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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Exposure of animals to airborne particulates is associated with pulmonary injury and inflammation. In the
studies described here, primary cultures of rat tracheal epithelial (RTE) cells were exposed to suspensions
of residual oil fly ash (ROFA). ROFA exposure resulted in progressive cytotoxicity whereby the amount of lactate dehydrogenase (LDH) released was significantly greater at 24 h than at 6 h after exposure. In a
dose-dependent manner, exposure to 5, 10, or 20 µg/cm2 of ROFA for 24 h resulted in cytotoxicity and detachment of cells from the collagen matrix, along with altered permeability of the RTE cell layer. ROFA
exposure caused cellular glutathione levels to decrease, producing a condition of oxidative stress in the RTE cells. Treatment of RTE cells with buthionine sulfoxamine, an inhibitor of 
-glutamyl cysteine synthetase, was found to augment ROFA-induced cytotoxicity. Treatment with dimethylthiourea (DMTU) inhibited ROFA-induced LDH release and permeability changes in a dose-dependent manner. Treatment
with the nitric oxide synthase inhibitor NG-monomethyl-D-arginine (D-NMA) for 24 h was without effect.
In rats intratracheally instilled with ROFA (500 µg/rat), intraperitoneal administration of DMTU (500 mg/
kg) significantly ameliorated the degree of pulmonary neutrophilic inflammation present at 24 h. Overall,
these in vitro findings suggest that ROFA-induced RTE cell injury may be mediated by hydroxyl-radical-like reactive oxygen species (i.e., species scavenged by DMTU) that are generated via non-nitric oxide
pathways. The delay in induction of maximal RTE cell injury may reflect the time necessary to produce an
oxidative burden by depleting antioxidant defenses such as cellular glutathione.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Epidemiologic studies have demonstrated positive associations between particulate air pollution and daily mortality (1, 2). Positive associations have also been reported between particulate air pollution and daily morbidity as measured by increased respiratory symptoms, exacerbations of asthma, decreased pulmonary function, and increased emergency room visits and hospitalizations related to respiratory (3) and cardiovascular disease (4). These observations are particularly disturbing because they suggest that adverse health effects associated with air particulate pollution exposure are occurring at levels below the current National Ambient Air Quality Standard for particulate matter. However, plausible biologic mechanisms for these epidemiologic associations have yet to be determined.
We investigated the biologic effects of an emission-source particle, residual oil fly ash (ROFA), because emission sources contribute to the overall ambient air particulate burden (5). As a model for differentiated airway
epithelial cells, primary cultures of rat tracheal epithelial
(RTE) cells were established under conditions that produce a pseudostratified mucociliary epithelium containing
secretory and ciliated cell types, not unlike tracheal epithelium in vivo (6). These cells were used as a target cell population to examine the acute toxicity of ROFA because epithelial cells are among the first cells to interact
with respirable particles and hence may be at greater risk
of injury. Additionally, airway epithelial cells may act as
effector cells that respond to certain stimuli by releasing
inflammatory mediators or altering the expression of cell
adhesion molecules
processes that can initiate airway inflammation or perpetuate preexisting inflammation (7).
Thus, studies examining the effects of ROFA on RTE cells
may have important physiologic consequences relevant to
the morbid respiratory effects identified in recent epidemiologic studies.
In our study, we examined for the first time the acute
toxicity of ROFA in primary cultures of airway epithelial
cells. We evaluated the effect of ROFA exposure on
changes in cellular glutathione, and assessed the protective
role of cellular glutathione by pretreating cells with an inhibitor of -glutamyl cysteine synthetase prior to ROFA
exposure. Further, we examined the potential role and pathways of ROFA-associated generation of reactive oxygen species. We also extended our in vitro findings to the
pulmonary system as a whole by examining the effect of in
vivo scavenging of reactive oxygen species on ROFA-induced pulmonary injury and inflammation in the rat.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Materials
Tissue-culture media and reagents were obtained from Sigma Chemical Co. (St. Louis, MO) with the following exceptions: insulin, transferrin, and amphotericin B were obtained from Gibco Laboratories (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-NMA) 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 the study 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 (8). Mount St. Helens (MSH) volcanic ash was collected for the U.S. Environmental Protection Agency from an open field near Ritzville, Washington, following the 1980 eruption of MSH (8).
