicology Division, Pulmonary Toxicology Branch, United States Environmental Protection Agency, Research Triangle Park 27711; and 2 Department of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606


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Particulate matter (PM) metal content and bioavailability have been hypothesized to play a role in the health effects epidemiologically associated with PM exposure, in particular that associated with emission source PM. Using rat tracheal epithelial cells in primary culture, the present study compared and contrasted the acute airway epithelial effects of an emission source particle, residual oil fly ash (ROFA), with that of its principal constitutive transition metals, namely iron, nickel, and vanadium. Over a 24-h period, exposure to ROFA, vanadium, or nickel plus vanadium, but not to iron or nickel, resulted in increased epithelial permeability, decreased cellular glutathione, cell detachment, and lytic cell injury. Treatment of vanadium-exposed cells with buthionine sulfoximine further increased cytotoxicity. Conversely, treatment with the radical scavenger dimethylthiourea inhibited the effects in a dose-dependent manner. RT-PCR analysis of RNA isolated from ROFA-exposed rat tracheal epithelial cells demonstrated significant macrophage inflammatory protein-2 and interleukin-6 gene expression as early as 6 h after exposure, whereas gene expression of inducible nitric oxide synthase was maximally increased 24 h postexposure. Again, vanadium (not nickel) appeared to be mediating the effects of ROFA on gene expression. Treatment with dimethylthiourea inhibited both ROFA- and vanadium-induced gene expression in a dose-dependent manner. Corresponding effects were observed in interleukin-6 and macrophage inflammatory protein-2 synthesis. In summary, generation of an oxidative stress was critical to induction of the ROFA- or vanadium-induced effects on airway epithelial gene expression, cytokine production, and cytotoxicity.

particulate matter; airway epithelial cells; gene induction; oxidative stress


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INTRODUCTION
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PARTICULATE AIR POLLUTION has been positively associated with daily morbidity and mortality in numerous recent epidemiologic studies (for reviews, see Refs. 45, 53). Morbidity effects have included increased respiratory symptoms, asthma exacerbations, pulmonary function decrements, and increased emergency room visits or hospitalizations for respiratory (13) and cardiovascular (55) diseases. These observations have led to considerable debate over whether or not the National Ambient Air Quality Standard for particulate matter (PM) is sufficiently stringent to protect human public health and whether it should be changed. However, plausible biological mechanisms underlying these epidemiologic associations have yet to be firmly established. In addition, experimental evidence is lacking to definitively identify specific characteristics of particulate pollution that may be mediating these associations (e.g., physical attributes, biological or chemical constituents). One hypothesis is that PM metal content and metal bioavailability play a role in the health effects associated with PM exposure, in particular that associated with exposure to emission source PM (9, 14, 22, 46). Overall, ambient air PM, the term used for solid or liquid particles in air, is a composite of PM arising from natural sources (e.g., wind erosion), man-made sources (e.g., automobile exhaust, power plants, woodstoves), and particles formed in the atmosphere during condensation of gaseous pollutants (e.g., SO2 and NO-containing compounds) (5). Because emission sources contribute to the overall ambient air particulate burden (57), in these studies, we investigated the biological effects of a fugitive emission source particle, residual oil fly ash (ROFA). This fly ash contains several transition metals, constituting 10% of its mass. Moreover, the three principal ROFA-associated metals, namely iron (Fe), nickel (Ni), and vanadium (V), are readily soluble in water (9, 22).

Because airway epithelial cells are one of the first cell populations to come in contact with respirable PM, in these studies, primary cultures of rat tracheal epithelial (RTE) cells were used as a model for assessing PM effects on the airways. Established under conditions that produce a pseudostratified mucociliary epithelium (26), the RTE cultures were exposed to fly ash suspensions or metal-containing solutions on their apical (i.e., luminal) airway surface. Initial studies compared the epithelial cytotoxicity induced during exposure to ROFA with that induced during exposure to comparable amounts of individual constitutive metals. Airway epithelial cells, however, not only function as a protective barrier to the external environment, they can also initiate and amplify airway inflammation by producing a number of proinflammatory mediators (1, 32). In additional studies, we examined the effects of ROFA or transition metal exposure on RTE cell gene expression or synthesis of three potential inflammatory mediators: the pleiotropic cytokine interleukin (IL)-6 (12, 39), the potent rodent neutrophil chemoattractant macrophage inflammatory protein (MIP)-2 (15), and the inflammatory enzyme inducible nitric oxide (NO) synthase (iNOS) (8).

Because ROFA-associated transition metals could elicit their pulmonary effects by generating reactive oxygen species (ROS) through catalysis of Fenton-like reactions or, alternatively, they could act via mechanisms independent of their oxidant catalytic activity, we also examined the role of oxidative stress in fly ash- and metal-induced epithelial cell responses. Results demonstrated that in cultured airway epithelial cells, vanadium, but not iron or nickel, was largely responsible for mediating the overall effects of ROFA (e.g., gene expression changes, cytokine production, cytotoxicity). These results were consistent with the clinical syndrome of airway injury and inflammation observed in workers occupationally exposed to vanadium-containing fumes. Results also indicated that oxidative stress was critically involved in the RTE cell effects observed after exposure to ROFA or vanadium. Thus, to the extent that emission sources contribute to the overall ambient air PM and to the extent that these in vitro airway responses reflect pulmonary effects in vivo, our findings may have important pathophysiological consequences relevant to the morbid respiratory effects noted in recent epidemiologic studies, especially in individuals with preexisting airway inflammation (e.g., asthmatic patients) (47).


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Materials. Tissue culture media and reagents were obtained from Sigma (St. Louis, MO) with the following exceptions: insulin, transferrin, and amphotericin B were obtained from GIBCO BRL (Life Technologies, Grand Island, NY); rat tail collagen was obtained from Collaborative Research (Bedford, MA); bovine pituitaries were obtained from Pel Freeze (Rogers, AR); Transwell tissue culture inserts (24-mm diameter, 0.4-µm pore size) were purchased from Costar (Cambridge, MA); NG-monomethyl-D-arginine (D-NMMA) was purchased from Calbiochem (La Jolla, CA); and sterile, preservative-free 0.9% sodium chloride used to suspend particles was obtained from Lyphomed (Deerfield, IL).

