Differential Regulation of the Lung Endothelin System by Urban Particulate Matter and Ozone

Errol Thomson*,{ddagger}, Prem Kumarathasan*, Patrick Goegan*, Rémy A. Aubin{dagger},{ddagger} and Renaud Vincent*,{ddagger},1

* Healthy Environments and Consumer Safety Branch, and {dagger} Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada, K1A 0K9, and {ddagger} Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada, K1N 6N5

1 To whom correspondence should be addressed at Inhalation Toxicology and Aerobiology Section, Health Canada, 0803C Tunney's Pasture, Ottawa, Ontario, K1A 0K9, Canada. Fax: (613) 946-2600. E-mail: Renaud_Vincent{at}hc-sc.gc.ca.

Received May 13, 2005; accepted July 28, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Periodic elevation of ambient particulate matter and ozone levels is linked to acute cardiac morbidity and mortality. Increased plasma levels of the potent vasoconstrictor endothelin (ET)-1, a prognostic indicator of cardiac mortality, have been detected in both animal models and humans after exposure to air pollutants. The lungs are the primary source of circulating ET-1, but the direct effects of individual air pollutants and their interaction in modulating the pulmonary endothelin system are unknown. Fischer-344 rats were exposed to particles (0, 5, 50 mg/m3 EHC-93), ozone (0, 0.4, 0.8 ppm), or combinations of particles and ozone for 4 h. Changes in gene expression were measured using real-time reverse transcription polymerase chain reaction immediately after exposure and following 24 h recovery in clean air. Both pollutants individually increased preproET-1, endothelin converting enzyme-1, and endothelial nitric oxide synthase mRNA levels in the lungs shortly after exposure, consistent with the concomitant increase in plasma of the 21 amino acid ET-1[1-21] peptide measured by HPLC-fluorescence. PreproET-1 mRNA remained elevated 24 h after exposure to particles but not after ozone, in line with previously documented changes of the peptide in plasma. Both pollutants transiently increased endothelin-B receptor mRNA expression, while ozone decreased endothelin-A receptor mRNA levels. Coexposure to particles plus ozone increased lung preproET-1 mRNA but not plasma ET-1[1-21], suggesting alternative processing or degradation of endothelins. This coincided with an increase in the lungs of matrix metalloproteinase-2 (MMP-2), an enzyme that cleaves bigET-1 to ET-1[1-32]. Taken together, our data indicate that ozone and particulate matter independently regulate the expression of lung endothelin system genes, but show complex toxicological interaction with respect to plasma ET-1.

Key Words: lung; pollution; particles; ozone; endothelin; cardiovascular.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidemiological evidence indicates that particulate and gaseous air pollutants are linked to higher rates of cardiovascular morbidity and mortality (Burnett et al., 2000Go; Goldberg et al., 2001aGo,bGo; Pope et al., 2004Go). Cardiac effects (e.g., decreased heart rate variability, increased myocardial infarction) have been reported within a few hours of increases of ambient ozone or respirable particulate matter (Gold et al., 2000Go; Peters et al., 2001Go), indicating that processes responsible for health effects are rapid. Individuals with chronic artery diseases and congestive heart failure are at higher risk of dying after an air pollution episode (Goldberg et al., 2001aGo). Such findings have led to the suggestion that air pollutants may exert their effects through perturbations of vascular homeostasis (Bouthillier et al., 1998Go; Vincent et al., 2001Go). In line with this hypothesis, inhalation exposure of healthy adults to ambient fine particulate matter and ozone resulted in arterial vasoconstriction within 2 h of exposure (Brook et al., 2002Go), consistent with the dynamics of acute health effects indicated by epidemiological studies. Experimental evidence of an elevation of circulating levels of the vasoconstrictor peptide endothelin (ET)-1 by air pollution provides a biologically plausible explanation for such effects (Bouthillier et al., 1998Go; Kang et al., 2002Go; Thomson et al., 2004Go; Ulrich et al., 2002Go; Vincent et al., 2001Go), and field work has confirmed the association between urban pollution and elevated plasma ET-1 in humans (Calderon-Garciduenas et al., 2003Go). However, the mechanisms governing the elevation of plasma endothelin and the respective contribution of ozone and particulate matter are not clear.