Cell Culture
Primary cultures of RTE cells were established under conditions that produce pseudostratified mucociliary epithelium (6). Briefly, for these experiments, RTE cells were
obtained from male Sprague-Dawley rats (11 to 16 wk old;
Charles River Laboratories, Raleigh, NC) by overnight digestion with pronase at 4°C. 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 (FBS) and
3 mg/ml bovine serum albumin (BSA). After day 0, the
CM was composed of Dulbecco's modified Eagle's medium (DMEM)/ Ham's F12 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
(EGF; 25 ng/ml), 4-(2-hydroxyethyl)-1-piperazine-N'-2-ethanesulfonic acid (Hepes; 30 mM, pH 7.2), BSA (0.5 mg/
ml), phosphoethanolamine (50 µM), ethanolamine (80 µM),
penicillin-streptomycin (50 U/ml-50 µg/ml), 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). 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. Cultures were fed basally, once daily, for an
additional 6 days. Mature 13-day-old cultures were used in
all experiments.
Exposure of RTE Cultures
The ROFA suspensions were prepared in saline or in saline acidified to pH 3.0 with H2SO4 ("acid-saline"). Just prior to application of saline or particle suspensions, each culture was fed 2.5 ml of CM basally and the apical surface was washed twice with Hanks' balanced salt solution (HBSS) (1 ml/wash) to remove secretions, media, or nonadherent cells. Newly prepared particle suspensions were applied to RTE cells within 2 h of preparation. For the exposure, 0.5 ml of saline, acid-saline, an ROFA suspension, or an MSH ash suspension was applied to the apical (i.e., luminal) surface, in analogy to the in vivo situation. In one study, cells were pretreated for 18 h with buthionine sulfoxide (BSO; 500 µM dissolved in the CM). The BSO-containing medium was then removed and cells were given CM (without BSO) and then exposed to ROFA for 24 h. In another study, RTE cells were treated with BSO (50 or 150 µM in CM) for 18 h prior to as well as during the 24-h ROFA exposure. In additional studies, cells were treated with dimethylthiourea (DMTU; 4, 15, and 40 mM in the CM) for 30 min prior to and during the 24-h ROFA exposure. Cells also were treated with NG-monomethyl-L-arginine (L-NMA; 0.5 and 5.0 mM) or D-NMA (5.0 mM) for 30 min prior to and during the 24-h ROFA exposure.
Cellular Toxicity Assessments
Cultures were examined visually, using an inverted microscope, for overt changes in cellular adhesion and morphology. Cytotoxicity was quantified by measuring release of
lactate dehydrogenase (LDH), a stable cytosolic enzyme,
into the apical (A) compartment (which was done by adding an additional 0.5 ml of HBSS, swirling for 20 s, and removing the entire quantity of liquid present apically) or
into the basolateral (B) compartment of the Transwell
unit. LDH activity of the cells still attached to the collagen
membrane was measured following lysis with 0.5% Triton-X
100 in phosphate-buffered saline (PBS) (cell lysate; L).
LDH, total protein, albumin, and select enzymes involved
in the maintenance of cellular glutathione were measured
in the apical, basolateral, and lysate samples with a Cobas
Fara II centrifugal spectrophotometer (Hoffman-La Roche, Branchburg, NJ). The activities of LDH and -glutamyl-transferase (GGT; EC 2.3.2.2) were determined with commercially available kits from Sigma; total protein concentrations were determined with the Coomassie Plus Protein
Assay from Pierce Chemical Co. (Rockland, IL); albumin
concentrations were determined with the MALB SPQ kit
from Incstar Corporation (Stillwater, MN), using a standard curve prepared with BSA. The following assays were performed according to previously described methods
adapted for the centrifugal spectrophotometer: glucose-6-phosphate dehydrogenase (G-6-PDH) (9), glutathione reductase (10), and glutathione S-transferase (11). The total
percent of LDH and other enzymes released was calculated by the equation: [(A + B) / (A + B + L)] × 100%.