The ROFA particles used in these studies were collected by the Southern Research Institute (Birmingham, AL) at a temperature of 204°C on a Teflon-coated glass-fiber filter downstream from the cyclone of a power plant burning low-sulfur no. 6 residual oil (22). Mount St. Helens (MSH) volcanic ash was collected from an open field near Ritzville, WA, after the 1980 eruption (22). Ferric sulfate [Fe2(SO4)3] and nickel sulfate (NiSO4) were purchased from Sigma, and vanadium sulfate (VOSO4) was purchased from K & K Laboratories (Plainview, NY).

Cell culture. Primary cultures of RTE cells were established under conditions that produce pseudostratified mucociliary epithelium (26). Briefly, for these experiments, RTE cells were obtained from male Sprague-Dawley rats (10-14 wk old; Charles River Laboratories, Raleigh, NC) by overnight digestion with Pronase at 4°C. The cells were plated on rat tail (type I) collagen gel-coated membranes at a density of 3 × 104 cells/cm2 in complete medium (CM) at 35°C in a humidified environment of 3% CO2. On the day of RTE cell plating (day 0), CM in the basolateral compartment was supplemented with 10% fetal bovine serum and 3 mg/ml of bovine serum albumin (BSA). After day 0, the CM was composed of Dulbecco's modified Eagle's medium (DMEM)-Ham's F-12 medium supplemented with L-leucine (0.45 mM), L-lysine (0.5 mM), L-glutamine (6.5 mM), L-methionine (0.12 mM), MgCl2 (0.30 mM), MgSO4 (0.40 mM), CaCl2 (1.05 mM), NaHCO3 (1.2 mg/ml), insulin (10 µg/ml), hydrocortisone (0.1 µg/ml), cholera toxin (0.1 µg/ml), transferrin (5 µg/ml), epidermal growth factor (25 ng/ml), HEPES (30 mM, pH 7.2), BSA (0.5 mg/ml), phosphoethanolamine (50 µM), ethanolamine (80 µM), penicillin-streptomycin (50 U/ml and 50 µg/ml, respectively), bovine pituitary extract (1% vol/vol), all trans-retinoic acid (5 × 10-8 M), and amphotericin B (1.0 µg/ml, decreasing to 0.4 µg/ml). The cells were grown submerged in CM for the first 7 days, at which time an air-liquid interface was established by removing medium from the apical surface of each culture. The cultures were fed basally once a day for an additional 6 days. Mature, highly confluent 13-day-old cultures were used in all experiments.

Exposure of RTE cultures. Just before exposure, each culture was fed 2.5 ml of CM basally, and the apical surface was washed twice with Hanks' balanced salt solution (1 ml/wash) to remove secretions, media, and nonadherent cells. The ROFA suspensions or metal-containing solutions were prepared in saline, with the final pH adjusted to 3.0 with H2SO4 (acid-saline). Newly prepared particle suspensions or metal solutions were applied to the RTE cells within 2 h of preparation. For the actual exposure, 0.5 ml of either saline, acid-saline, a ROFA suspension, an MSH ash suspension, or a metal-containing solution was applied to the apical surface. In one study, the ROFA suspension was centrifuged for 15 min, and the clear supernatant or "leachate" fraction was separated. The pellet fraction was washed three times to ensure that the supernatant fraction recovered was of neutral pH. The remaining "washed" particles were then resuspended in the original volume of saline. In another study, the ROFA suspension and leachate fraction were neutralized before application on the RTE cells. In an additional study, the cells were pretreated for 18 h with buthionine sulfoxide (BSO; 500 µM dissolved in CM). The BSO-containing medium was then removed, and the cells were given CM without BSO, and then exposed to a vanadium solution for 24 h. In other experiments, the cells were treated with dimethylthiourea (DMTU; 4 or 40 mM in CM) for 30 min before and during a ROFA or vanadium exposure. The cells also were treated with NG-monomethyl-L-arginine (L-NMMA; 0.5 and 5.0 mM) or D-NMMA (5.0 mM) for 30 min before and during a ROFA exposure.

Cellular toxicity assessments. The cultures were examined visually with an inverted microscope for overt changes in cellular adhesion and morphology. Cytotoxicity was quantified by measuring the release of lactate dehydrogenase (LDH), a stable cytosolic enzyme, into the apical compartment (which was done by adding an additional 0.5 ml of Hanks' balanced salt solution, swirling for 20 s, and removing the entire quantity of liquid present apically) or into the basolateral compartment of the Transwell unit. LDH activity of the cells still attached to the collagen membrane was measured after lysis with 0.5% Triton X-100 in PBS (cell lysate). LDH, total protein, and albumin were measured in the apical, basolateral, and lysate samples with a Cobas Fara II centrifugal spectrophotometer (Hoffman-La Roche, Branchburg, NJ). Activity for LDH was determined with commercially available kits from Sigma; total protein concentrations were determined with the Coomassie Plus Protein Assay from Pierce Chemical (Rockland, IL); albumin concentrations were determined with the MALB SPQ kit from INCSTAR (Stillwater, MN) with a standard curve prepared with BSA. The total percentage of LDH released was calculated with the equation [(A + B)/(A + B + L)] × 100%, where A is the apical compartment, B is the basolateral compartment, and L is the cell lysate. In separate cultures, cells remaining attached to the collagen membrane were lysed with perchloric acid (PCA) and analyzed for total cellular glutathione with Anderson's (3) 5,5'-dithio-bis(2-nitrobenzoic) acid-oxidized glutathione reductase recycling assay adapted for the centrifugal spectrophotometer. Again, for each culture, total cellular glutathione was normalized to the lysate cellular protein (i.e., micrograms per milligram of protein). For each PCA-lysed sample, the cellular protein was determined by subtracting the amount of protein in the cured rat tail collagen layer from the total protein content in the PCA-lysed sample.

Evaluation of gene expression. Semiquantitative reverse transcriptase-polymerase chain reactions (RT-PCRs) were carried out on RNA isolated from RTE cells after exposure to saline, acid-saline (pH 3.0), a ROFA suspension (pH 3.0), or an equivalent metal-containing solution (pH 3.0). RNA was isolated with RNAzol (Cinna/Biotecx Laboratories, Houston, TX) followed by chloroform extraction and precipitation with 75% ethanol. Total RTE cell RNA was quantified on the basis of absorbance at 260 nm, and 0.5 µg of RNA was reverse transcribed for 45 min in a total volume of 10 µl with reagents from Perkin-Elmer (reagents manufactured by Roche Molecular Systems, Branchburg, NJ). Reagents and final concentrations used were 5 mM MgCl2, 1 mM deoxyguanosine 5'-triphosphate, 1 mM deoxyadenosine 5'-triphosphate, 1 mM deoxythymidine 5'-triphosphate, 1 mM deoxycytidine 5'-triphosphate, 1 U/µl of RNase inhibitor, 2.5 µM random hexamers, and 2.5 U/µl of murine leukemia virus RT in 1× PCR buffer.