Endothelin-1 is a potent vasoconstrictor peptide involved in the homeostatic control of vascular smooth muscle tone (Haynes et al., 1995Go). Circulating and tissue ET-1 levels are elevated in many cardiovascular diseases, including atherosclerosis, congestive heart failure, and hypertension (Luscher and Barton, 2000Go). The precursor preproET-1 peptide is processed by endoproteases to yield bigET-1, which is cleaved by endothelin-converting-enzymes (ECEs) to produce the mature vasoactive 21-amino acid ET-1[1-21]. Endothelin-1 acts through specific G-protein coupled receptors, the ETA-receptor and ETB-receptor, and is cleared from circulation through the latter (Bremnes et al., 2000Go) and in tissue through degradation by neutral endopeptidases (D'Orléans-Juste et al., 2003Go). Big ET-1 and mature ET-1[1-21] produced by endothelial cells are primarily secreted basolaterally into the interstitium toward smooth muscle cells, and circulating levels of the peptides reflect luminal spill-over from basolateral secretion. PreproET-1 mRNA has a half-life of approximately 15 min (Inoue et al., 1989Go), and bigET-1 and ET-1 have half-lives in the blood of rats of 4 min and less than 1 min, respectively (Burkhardt et al., 2000Go). Consequently, increased steady-state levels of the peptides in plasma represent a sustained increase of de novo synthesis, a reduced clearance from circulation, or both. While ECE-dependent processing of bigET-1 is considered the dominant pathway in the endothelium, bigET-1 can be cleaved through a number of alternate pathways, such as by chymase to form the peptide ET-1[1-31], which is itself a substrate for ECEs (D'Orléans-Juste et al., 2003Go), and matrix metalloproteinase-2 (MMP-2) to form the vasoactive ET-1[1-32] peptide (Fernandez-Patron et al., 1999Go). This alternate processing pathway may be notably significant in tissue injury (Fernandez-Patron et al., 2001Go).

We have reported that inhaled urban particles, while not directly injurious to normal lungs (Adamson et al., 1999Go; Vincent et al., 1997aGo), nevertheless increased the circulating levels of ET-1[1-21] (Bouthillier et al., 1998Go; Vincent et al., 2001Go). Measurements in Wistar rats after inhalation of urban particles showed progressive increases of plasma ET-1[1-21] and blood pressure with maximal values at 36 h post-exposure (Vincent et al., 2001Go). In Fischer-344 rats, plasma ET-1[1-21] was elevated 24 h after inhalation of urban particles alone and after exposure to urban particles plus ozone, but not after ozone alone (Bouthillier et al., 1998Go). However, lung preproET-1 mRNA levels were elevated as early as 2 h after coexposure of Fischer-344 rats to urban particulate matter and ozone (Thomson et al., 2004Go), suggesting that the peptide might be up-regulated at an earlier time.

By factoring doses of both particulate matter and ozone, we undertook here to clarify the early effects of the individual pollutants, as well as their toxicological interaction vis-à-vis regulation of the pulmonary endothelin system genes in the lungs of Fischer-344 rats. Real-time RT-PCR was used to evaluate and quantify subtle changes in the gene expression of preproET-1, ECE-1, the endothelin receptors ETA and ETB, and the endothelial (eNOS) and inducible (iNOS) nitric oxide synthases immediately after inhalation exposure to the pollutants and following a 24-h recovery in clean air. The changes in gene expression were then contrasted with plasma ET-1[1-21] and bigET-1 levels, measured by high-performance liquid chromatography (HPLC) with native fluorescence detection. We show that particulate matter and ozone independently regulate lung endothelin system genes and interact toxicologically with respect to their impact on circulating ET-1[1-21].


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Specific pathogen-free Fischer-344 male rats (200–250 g) were obtained from Charles River (St. Constant, QC, Canada). The animals were housed in individual Plexiglass cages on wood-chip bedding under HEPA-filtered air and held to a 12-h dark/light cycle. Food and water were provided ad libitum. All experimental protocols were reviewed and approved by the Animal Care Committee of Health Canada. Distribution of the animals in experimental groups is summarized in Table 1.


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TABLE 1 Distribution of animals for gene expression analyses (subset number of animals for peptide analyses in parentheses)

 
Inhalation exposure to air pollutants.
The ambient urban particles EHC-93 consist of total suspended particulate matter recovered from filters of the single-pass air-purification system at the Environmental Health Centre (Tunney's Pasture, Ottawa, ON, Canada) and mechanically sieved using a 36-µm mesh filter. The chemical composition, biological reactivity of the particles in cell culture models, and applications in inhalation studies have been described elsewhere (Bouthillier et al., 1998Go; Vincent et al., 1997aGo,bGo, 2001Go). Animals were exposed to EHC-93 urban particles, ozone, or the combined pollutants using a nose-only inhalation system. Rats were trained in nose-only exposure tubes over five consecutive days, and then exposed for 4 h to clean air or to combinations of the individual pollutants EHC-93 (0, 5, 50 mg/m3) and ozone (0, 0.4, 0.8 ppm) essentially as described previously (Thomson et al., 2004Go; Vincent et al., 1997aGo). The particle size distribution of resuspended EHC-93 in our flow-past nose-only exposure system was multimodal, with two respirable modes at 1.3 µm aerodynamic diameter (DAE) and 3.6 µm DAE that together comprised 55% of the mass of the aerosol, and a nonrespirable mode at 15 µm DAE that comprised 45% of the mass (Vincent et al., 2001Go). Animals were euthanized immediately after exposure, or following 24 h recovery in filtered air.