As an indirect means of assessing whether the enzyme activity (and possibly glutathione recycling ability) was reduced on a per-cell basis, the cellular lysate enzyme content for each culture was normalized to the protein
content of the lysed cells (i.e., mU/mg protein). In separate cultures, cells remaining attached to the collagen
membrane were lysed with perchloric acid (PCA) and analyzed for total cellular glutathione (GSHtot), using Anderson's 5,5'-dithio-bis-(2-nitrobenzoic) acid-oxidized glutathione (DTNB-GSSG) reductase recycling assay (12)
adapted for the centrifugal spectrophotometer. Again for
each culture, GSHtot was normalized to the lysate cellular protein (i.e., µg/mg 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 of the PCA-lysed sample.
In Vivo ROFA Exposures
Sixty five-day-old male Sprague-Dawley rats (Charles River Laboratories, Raleigh, NC) were briefly anesthetized with halothane, transorally intubated, and instilled intratracheally with a 0.3-ml bolus of either acid-saline (pH 3.3) or a suspension of ROFA (500 µg /0.3 ml, pH 3.3). Thirty minutes prior to instillation, saline or DMTU (500 mg /kg, pH 6.0) was administered intraperitoneally. Rats were returned to their cages after instillation and were provided with commercial rat food in pellet form and water ad libitum.
Twenty-four hours after instillation, rats were anesthetized with pentobarbital sodium (Abbott Laboratories,
Chicago, IL) and exsanguinated via the abdominal aorta.
Necropsies were performed to assess gross pathologic effects of ROFA or DMTU administration. Tracheas were
cannulated and the lungs lavaged with Mg2+/Ca2+-free
PBS at 28 ml /kg body weight, based on the animal's
weight just prior to instillation. The bronchoalveolar lavage fluid (BALF) white blood cell counts were determined
with a Coulter Counter (Coulter, Inc., Miami, FL). Cytologic preparations of the lavage fluid samples were made
with a cytocentrifuge (Shandon Cytospin 3; Shandon Instruments, Pittsburgh, PA). Cells were stained (LeuKoStat; Fisher Chemical Co., Pittsburgh, PA) and differential
white cell counts were determined on the basis of 
500 cells per specimen. Ratios of the number of red blood cells
to white blood cells were also determined. Ratios were
used to estimate the number of red blood cells/ml of
BALF. Following centrifugation of the BALF (850 × g for
10 min), the cell-free supernatant was analyzed for LDH, total protein, albumin, and total antioxidant activity. Total antioxidant activity assessments were done with previously described methods (13). In a separate group of rats
receiving intraperitoneal injections of saline or DMTU,
the abdominal cavities were lavaged (80 ml/kg body weight)
24 h later. Abdominal lavage fluid samples were analyzed
as described earlier.
Statistical Analysis
Data were analyzed with a t test for single comparisons or analysis of variance (ANOVA) with Scheffe's posttest correction for multiple comparisons. For all analyses, group differences were considered significant if the test statistical Type I error was less than 0.05 (i.e., P < 0.05) (14).
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Abstract
Introduction
Materials & Methods
Results
Discussion
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In Vitro Exposures
In a preliminary study, RTE cells were exposed apically to
either saline or ROFA particles suspended in saline, applied at 30 µg/cm2. After 6 h of exposure, microscopic
evaluation indicated that neither saline nor ROFA-exposed
cultures had undergone gross alterations in cellular morphology, adhesion, or cytotoxicity (based on 
5% LDH
release in either treatment group). However, after a 24-h exposure to ROFA, extensive injury was evident. In some
areas on the membrane, numerous detached individual
cells or small clumps of cells were observed, many of which
appeared excessively spherical and hyperlucent (i.e., prelytic swelling). In other areas, rafts of epithelial cells were
lifting completely off the collagen-coated membrane (i.e.,
epithelial desquamation). Pigmented agglomerations of
ROFA particles were visible, primarily adjacent to or
within the areas of lifting. Accordingly, after a 24-h exposure, the mean (± SEM) percent LDH released from saline-exposed cells was 2.1 ± 0.3%, whereas in ROFA-exposed cells it was significantly increased at 21.1 ± 3.6%
(n = 3/group). Most of the LDH was in the apical samples
(16.8 ± 3.9%), with lesser quantities present basally (4.2 ± 0.6%).