Primer sequences for IL-6 (37), MIP-2 (15), iNOS (40), and beta -actin (30) were obtained from the published literature. All oligonucleotide primer pairs used were designed to amplify segments of mRNA derived from two or more gene exons. The primer sequences used were IL-6 (rat) forward primer 5'-gactgatgttgttgcagccactgc-3' and reverse primer 5'-tagccactccttctgtgactctaact-3', MIP-2 (murine) forward primer 5'-ggcacatcaggtacgatccag-3' and reverse primer 5'-accctgccaagggttgacttc-3', iNOS (rat) forward primer 5'-agatcgagccctggaagacc-3' and reverse primer 5'-tgtcatgagcaaaggcacaga-3', and beta -actin (rat) forward primer 5'-ctgatccacatctgctggaaggtgg-3' and reverse primer 5'-accttcaacaccccagccatgtacg-3'. To avoid saturation of the PCR product, for each primer pair, amplification conditions were optimized for the RTE cell cDNA with respect to cycle number and final MgCl2 concentration. These primer pairs generate a 509-bp PCR product for IL-6, a 287-bp product for MIP-2, a 447-bp product for iNOS, and a 703-bp product for beta -actin. PCR was performed with a 10-µl aliquot of cDNA in a total volume of 50 µl. A "hot-start" procedure (Perkin-Elmer Technical Bulletin BIO-66) was used to initiate the PCR. A DNA thermal cycler 480 (Perkin-Elmer Cetus, Foster City, CA) was used, with final amplification conditions as follows: initial denaturation at 94°C for 2 min, followed by varying cycle numbers (i.e., IL-6, 36 cycles; MIP-2, 28 cycles; iNOS, 25 or 28 cycles; and beta -actin, 25 cycles) under the following conditions: denaturation at 92°C for 45 s, annealing at 60°C for 1 min, and extension at 72°C for 2 min, with a final extension performed at 72°C for 10 min. Before amplification of beta -actin, cDNA was diluted 1:10. DNA amplification products were separated on a 1.5% Separide gel matrix (GIBCO BRL), and the bands were stained with SYBR Green I nucleic acid stain (Molecular Probes, Eugene, OR). DNA was visualized by ultraviolet illumination and recorded with a Polaroid MP-4 Land Camera (Fotodyne, New Berlin, WI) on positive/negative no. 55 film (Polaroid, Cambridge, MA). The band densities were quantified with National Institutes of Health Image (version 1.58) software program. For final analysis, for each well, the band density of the gene of interest was normalized relative to the corresponding parallel-amplified band of beta -actin (a housekeeping gene).

Quantification of IL-6 and MIP-2 proteins. At 6 or 24 h after the application of saline, a ROFA or MSH ash suspension, or a metal solution, conditioned apical or basal samples were collected, cell debris was pelleted by centrifugation, and the supernatants were frozen for subsequent determination of IL-6 and MIP-2 concentrations with ELISA kits for the rat (Biosource International, Camarillo, CA). The assays were done following the instructions of the manufacturer.

Statistical analysis. All data were analyzed with a t-test for single comparisons or analysis of variance with Scheffé's posttest correction for determination of multiple comparisons. For all analyses, group differences were considered significant if the test statistic type I error was <0.05 (i.e., P < 0.05) (18).


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Cytotoxicity assessments. In an effort to determine what component(s) of ROFA was biologically active in airway epithelial cells, in the first of a series of experiments, RTE cells were exposed apically to saline, acid-saline (pH 3.0), or ROFA particles suspended in saline (pH 3.0) applied at 5, 10, or 20 µg/cm2 for 24 h. When ROFA is suspended in saline at the concentrations used here, the suspension becomes relatively acidic (pH of 3-4). We (16) have previously determined that acidity does not contribute significantly toward ROFA-induced RTE cell injury; therefore, in these studies, we continued to use acid-saline (pH 3.0) as our "vehicle control" treatment. In this same experiment, ROFA suspensions were centrifuged, and the "particle-free" supernatants (pH 3.0) were applied in the same volumes as the suspensions. The remaining pelleted PM was washed repeatedly with neutral saline until the recovered supernatant was also of neutral pH. The pellets were resuspended in saline and applied in the same volume as the original suspension. Microscopic evaluation 24 h later indicated that the cells exposed to the ROFA suspension or the particle-free fraction had extensive injury as evidenced by numerous detached individual cells or clumps of cells, many of which appeared hyperlucent and excessively spherical (i.e., prelytic swelling). In some areas of the culture, rafts of epithelial cells were lifted completely off the collagen-coated membrane. Accordingly, after the 24-h exposure, LDH release from cultures exposed to the particle-free ROFA supernatant was equal to or greater than that of cultures exposed to the intact suspension. The particle-free fraction of the suspension has been termed the leachate fraction. Conversely, the washed PM elicited only mild cytotoxicity even at the highest dose (Fig. 1). To further determine whether the acidity of the ROFA suspension or leachate influenced its cytotoxicity, in a second experiment, ROFA suspensions and leachate solutions were neutralized with NaOH before application. The resultant toxicity was comparable to that observed in the first study with acidic preparations (data not shown).


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Fig. 1.   Effects of exposure on release of lactate dehydrogenase (LDH). Rat tracheal epithelial (RTE) cells were exposed for 24 h to either saline, acid-saline (pH 3.0), a suspension of residual oil fly ash (ROFA) at 5, 10, and 20 µg/cm2 (pH 3.0), a particulate matter-free leachate of ROFA suspension at the corresponding exposure (pH 3.0), or washed particulate material alone, again at the corresponding exposure. Values are means ± SE; n = 4 samples/point. * Significantly different from saline-exposed cultures.

Seemingly, the epithelial toxicity of ROFA was being mediated by the leachate fraction of the suspension. This fraction presumably contained the majority of the water-soluble metals originally present in or on the intact fly ash particles. Therefore, in the next set of experiments, we compared the cytotoxicity induced by exposure of RTE cells to a ROFA suspension with that induced by exposure to water-soluble amounts of individual constitutive metals. On the basis of acid digestion, ROFA contains ~23 µg iron/mg fly ash, of which 68% is readily soluble in water; 38 µg nickel/mg ash, of which 92% is water soluble; and 42 µg vanadium/mg ash, of which 84% is water soluble (9). First, we examined the toxicity of iron, the transition metal classically involved in Fenton chemical reactions. Cells were exposed for 24 h to either acid-saline, a ROFA suspension (pH 3.0) at 10 µg/cm2, or a ferric sulfate solution (pH 3.0) containing an equimolar quantity of iron to that present in the ROFA suspension on a water-soluble basis. LDH release in iron-exposed cells was not increased (3.2 ± 0.27%) compared with that in acid-saline-exposed cells (2.5 ± 0.24%), whereas in ROFA-exposed cells, it was significantly increased (55.0 ± 5.5%; n = 3 samples/group).

Because under these in vitro conditions, iron exposure elicited negligible cytotoxicity compared with the ROFA suspension, in the remainder of the studies, we focused on the epithelial effects of the two other most prevalent ROFA-associated metals, nickel and vanadium. Cells were again exposed to metal solutions containing equimolar quantities of metal to those present in the ROFA suspension on a water-soluble basis. Vanadium sulfate or nickel sulfate solutions were prepared at an acidic pH (3.0). Results demonstrated that over a 24-h period, exposure to vanadium either alone or in combination with nickel resulted in similar morphological effects (i.e., cell detachment and swelling) as did exposure to the ROFA suspension. Accordingly, cells exposed to vanadium or vanadium plus nickel had ~50% of the LDH release induced by exposure to the ROFA suspension, whereas nickel exposure alone resulted in minimal LDH release (Fig. 2). Identical results were obtained in a replicate of this experiment (data not shown). We further evaluated ROFA- or metal (nickel, vanadium, or nickel plus vanadium)-induced effects on solute permeability of the epithelial layer. We (16) have previously demonstrated that "leakage" of BSA (from medium in the basolateral compartment onto the apical culture surface) is a reliable indicator of the solute permeability of the cultured RTE layer. In this study, negligible changes in permeability were observed 6 h postexposure in any treatment group. However, by 18 and 24 h postexposure, solute permeability was significantly increased in cultures exposed to vanadium, nickel plus vanadium, or ROFA but not in cultures exposed to nickel alone (compared with acid-saline-exposed cultures; data not shown).


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Fig. 2.   Effect of ROFA or metal exposure on LDH release. RTE cells were exposed for 24 h to either acid-saline (pH 3.0), acidified metal solutions (pH 3.0) of nickel (Ni), vanadium (V), a combination of Ni and V (Ni+V), or a suspension of ROFA (pH 3.0) at 10 µg/cm2. Metal solutions contained amount of metal present in a water-soluble extract of ROFA equivalent to a 10 µg/cm2 exposure. Values are means ± SE; n = 3 samples/point. * Significantly different from acid-saline-exposed cultures. dagger  Significantly different from ROFA-exposed cultures.

In a subsequent study, cells were exposed to metal solutions containing a quantity of metal equivalent to the "total" amount present in (or on) ROFA (i.e., the amount of metal present in an acid-extracted leachate of fly ash). In this study, vanadium exposure alone elicited ~80% of the cytotoxicity induced by exposure to the ROFA suspension, whereas nickel exposure alone induced ~14% (n = 3-4 samples/group; data not shown). Seemingly, if RTE cells are exposed to the total metal present in a ROFA suspension and one assumes simple additivity of the effects of nickel and vanadium, then these two metals could account for nearly all of the cytotoxicity induced by the suspension itself.

In the next set of experiments, we compared changes in total intracellular glutathione induced by exposure of cells to ROFA with that induced by exposure to water-soluble quantities of metal (nickel, vanadium, or nickel plus vanadium). In one study, glutathione levels were significantly depleted in cells exposed to vanadium, nickel plus vanadium, or ROFA, with vanadium exposure eliciting nearly 50% of the depletion induced by exposure to the ROFA suspension (Fig. 3A). In a replicate of this study, similar trends were observed; however, the degree of glutathione depletion in vanadium-exposed cells was as severe as that induced in the ROFA-exposed cells (Fig. 3B). In both experiments, on the other hand, nickel exposure was associated with minor increases in intracellular glutathione.


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Fig. 3.   Comparison of exposure to ROFA or corresponding metal solutions on total intracellular glutathione in RTE cells in initial experiment (A) and a replicate of this experiment (B). RTE cells were exposed for 18 h to either acid-saline (pH 3.0); acidified metal solutions (pH 3.0) of Ni, V, or Ni+V; or a suspension of ROFA (pH 3.0) at 10 µg/cm2. Metal solutions contained amount of metal present in a water-soluble extract of ROFA equivalent to a 10 µg/cm2 exposure. Values are means ± SE; n = 3 samples/point. * Significantly different from acid-saline-exposed cultures. dagger  Significantly different from ROFA-exposed cultures.

The results suggested that similar to a ROFA exposure, vanadium (but not nickel) exposure was associated with an oxidative stress on RTE cells as intracellular glutathione concentrations were significantly depleted. To further substantiate this finding, RTE cells were pretreated with BSO, an irreversible inhibitor of gamma -glutamyl cysteine synthetase (21) to deplete intracellular glutathione before vanadium exposure. Accordingly, LDH release after 24 h in vanadium-exposed BSO-pretreated cells (equivalent to 5 µg/cm2 of ROFA) was significantly increased (58 ± 4.0%) compared with that in vanadium-exposed untreated cells (22.3 ± 2.8%). However, LDH release was also significantly increased in acid-saline-exposed BSO-pretreated cells (23.6 ± 2.8%) compared with that in acid-saline-exposed untreated cells (4.0 ± 0.1% LDH release; n = 3 samples/group). Overall, the results demonstrate the importance of adequate intracellular glutathione concentrations in maintaining epithelial cell integrity. Furthermore, the results are consistent with a protective effect of intracellular glutathione against the cytotoxicity induced by vanadium exposure.