The ROFA suspension was found to be relatively acidic, at pH 3.8. To determine whether acidity contributed to ROFA-induced RTE injury, cells were exposed to saline acidified to a pH of 3.0 with H2SO4. Microscopic evaluation revealed no alterations in cellular morphology or adhesion after a 6- or 24-h exposure to acid-saline, and there was no difference in LDH release from saline- or acid-saline-treated cells after a 24-h exposure (Figure 1A).
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Significantly different from acid-saline-exposed cultures.
In subsequent studies of in vitro ROFA toxicity, doses of 5, 10, or 20 µg/cm2 were used, with LDH determinations made at 24 h after exposure. In these studies, as in the preliminary study, there were minimal morphologic alterations in ROFA-exposed cells by 6 h after exposure. However, after 24 h, microscopic evaluation revealed a dose-dependent increase in the number of hyperlucent adherent and nonadherent cells. LDH release increased correspondingly (Figure 1A). Similar patterns of release were observed for G-6-PDH, glutathione reductase, and glutathione S-transferase (Figure 1B). In addition, the lysate content (i.e., mU/mg protein) of the latter enzymes was significantly decreased at the higher ROFA exposure doses (data not shown), possibly indicating defective glutathione synthesis or recycling.
To assess whether ROFA-induced injury was related to particle exposure in conjunction with an acidic insult, RTE cultures were again exposed to acid-saline or ROFA (5, 10, or 20 µg/cm2), whereas other cultures were exposed to a relatively inert particle, MSH ash (5, 10, or 20 µg/cm2). All particle suspensions were prepared at a final pH of 3.0. Extensive injury occurred in ROFA-exposed cultures, but minimal cytotoxicity (as measured by LDH release) occurred in acid-saline- or MSH ash-exposed cultures after 24 h (Figure 2A). The release of GGT, an enzyme involved in transporting substrates into the cell for glutathione synthesis (15), was also evaluated in these studies. There was a significant overall treatment effect (P = 0.003) on release of GGT; however, the release from ROFA-exposed cells was not statistically different from that from acid-saline-exposed cells, owing in part to large quantities of GGT in the apical samples from control cells (Figure 2A). Essentially, no GGT was detected in the basolateral compartment.
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Because RTE cells are exclusively fed basally after reaching confluence, we used retention of BSA within the basolateral compartment as an index of solute permeability of the cultured RTE cell layer. As seen in Figure 2B, when 2.5 ml of CM (containing 500 µg/ml BSA) was placed in the basolateral compartment of a collagen gel-coated membrane (without RTE cells), hydrostatic pressure displaced 158 ± 10 µg of albumin onto the apical surface over a 24 h period. This was equivalent to approximately 320 µl of CM "leaking" onto the apical surface. In saline-exposed RTE cultures, however, only 33 ± 18 µg of albumin was detected apically after 24 h, which was equivalent to approximately 70 µl of CM. Thus, under control conditions, the cultured RTE layer was relatively impervious to this degree of hydrostatic pressure.
After a 24-h exposure to MSH ash, the amount of albumin present in the apical compartment was not significantly different than in the acid-saline-exposed cultures.
However, after a 24-h exposure to ROFA at all doses, the
amount of albumin present apically was significantly increased, reaching a plateau near that of the collagen-coated membrane (
RTE cells) (Figure 2B). In related studies, changes in albumin retention were already developing by 20 h. After exposure to 5, 10, or 20 µg/cm2 of
ROFA for 20 h, apical albumin quantities increased significantly to 120 ± 6, 170 ± 4, and 170 ± 13 µg, respectively,
as compared with only 9.7 ± 3.7 µg in acid-saline-exposed
cultures. No increases in apical albumin quantities were
detected after a 6-h exposure to 5 µg/cm2 of ROFA.