Effects on gene expression and cytokine production. To assess the potential contribution of airway epithelial cells in the development of the pulmonary inflammatory response that occurs after in vivo exposure to ROFA, we used RT-PCR analysis to evaluate changes in steady-state gene expression of IL-6, MIP-2, and iNOS in RTE cells exposed to 5, 10, or 20 µg/cm2 of ROFA (applied as a suspension) for 3, 6, or 24 h. For each primer pair used, the resultant products were of the size anticipated. No bands were observed in samples from which mRNA or RT enzyme was withheld before thermocycling. The results demonstrated that as early as 6 h, IL-6 expression was present in all cells exposed to ROFA (Fig. 4A). MIP-2 expression followed a similar pattern except that the most intense bands were present at 24 h in the highest exposure group (Fig. 4B). On the other hand, for iNOS expression, only faint bands were visible at 6 h. At 24 h, however, prominent iNOS bands were observed in all cells exposed to ROFA (Fig. 4C).




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Fig. 4.   Effects of ROFA exposure on steady-state gene expression of interleukin (IL)-6 (A), macrophage inflammatory protein (MIP)-2 (B), and inducible nitric oxide synthase (iNOS; C). RTE cells were exposed for 3, 6, or 24 h to saline, acid-saline (pH 3.0), or a suspension of ROFA at 5 (ROFA 5), 10 (ROFA 10), or 20 (ROFA 20) µg/cm2 (pH 3.0). Delta , Change in respective time period. Values are mean of densitometry measurements normalized to housekeeping gene beta -actin; n = 3-4 samples/point. * Significantly different from saline-exposed cultures.

Using enzyme-linked immunosorbent assays (ELISAs), we further evaluated IL-6 and MIP-2 protein production and release into the apical and basolateral compartments of cells exposed to acid-saline or ROFA (10 µg/cm2) for 24 h. IL-6 concentrations in apical samples from acid-exposed cells were below detectable limits. However, in ROFA-exposed cells, a mean (±SE) of 760 ± 50 pg of IL-6 was detected in the apical samples (n = 3-4 samples/group). Due to inconsistent spurious detection of low levels of "IL-6" in the unconditioned CM, IL-6 concentrations in the medium-containing basolateral samples were not determined. The quantity of MIP-2 in the apical compartment of acid-saline-exposed cells was 1,080 ± 70 pg. In ROFA-exposed cells, MIP-2 was significantly increased to 15,500 ± 1,400 pg/apical compartment (n = 3-4 samples/group). Because at 24 h, comparable concentrations of MIP-2 were measured in the basolateral samples compared with those in the apical samples, in subsequent studies, only apical samples were evaluated for cytokine content.

We further evaluated cytokine production in RTE cells exposed to a relatively inert particle, MSH ash. The cultures were exposed to acid-saline (pH 3.0), a ROFA suspension (10 µg/cm2, pH 3.0), or an acidified suspension of MSH ash (10 µg/cm2, pH 3.0) for 24 h. We (16) had previously demonstrated that at this dose, MSH ash was not cytotoxic to RTE cells. In this experiment, IL-6 concentrations in apical samples from acid-saline-exposed and MSH ash-exposed cells were below detectable limits. Unlike the previous study, however, IL-6 was not detectable in the ROFA-exposed cells either, possibly due to the severe cytotoxicity induced by ROFA in this study (i.e., >50% LDH release). MIP-2 production, on the other hand, was again significantly increased in ROFA-exposed cells (11,800 ± 280 pg/apical compartment), whereas MIP-2 production by MSH ash-exposed cells (1,430 ± 200 pg/apical compartment) was not significantly different from that of acid-saline-exposed cells (1,250 ± 360 pg/compartment; n = 3 samples/group).

To assess whether oxidative stress was also involved in the ROFA-induced effects on epithelial cytokine expression or production, RTE cells were coexposed to ROFA (5 µg/cm2) and the potent reactive oxygen radical scavenger DMTU at 4, 15, or 40 mM concentrations in CM for 6 h. This exposure time period was selected to evaluate all three genes of interest. However, to enhance detection of iNOS expression by 6 h, thermocycling for iNOS was increased from 25 to 28 cycles. The results demonstrated that, in a dose-dependent manner, coexposure with DMTU significantly inhibited ROFA-induced gene expression of all three genes evaluated (Fig. 5). Similarly, DMTU treatment inhibited MIP-2 production and/or release in ROFA-exposed cells. Specifically, at 6 h postexposure, samples from acid-saline-exposed untreated cells contained 730 ± 130 pg/apical compartment, acid-saline-exposed cultures pretreated with 40 mM DMTU contained 770 ± 120 pg/apical compartment, and ROFA (5 µg/cm2)-exposed untreated cells contained significantly more MIP-2 (2,750 ± 530 pg/apical compartment), whereas ROFA-exposed cells pretreated with 40 mM DMTU contained only 510 ± 150 pg/apical compartment (n = 3 samples/group). The 6-h time period was seemingly too early to detect changes in IL-6 production by ROFA-exposed RTE cells because IL-6 concentrations were below detectable limits in the apical samples from all exposure groups.


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Fig. 5.   Effect of dimethylthiourea (DMTU) on ROFA-induced steady-state gene expression of IL-6 (A) and MIP-2 (B). Similar trends were observed for iNOS expression (data not shown). RTE cells were exposed to acid-saline or a suspension of ROFA at 5 µg/cm2 (pH 3.0) for 6 h with and without pre- and coexposure to DMTU at indicated concentrations ([DMTU]) dissolved in complete medium. Values are means ± SE; n = 3 samples/point. * Significantly different from acid-saline-exposed cultures without DMTU treatment. dagger  Significantly different from ROFA-exposed cultures without DMTU treatment.

We further evaluated gene expression changes in cells coexposed to ROFA at 5 µg/cm2 for 6 h along with L-NMMA, a competitive inhibitor of NOS enzymes. Relatively high concentrations of L-NMMA were used (0.5 and 5.0 mM) because the specialized RTE cell CM contained arginine. As demonstrated above, ROFA exposure again resulted in increased steady-state expression of IL-6, MIP-2, and iNOS (data not shown). ROFA-induced expression of MIP-2 and iNOS was not significantly affected by coexposure with L-NMMA even at the 5.0 mM concentration. However, IL-6 expression was significantly inhibited in the ROFA-exposed cells that were coexposed to 5.0 mM L-NMMA. This inhibition was not observed in cells coexposed to 0.5 mM L-NMMA or in cells coexposed to D-NMMA (5.0 mM), the inactive form of L-NMMA (n = 3 samples/group; data not shown).

To determine which of the ROFA-associated metals may be mediating these gene expression changes, in the next set of experiments, gene expression changes in ROFA-exposed RTE cells (5 µg/cm2 for 6 h) were compared with changes induced in RTE cells exposed to comparable quantities of metals (nickel, vanadium, or nickel plus vanadium) on a water-soluble basis. The results demonstrated increased steady-state expression of IL-6, MIP-2, and iNOS in cells exposed for 6 h to vanadium, nickel plus vanadium, or ROFA. However, gene expression in nickel-exposed cells was not different from that in acid-saline-exposed cells (Fig. 6). Similar trends were observed for apical release of IL-6 and MIP-2 by RTE cells exposed to ROFA or vanadium for 24 h (Fig. 7).


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Fig. 6.   Effect of ROFA or metal exposure on steady-state gene expression of IL-6 (A) and MIP-2 (B). Similar trends were observed for iNOS expression (data not shown). RTE cells were exposed to acid-saline (pH 3.0), a suspension of ROFA (pH 3.0) at 5 µg/cm2, or water-soluble equivalent metal solutions (pH 3.0) for 6 h. Values are means ± SE; n = 3 samples/point. * Significantly different from acid-saline-exposed cultures.



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Fig. 7.   Effect of ROFA or metal exposure on RTE cell production and apical release of IL-6 (A) and MIP-2 (B). RTE cells were exposed for 24 h to acid-saline (pH 3.0), a ROFA suspension (pH 3.0) at 10 µg/cm2, or water-soluble equivalent metal solutions. BDL, below detectable limits of assay. Values are means ± SE; n = 3 samples/point. * Significantly different from acid-saline-exposed cultures. dagger  Significantly different from ROFA-exposed cultures.

Although the gene expression changes due to ROFA exposure appeared to involve development of an oxidative stress, it was not clear whether the effects observed after a vanadium exposure were also being elicited through redox changes in the RTE cells. Therefore, in the following studies, we evaluated the effect of coexposure with DMTU on vanadium-induced LDH release and changes in steady-state gene expression. The results demonstrated that, similar to its effects on ROFA-exposed cells, DMTU coexposure significantly inhibited both the vanadium-induced LDH release and gene expression changes in a dose-dependent manner (Fig. 8).


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Fig. 8.   Effect of DMTU on V-induced LDH release (A) and steady-state gene expression of iNOS (B). Similar trends were observed for IL-6 and MIP-2 (data not shown). RTE cells were exposed to acid-saline (pH 3.0) or a V-containing solution (pH 3.0). Quantity of V present was equivalent to water-soluble V present in ROFA suspension applied (10 µg/cm2 in A and 5 µg/cm2 in B). Values are means ± SE; n = 3 samples/point. * Significantly different from acid-saline-exposed cultures without DMTU treatment. dagger  Significantly different from V-exposed cultures without DMTU treatment.

And finally, we wanted to assess whether airway epithelial cells were generally sensitive to the effects of vanadium or if there was something unique about the vanadium present in this particular ROFA sample. In this study, RTE cells were exposed for 24 h to assorted oil fly ash samples that had been collected on the effluent emission control systems downstream from the furnace of an oil-burning power plant (29). Three oil fly ash samples that contained increasing amounts of vanadium but little or no iron or nickel were used. As shown in Fig. 9, the cytotoxicity resulting from exposure of RTE cells to 10 µg/cm2 of the oil fly ash samples increased proportionately with the amount of water-soluble vanadium present in the oil fly ash samples. By comparison, the cells exposed to the intact ROFA suspension had somewhat less injury than would have been predicted simply on the basis of the water-soluble vanadium content of ROFA. These results suggest that the other metals present in ROFA (i.e., iron) were in some manner influencing or interfering with the overall airway toxicity of the vanadium present in ROFA.


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Fig. 9.   Correlation between LDH release in RTE cells exposed to assorted oil fly ash (OFA) samples with amount of water-soluble V present in OFA samples. RTE cells were exposed for 24 h to acid-saline (pH 3.0), a ROFA suspension (pH 3.0) at 10 µg/cm2, or to other OFA samples (pH 3.0) at 10 µg/cm2. OFA samples contained increasing amounts of water-soluble V, with little or no iron and Ni. Values are means ± SE; n = 3 samples/point.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ambient air PM is composed of a complex mixture of crustal and anthropogenic material. Crustal PM largely falls within the "coarse-mode" PM (i.e., PM with a mass median aerodynamic diameter between 2.5 and 10 µm). The fraction of ambient air PM that is <= 2.5 µm is referred to as "fine-mode" PM. Emission or combustion sources are thought to contribute primarily to the fine mode (57). Over the past several years, a robust epidemiologic data base has been generated that repeatedly demonstrated statistical associations between ambient air PM and mortality/morbidity in exposed human populations (45, 53). These associations were identified with a mass-based metric as an index of PM exposure. However, in some instances (54), because the mass metric for PM was restricted to smaller particulate size ranges, the strength of the statistical correlation appeared to increase (i.e., 2.5-µm PM > 10-µm PM > total suspended particulates). Furthermore, it is this fine mode that is of particular concern in terms of human health risks because this fraction may remain airborne for long periods, may be transported over long distances, is capable of penetrating indoor air environments, and, most importantly, this fraction is of the appropriate size to enter the respiratory tract and come in direct contact with airway and deep lung surfaces (9). Although one cannot equate emission source PM directly with ambient air <= 2.5-µm PM, emission source PM shares a number of potentially key features with PM of the fine mode. These features include but are not limited to high sulfate content, acidity, and a relatively rich metal contamination or content.

Still, specific characteristics of PM and related mechanisms by which PM may be mediating the health effects epidemiologically associated with particulate exposure remain to be defined. Insofar as the hypothesis that PM metal content and bioavailability are involved in the pathophysiological consequences of particulate exposure, the present in vitro experiments in rat airway epithelial cells were designed to complement previous in vivo studies in which rodents were exposed to model emission source PM (9, 14, 19, 20, 29, 46, 60). The initial objective of the present study was to use RTE cells in primary culture as a model target cell population to compare and contrast the acute airway epithelial toxicity of an emission source PM (e.g., ROFA) with that of its constitutive transition metals. The results demonstrated that exposure of cells to a water-soluble leachate of ROFA resulted in as much or more toxicity as exposure to the intact suspension, indicative that soluble components (e.g., metals) were mediating the cytotoxic effects of ROFA. Of the three predominant metals contained in (or on) ROFA, exposure to vanadium sulfate, but not to ferric sulfate or nickel sulfate, resulted in comparable cytotoxic responses occurring over an 18- to 24-h period. The effects included increased epithelial solute permeability, epithelial cell detachment, and lytic cell injury.