Because ROFA exposure was associated with increased release of several enzymes involved in glutathione recycling, we investigated the effect of ROFA exposure on total cellular glutathione. Initially, RTE cells were exposed to 5, 10, or 20 µg/cm2 of ROFA for 24 h, after which we found that GSHtot concentrations in the adherent cells were depleted below detectable limits. In a subsequent study, cells were assessed after a 20-h exposure. In acid-saline-exposed cells, GSHtot was 5.7 ± 0.2 µg/mg, whereas it was significantly decreased to 2.4 ± 0.4, 2.0 ± 0.1, and 1.8 ± 0.2 µg/mg, respectively (n = 3/group) in cells exposed to 5, 10, or 20 µg/cm2 of ROFA. In a third experiment, after an 18-h exposure, the GSHtot of acid-saline- exposed cells was 8.4 ± 0.5 µg/mg, whereas it was significantly decreased to 3.2 ± 0.3 µg/mg (n = 3/group) in cells exposed to 10 µg/cm2 of ROFA. Even greater glutathione depletion may have occurred in cells that had already detached or lysed.
These results indicated that ROFA exposure was associated with an oxidative stress on the RTE cells through
depletion of cellular glutathione concentrations. In order
to substantiate this finding, RTE cells were pretreated
with BSO, an irreversible inhibitor of -glutamyl cysteine
synthetase (16), the rate-limiting enzyme involved in glutathione synthesis (17). Initially, cells were pretreated with
500 µM BSO for 18 h, after which we confirmed that GSHtot
decreased to ~ 10% of that in control cultures without affecting LDH release. After this pretreatment period,
BSO-containing medium was removed and cells were exposed to 5 µg/cm2 ROFA for 24 h. LDH release was significantly increased in ROFA (+ BSO)-exposed cells (i.e.,
44.4 ± 2.0%) as compared with ROFA ( BSO)-exposed
cells (i.e., 17.0 ± 0.8%) (n = 6/group). In the acid-saline-exposed cells, glutathione levels recovered when BSO was
removed from the medium during the 24-h exposure period, possibly owing to de novo synthesis of -glutamyl
cysteine synthetase. Therefore, in subsequent studies, cells
were pretreated with BSO (50 or 150 µM in CM) for 18 h
prior to as well as during the 24-h exposure. As in the earlier case, cells exposed to ROFA (+ BSO) had significantly
greater LDH release than ROFA ( BSO)-exposed cells
(Figure 3). These results are consistent with a protective effect of glutathione against the toxicity of ROFA, as well
as with the ability of ROFA to induce an oxidative burden
on RTE cells.
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To obtain further evidence that an oxidative burden produced by the generation of reactive oxygen species may be involved in ROFA-induced RTE cell injury, cells were coexposed to ROFA at 10 µg/cm2 and the potent reactive oxygen metabolite scavenger DMTU at 4, 15, or 40 mM concentrations in CM for 24 h. In a dose-dependent manner, coexposure with DMTU significantly inhibited ROFA-induced LDH release (Figure 4A) and changes in albumin retention (Figure 4B). Comparable inhibition occurred in a replicate of this experiment (Figure 4C).
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These results suggested that reactive oxygen species, possibly including hydroxyl radicals, were critical mediators of ROFA-induced RTE cell injury. Hydroxyl radicals, however, can be generated via Fenton pathways or via nitric oxide pathways (18). Therefore, cells were exposed to L-NMA, a competitive inhibitor of nitric oxide synthase (NOS), to determine whether hydroxyl-radical generation by ROFA was produced via nitric oxide pathways. Coexposure to relatively high concentrations of L-NMA had no effect on LDH release or altered albumin retention in cells exposed to ROFA at 10 µg/cm2 for 24 h (e.g., LDH release in ROFA exposed cells [without L-NMA] was 45.0 ± 3.8%, whereas in cells exposed to 5.0 mM D-NMA, 0.5 mM L-NMA, or 5.0 mM L-NMA, it was 42.9 ± 1.6%, 43.5 ± 0.6%, and 41.6 ± 1.5%, respectively).