A second objective of this study was to better elucidate the role of airway epithelial cells in the generation of the overall pulmonary inflammatory response to ROFA. Airway epithelial cells are in a key position to influence the development of pulmonary inflammation after exposure to particulate air pollutants. Furthermore, it is well established that airway epithelial cells are capable of producing a wide variety of proinflammatory mediators (11). Therefore, by means of RT-PCR analysis, we evaluated the changes in steady-state RTE cell gene expression of the proinflammatory enzyme iNOS and the cytokines IL-6 and MIP-2. The results demonstrated that at all doses, MIP-2 and IL-6 gene expression were increased as early as 6 h after ROFA application, whereas maximal iNOS expression was observed 24 h after ROFA application. We confirmed that the cytokine gene induction changes correlated with increases in IL-6 and MIP-2 synthesis, although as measured, the quantities of MIP-2 far exceeded those of IL-6. In fact, over a 24-h period, ROFA-exposed cultures produced nanogram quantities of MIP-2, a 10- to 15-fold increase over those of acid-saline-exposed cells. Similarly, Carter et al. (6) demonstrated that exposure of normal human bronchial epithelial cells to ROFA or vanadium, but not to iron or nickel, increased production of IL-6 and IL-8 (a functional homologue to MIP-2 in the rat), with corresponding changes in mRNA. In addition, Samet et al. (50) demonstrated that exposure of an immortalized bronchial epithelial cell line to ROFA or vanadium, but not to iron or nickel, resulted in tyrosine phosphatase inhibition, with cellular accumulation of protein phosphotyrosines. In the present study, the results further demonstrated that exposure of RTE cells to MSH ash, a PM sample with a very low soluble metal content, was not associated with increased cytokine production. On the aggregate, current and previous data suggest that airway epithelial cells potentially play a key role in the overall pulmonary inflammatory response that develops after an in vivo exposure to ROFA and that, moreover, vanadium, not iron or nickel, appears to be mediating the majority of the cellular and molecular effects of ROFA in epithelial cells.

With regard to vanadium-induced effects on the airways, the results of the present in vitro study are also largely consistent with published in vivo observations. Experimental exposure of laboratory rats to vanadium resulted in development of acute neutrophilic airway and pulmonary inflammation (29, 42). Before neutrophil influx into the airways, bronchoalveolar lavage fluid analysis revealed significant increases in MIP-2 expression consistent with the neutrophilic chemotactic activity of MIP-2 in the rat (42). In addition, a clinical database exists regarding occupational exposure such as occurs in workers of the petrochemical, mining, steel, or power-generating industries to vanadium-containing fumes or respirable dust. Associated respiratory effects are largely confined to inflammatory changes of the upper and lower airways (24, 61), although decrements in peak airflow assessments (23) and increased airway responsiveness have also been reported (44). Less commonly, severe respiratory tract irritation and inflammation develops, clinically termed "boilermaker's bronchitis." Symptoms include cough, dyspnea, and chest pain primarily related to severe airway inflammation (33, 58). In addition, there are isolated case reports of individuals exposed to massive doses of fly ash that developed severe dyspnea and interstitial fibrosis (7) or bronchiolitis obliterans with partially reversible airway obstruction (4).

Although the observations outlined above indicate that the vanadium content was largely responsible for the effects of ROFA on cultured airway epithelial cells, it was still unclear as to how these cellular effects were being elicited. As reviewed recently (34, 49), one proposed mechanism by which exposure to particulate air pollution results in the development of lung inflammation is through generation of oxygen and nitrogen radicals. Furthermore, a previous study (16) in RTE cells indicated that oxidative stress, likely related to Fenton-like (transition metal-catalyzed) pathways for the generation of ROS, was involved in ROFA-induced cytotoxicity. However, transition metals such as vanadium not only participate in Fenton-like chemical reactions to produce reactive species such as hydroxyl radicals (56); alternatively, they can act through pathways independent of their ability to catalyze such reactions [e.g., interactions with "metal response" elements (10) or metal-associated inhibition of tyrosine phosphatase activity (31)]. The present data suggest that the cytotoxic effects of vanadium were, in fact, associated with generation of an oxidative stress as evidenced by depletion of RTE cell glutathione, augmentation of injury in cells pretreated with BSO, and complete abolition of injury and permeability changes in cells coexposed with the radical scavenger DMTU. Similarly, the effects of vanadium on RTE cell gene expression and cytokine synthesis were completely abolished by coexposure of the cells with DMTU.

As with strictly in vitro observations, however, one may question whether the oxidative stress mechanisms underlying the in vitro effects of ROFA are representative of cellular events occurring during an in vivo exposure to ROFA or other fly ash samples. Clearly, the in vivo situation is more complicated because it involves multiple cell types, their products, and complex metal pharmacokinetic considerations. Still, the results of the present in vitro study are largely consistent with an in vivo study (27) using electron spin resonance techniques to detect radical adduct formation in the lungs of ROFA- or metal-exposed rats. Kadiiska et al. (27) reported that prominent electron spin resonance signals were present in lung tissue from rats exposed to ROFA or vanadium (VOSO4) in vivo, whereas lungs from saline- or nickel-exposed rats had much weaker signals. Furthermore, we (16) have previously demonstrated that in rats intratracheally exposed to ROFA, systemic administration of DMTU significantly decreased the number of neutrophils present in bronchoalveolar laveage fluid by nearly 80%. We postulated that DMTU treatment resulted in either scavenging or diminished production of key reactive oxygen species, which, in turn, prevented cellular redox changes from occurring in pulmonary "effector" cell populations (e.g., epithelial cells, macrophages, lymphocytes). Changes in the redox status of a cell have been proposed to modulate activity of several transcription factors, including nuclear factor (NF)-kappa B and activator protein-1 (43, 52); however, the upstream, intracytoplasmic processes leading to transcription factor activation are not well understood. Such processes appear to include complex cascades of phosphorylation-dephosphorylation reactions that are individually modulated by specific redox changes (17). Of relevance to the present study, binding sites for NF-kappa B are located in the promoter region of iNOS (2), IL-6 (63), IL-8 (35), and, possibly, MIP-2 (62), all genes that may serve to initiate, amplify, or perpetuate an acute inflammatory response.

Somewhat unexpected was the relative lack of cytotoxicity induced in RTE cells exposed to iron or nickel. Individually, these metals have been shown to elicit pulmonary injury and inflammation in rats after intratracheal instillation (14, 29). In fact, nickel-exposed rats developed significant lung pathology that included severe pulmonary edema and hemorrhage (14, 29). These data bring into light some of the unique cellular sensitivities (i.e., sensitive target cell populations of the alveolar space) that underlie the overall pulmonary response to PM exposure in vivo.

Not only are multiple cell types and their products involved in the overall lung response to PM, the potential for unlimited "cross talk" between these cell types and their associated products (e.g., cytokines, ROS, NO) exists. For example, ROS generation has been associated with gene induction of iNOS and inflammatory cytokines (48, 51). However, as shown here, ROFA-exposed RTE cells required additional time for maximal expression of iNOS (relative to IL-6 and MIP-2), suggesting that multiple factors were involved in iNOS regulation, possibly the combination of ROS generation with IL-6 or MIP-2 production. Similarly, Kinugawa et al. (28) demonstrated that IL-6 treatment of cardiac myocytes significantly enhanced lipopolysaccharide (LPS) induction of iNOS expression, whereas the combination of IL-6 treatment with tumor necrosis factor-alpha also induced iNOS expression. Of note, however, transcriptional regulation of these interactions was exceedingly complex, involving not only NF-kappa B activation but also binding of complexes to an interferon-gamma regulatory factor-1 site and induction of a novel binding complex for the interferon-gamma activation site (28).

A substantial literature implicating inflammatory cytokines in the regulation of iNOS expression and NO production exists (28). However, NO modulation of cytokine expression has yet to be as thoroughly investigated. In the present study, we observed that ROFA-induced IL-6 expression was significantly reduced in cells coexposed to the highest concentration of the competitive NOS inhibitor L-NMMA, suggesting that increased NO production was necessary for IL-6 expression in RTE cells. Preliminary data with a fluorescent assay to measure nitrite production by RTE cells (38) indicated that relative to acid-saline-exposed cells, ROFA-exposed RTE cells (20 µg/cm2) produce increased amounts of NO (measured as NO-containing compounds and thus presumably nitrate) as early as 6 h postexposure (Dye, unpublished observations). Although these observations require more detailed study, Villarete and Remick (59) similarly reported that IL-6 and IL-8 production in LPS-stimulated human whole blood was significantly inhibited by coexposure with the NOS inhibitor NG-nitro-L-arginine methyl ester. Conversely, when the NO concentrations in blood were increased, IL-8 production increased in a dose-dependent manner. This treatment, however, had no effect on IL-6 production (59). The in vivo relevance of such observations is supported by the work of Hierholzer et al. (25) demonstrating that in mice undergoing hemorrhagic shock, lung and liver injuries were markedly reduced in mice treated with NOS inhibitors. The investigators concluded that 1) increased NO production was essential for the upregulation of the inflammatory response and 2) increased NO production contributed to the end-organ damage occurring in the resuscitated animals. In contrast to the observations above, in LPS-stimulated alveolar macrophages (41) and enterocytes (36), coexposure with NOS inhibitors increased IL-6 production, whereas treatment with NO donors decreased IL-6 production (36). Thus the influence of NO on cytokine production (or vice versa) appears to be cell specific.

As investigations continue to attempt to define whether the metal content of ambient air PM contributes significantly toward the health effects epidemiologically associated with particulate exposure in addition to characterizing PM pulmonary deposition, it will be important to characterize the pharmacokinetics of soluble versus insoluble (i.e., particle-bound) metal translocation throughout the lung in terms of dissolution and absorption, distribution and sequestration on metal-binding proteins, and ultimately elimination. Similarly, the pharmacodynamic consequences of metal exposure will need to be better defined on a cell-specific basis. As suggested by the difference between RTE cell injury induced during exposure to ROFA and that induced by the other oil fly ash samples containing little or no iron and nickel, complex interactions between the various metals contained in particulate material are likely to occur. The RTE cell culture system, however, provides a useful model with which to begin to dissect such interactions at the airway level. We are currently using the RTE epithelial model to track cellular versus extracellular location of individual metals after exposure in an attempt to establish temporal relationships between metal translocation and generation of ROS or reactive nitrogen species with the development of airway epithelial injury and inflammatory mediator production.

In summary, to date, our findings in cultured airway epithelial cells exposed to ROFA or its constituent transition metal vanadium further support the hypothesis that, at least for emission source PM, metal content and bioavailability play a role in the adverse respiratory effects associated with PM exposure. The cultured airway epithelial cells appear to be particularly sensitive to the vanadium present in certain oil fly ash samples in that all of the effects induced during ROFA exposure could be temporally reproduced, at least qualitatively, by exposure to vanadium in amounts comparable to those present in a ROFA suspension. Furthermore, the increases observed in inflammatory mediator production (i.e., cytokines and enzyme systems) after ROFA or vanadium exposure suggest that airway epithelial cells play a central role in the pulmonary inflammatory response to emission source PM. Moreover, vanadium appeared to be eliciting its cellular effects, at least in part, through the generation of an oxidative stress. Although this observation does not eliminate involvement of other vanadium-induced pathways (e.g., tyrosine phosphatase inhibition) in the overall pulmonary effects of vanadium, it does suggest that, at some level, these processes also involve cellular redox changes or generation of reactive species.


    ACKNOWLEDGEMENTS

We thank Dr. Urmila Kodavanti for her expertise and for kindly providing the assorted oil fly ash samples; Dr. L. G. Rochelle, R. H. Jaskot, and J. Keith for assistance and technical expertise; and Dr. D. L. Costa and Dr. I. Pagan for critical review of this manuscript.


    FOOTNOTES

The information described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policy of the agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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: J. A. Dye, US Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, ETD, PTB, MD-82, Research Triangle Park, NC 27711.

Received 6 January 1999; accepted in final form 14 April 1999.


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METHODS
RESULTS
DISCUSSION
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