In Vivo Exposures
In an effort to determine the role of reactive oxygen species in the in vivo toxicity of ROFA, rats were given an intraperitoneal injection of saline or DMTU, followed 30 min later by intratracheal instillation of either acid-saline or a suspension of ROFA (500 µg total). As shown in Table 1, at 24 h after exposure, rats exposed to intratracheally administered ROFA and intraperitoneal saline developed acute pulmonary injury characterized by significant increases in BALF concentrations of LDH, total protein, and albumin. ROFA exposure also induced mild pulmonary hemorrhage, along with a neutrophilic, and to a lesser degree, an eosinophilic, inflammatory response. Systemic administration of DMTU alone had no effect on the BALF indices, except for increased antioxidant activity. Interestingly, at 24 h after exposure, DMTU administration reduced the number of neutrophils present in BALF by approximately 78%, whereas it appeared to increase the degree of pulmonary hemorrhage. DMTU administration also was associated with a 5 to 6% loss in body weight. Upon necropsy examination, the stomachs of these rats were overly distended with gas and excessive amounts of ingesta. In a separate group of rats receiving intraperitoneal injections of DMTU, abdominal lavage fluid analysis 24 h later revealed that total antioxidant activity was increased (1.5 mM, compared with 0.2 mM in saline-treated rats). Albumin concentrations were also increased, although LDH and total protein concentrations were not significantly different from those in rats given intraperitoneal saline. Qualitative assessment of cytologic preparations indicated that comparable numbers of white cells were present in lavage samples from both groups. These cells included numerous macrophages and eosinophils, moderate numbers of mast cells, and occasional neutrophils.
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Abstract
Introduction
Materials & Methods
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Discussion
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Previous studies of the in vivo effects of ROFA in the rat demonstrated that ROFA exposure resulted in acute pulmonary injury and inflammation (including an eosinophilic inflammatory response) (19, 20), increased airway responsiveness (21, 22), and pulmonary fibrosis (23). These effects are particularly relevant in light of the positive epidemiologic associations between particulate air pollution exposure and decreased pulmonary function and exacerbations of asthma (3). Furthermore, it has been suggested that one of the mechanisms of ROFA-induced in vivo injury may involve the development of an oxidative burden, owing in part to the high transition-metal content of ROFA (19). In the present study, we therefore employed in vitro methodologies to better examine the role of oxidative stress in ROFA-induced airway epithelial injury.
Exposure of RTE cells to ROFA resulted in a time- and dose-dependent cytotoxic response that included epithelial-cell detachment and lytic cell injury. We found a > 6-h delay between initial particle exposure and the development of significant injury, with maximal cytotoxic effects occurring 20 to 24 h after ROFA application. ROFA exposure also resulted in alterations in the permeability of the cultured RTE cell layer. Normally in the trachea, epithelial cells are joined by tight junctions that form a continuous barrier to solute movement (24). The integrity of analogous cell-cell junctions in cultured RTE cells was seemingly impaired by exposure to ROFA.
In rats, an earlier time-course study determined that after intratracheal instillation of 2.5 mg of ROFA, BALF neutrophilic influx peaked at 24 h, whereas albumin concentrations increased significantly at 6 h, after which they decreased slightly and then increased again at 48 h (20). Changes in BALF total protein and LDH concentrations followed a similar temporal pattern. We used a reduced ROFA exposure in our in vivo studies. By 24 h after exposure, instillation of 500 µg of ROFA also resulted in pulmonary injury and inflammation. Because surface-area estimates of the trachea and pulmonary airways in the rat range from approximately 25 cm2 (25) to 100 cm2 (26), the in vivo exposures in our rats would, if most of the instilled suspension was deposited onto and remained in contact with the airway surfaces, be "comparable" to the 20 or 5 µg/cm2 RTE cell exposures, respectively. It is likely, however, that in the rodent airways, particle residence time is relatively transient, owing to removal of the instillate by absorptive, phagocytic, and mucociliary clearance processes. One must also recognize that ROFA-induced pulmonary injury in vivo is a culmination of the direct toxic effects of ROFA along with the effects of the ensuing inflammatory response, and that changes in lung permeability are predominantly due to alveolar epithelial and endothelial effects. Still, in RTE cell cultures without the superimposed effects of airway inflammation, maximal cytotoxic effects did not occur until 20 to 24 h after ROFA application. The lag between initial ROFA exposure and the development of maximal injury in these in vitro and in vivo studies was not unlike that reported during the 1952 London air pollution disaster, when high particulate-based smog increased extremely rapidly. The rapid increase in air pollution was followed closely, with a lag of about 1 day or less, by a significant increase in the daily death rate (27).
The complete abolition of ROFA-induced injury and permeability changes produced by coexposure to DMTU provides strong evidence that generation of an oxidative stress was critical to ROFA-induced RTE cytotoxicity. It was previously shown that in a noncellular suspension, ROFA particles increased the absorbance of thiobarbituric acid-reactive products of deoxyribose, which is indicative of spontaneous oxidant generation by ROFA, and that DMTU was capable of inhibiting this effect (22). DMTU, originally thought to be a specific hydroxyl-radical scavenger (28), is actually a small, highly diffusible molecule that effectively scavenges a variety of reactive species including hydroxyl radical, H2O2, hypochlorous acid (29, 30), and possibly superoxide anion (31), although it has also been reported that thioureas do not react with superoxide (32). In these exposures, DMTU may have directly "scavenged" the hydroxyl-radical-like compounds produced, or alternatively, DMTU may have reacted primarily with H2O2 which, owing to the presence of transition metals associated with ROFA exposure, in effect limited subsequent hydroxyl-radical generation via Fenton pathways. It is therefore likely that ROFA-induced RTE cell injury resulted from the interaction of all the reactive oxygen molecules generated. Individually, however, H2O2 and superoxide radical are considered relatively weak oxidants that can be neutralized by the cellular antioxidants catalase and superoxide dismutase (SOD), respectively. Bronchial epithelial cells in primary culture have demonstrable catalase as well as MnSOD and CuZnSOD antioxidative capacity (33). Hydroxyl radical, on the other hand, is a highly cytotoxic reactive oxygen species that may initiate lipid peroxidation and other processes that lead to cell injury. Furthermore, hydroxyl radical has been previously implicated in the development of pulmonary disease associated with silica and asbestos exposure in both in vitro (34, 35) and in vivo studies (36, 37).
We therefore wanted to better characterize the potential source of hydroxyl radicals because they can be generated via classic Fenton pathways or via nitric oxide pathways (18). Under pathologic conditions, nitric oxide may be generated in large quantities by induced forms of NOS. Nitric oxide can react rapidly with superoxide to form a powerful oxidant, peroxynitrite. At a much slower rate, peroxynitrite spontaneously decomposes to form hydroxyl radicals (38) or dissociation products that closely mimic the effects of hydroxyl radical. Therefore, we inhibited nitric oxide formation in RTE cells by incubation with a nonmetabolizable L-arginine analogue, L-NMA. As expected with a competitive enzyme inhibitor, L-NMA alone had no effect on cytotoxicity, even at the high concentrations that were used. However, after 24 h, coexposure to L-NMA neither protected against nor potentiated ROFA-induced injury. We are relatively certain that at the highest concentration of L-NMA (i.e., 5 mM), the arginine present in CM did not interfere with NOS inhibition. Therefore, on the basis of these in vitro studies, nitric oxide-associated generation of hydroxyl radical via peroxynitrite does not appear to be the predominating pathway involved in ROFA-induced injury.
We pursued our in vitro findings with DMTU with limited in vivo studies in the rat. Previous studies with rats had shown that DMTU administration (250 mg/kg intraperitoneally) 30 min before reperfusion significantly improved contractile function in ischemic skeletal muscle (39), and that DMTU at 1,000 mg/kg intraperitoneally, given 10 min before induction of thermal skin trauma, significantly reduced the development of skin edema (40). In our study, systemic administration of DMTU impeded development of the pulmonary inflammatory response to ROFA, but did not ameliorate biochemical alterations in BALF. One possible explanation for this is that 30 min was not enough time to achieve effective DMTU concentrations in the airways, and that immediately after ROFA instillation there was incomplete scavenging of reactive oxygen species. However, the pulmonary inflammatory response required more time to develop, during which DMTU distribution to the airways may have been more complete. This possibility is supported by the increased total antioxidant activity in BALF at 24 h. Although not specific for DMTU, this assay reflects the overall antioxidant capacity of bodily fluids (13). Intraperitoneal administration of DMTU appeared to delay gastric emptying, resulting in decreased food and water ingestion and thus in mild dehydration and weight loss; however, it did not appear to be associated with a chemically induced peritonitis. Consequently, it is unlikely that the decrease in neutrophil chemotaxis to the lungs following ROFA instillation was simply due to the development of an inflammatory focus at a distant site.
We therefore believe that in these rats, oxidative stress
associated with ROFA exposure also played a role in initiating the pulmonary inflammatory response to ROFA,
and that systemic administration of DMTU resulted either
in scavenging of or in diminished production of key reactive oxygen species. This in turn ameliorated cellular redox changes and thus diminished the "effector cell" response to ROFA (i.e., decreased cytokine production by
airway epithelial cells, alveolar macrophages, or lymphocytes). It has been shown that the intracellular redox state
of the cell modulates the activity of several transcription
factors, including nuclear factor-
B (NF-B) (41), a critical step in the induction of a variety of proinflammatory
cytokine (42) and adhesion-molecule genes (43). Because
airway epithelial cells are in a key position to influence the
development of pulmonary inflammation after exposure to particulate air pollutants, we are currently using RTE
cells to elucidate the role of airway epithelial cells in initiating the inflammatory response to ROFA, and specifically to determine whether this response is related to the
development of an oxidative stress, focusing on Fenton
and/or Fenton-like (transition-metal-catalyzed) pathways
for the generation of reactive oxygen species.
Importantly, systemic DMTU administration also potentiated ROFA-induced pulmonary hemorrhage. Despite its protective effects and apparent lack of toxicity in a range of oxidant-related insults (28, 39, 40, 44, 45), DMTU administration has been previously shown to enhance pulmonary injury and mortality in rats exposed to the herbicide paraquat (46), through interactions at the alveolar level (47). Thus, in our study, DMTU treatment appeared to be both protective and deleterious, depending on the type of pulmonary cell involved. DMTU ameliorated ROFA-induced pulmonary inflammation, whereas at the alveolar level it potentiated ROFA-induced hemorrhage.
In summary, one important mechanism of ROFA-induced RTE cytotoxicity involves the development of an oxidative burden. The oxidative stress most likely involves production of hydroxyl radicals and H2O2 (± superoxide radicals), which in our study appeared to be generated via non-nitric oxide pathways. Experiments with BSO confirmed that cellular glutathione was involved in protecting RTE cells from ROFA-induced cytotoxicity. Other investigators have shown that early, sublethal oxidant-mediated renal epithelial cell injury involved the depletion of cellular adenosine triphosphate and increased release of [3H]adenine metabolites, whereas late effects (e.g., cell detachment and lytic injury) did not occur until 3 to 4 h after exposure to the oxidant stress (48). In our studies with RTE cells, there was an additional delay prior to the development of significant cytotoxicity, possibly owing to the time necessary to generate sufficient quantities of reactive oxygen species. Alternatively, if production of reactive metabolites was relatively constant and ongoing, the delay may have been due to the time required to significantly diminish RTE antioxidant defenses, such as cellular glutathione.
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Footnotes |
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Address correspondence to: Dr. Janice A. Dye, U.S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, ETD, PTB, MD-82, Research Triangle Park, NC 27711.
(Received in original form August 20, 1996 and in revised form February 10, 1997).
The information described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and has been 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.Acknowledgments: The authors wish to thank Dr. Scott Randell, Veronica Godfrey, and Nancy Akley for their expertise and assistance with the cell culture methodologies, and Drs. Sarah Gardner and Urmila Kodavanti for critical review of the manuscript.
Abbreviations BALF, bronchoalveolar lavage fluid; BSO, buthionine sulfoxamine; CM, complete medium; DMTU, dimethylthiourea; D-NMA, NG-monomethyl-D-arginine; EGF, epidermal growth factor; GGT, gamma-glutamyl transpeptidase; G-6-PDH, glucose-6-phosphate dehydrogenase; GSHtot, total cellular glutathione; LDH, lactate dehydrogenase; L-NMA, NG-monomethyl-L-arginine; MSH, Mount St. Helens; NOS, nitric oxide synthase; PCA, perchloric acid; ONOO·, peroxynitrite; RTE, rat tracheal epithelial; ROFA, residual oil fly ash; SOD, superoxide dismutase.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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