Environmental relevance of dose regimen.
Dosimetric relevance of the present experiment to an environmental exposure should be evaluated after scaling doses of pollutants within the lungs of rats and humans. To estimate deposition of particles in human lungs under an actual environmental exposure scenario, we have taken as model assumptions an average tidal volume of 875 ml and an average breathing frequency of 16 min–1 over the entire day (20.2 m3 air inhaled/d), oronasal breathing, and an alveolar surface area of 54 m2. Deposition rates for the 0.05–10 µm DAE size range of urban particulate matter with size cut-off of 10 µm DAE (PM10) containing a nucleation mode at 0.05 µm DAE (5% of mass), a condensation mode at 0.2 µm DAE (25% of mass) and coarse mode at 5 µm DAE (70% mass) were taken as 0.20 for all three modes (Schlesinger, 1989Go). Using these parameters, and assuming a 24-h exposure to an average PM10 concentration of 175 µg/m3 (Tellez-Rojo et al., 2000Go), a reference total dose in the pulmonary compartment of humans was estimated as 707 µg (175 µg/m3 x 20.2 m3 x 0.20), or 1.3 ng/cm2 alveolar surface area. Similarly, the peak centriacinar dose of ozone in the lungs of humans can be taken as 30 x 10–6 µg O3/cm2/h per µg ambient O3/m3 (Miller et al., 1988Go). Thus, exposure of a human subject to 0.12 ppm ozone (236 µg O3/m3) for 12 h (85 ng O3/cm2), followed by 0.06 ppm ozone for 12 h (42 ng O3/cm2) would lead to a total daily centriacinar peak dose estimated at 127 ng O3/cm2 (Vincent et al., 1997aGo).

Model assumptions for rats were a tidal volume of 2.1 ml, a breathing frequency of 102 min–1 (51.4 l air inhaled/4 h exposure), strict nasal breathing, and an alveolar surface area of 0.34 m2. Modeled deposition rates using the Multiple Path Particle Deposition software (MPPDep v1.11, RIVM Publications, Bilthoven, The Netherlands) were estimated at 0.081 for the 1.3 µm DAE mode (20% of aerosol mass), 0.047 for the 3.6 µm DAE mode (35% of aerosol mass), and 0.000 for the 15 µm DAE mode (45% of aerosol mass). Using these parameters, the pulmonary compartment dose of EHC-93 particles in the rats was estimated at 8.4 µg (5 µg/l x 51.4 l x {[0.20 x 0.081] + [0.35 x 0.047] + [0.45 x 0.000]}) or 2.5 ng/cm2 alveolar surface area, and 84 µg or 25 ng/cm2 alveolar surface area at the 5 mg/m3 and 50 mg/m3 exposure concentrations, respectively. Similarly, the peak centriacinar dose of ozone in the lungs of rats is taken as 68 x 10–6 µg O3/cm2/h per µg ambient O3/m3 (Miller et al., 1988Go). Exposure of our rats to 0.4 ppm (785 µg of O3/m3) or 0.8 ppm ozone (1570 µg of O3/m3) over 4 h should have translated into a total centriacinar peak dose of 214 ng O3/cm2 and 427 ng O3/cm2, respectively.

The ratio of an experimental particle EHC-93 dose within the respiratory compartment of the rats during the 5 mg/m3 exposure (2.5 ng/cm2) and 50 mg/m3 exposure (25 ng/cm2) to the particle dose calculated for a plausible human exposure scenario (1.3 ng/cm2) is 2-fold and 20-fold, respectively. The ratio of the centriacinar ozone dose in our animals at 0.4 ppm O3 (214 ng O3/cm2) and 0.8 ppm O3 (427 ng O3/cm2) to the estimated internal dose in a human subject under a plausible exposure scenario (127 ng O3/cm2) is only 1.7-fold and 3.4-fold, respectively. For ethical reasons, nose-only exposures should be kept to a minimum duration, and therefore the dose-rate in our study was obviously higher than for an environmental exposure spread over a 24-h period. Nevertheless, from the standpoint of evaluation toxicology, the pulmonary depositions of the pollutants in the current study are directly relevant to the human experience, including the experimental dose estimated for the high particle exposure concentration once a number of reasonable uncertainty factors are considered. These include the possible decay of the potency of EHC-93 by comparison to fresh particles, the known interspecies differences in sensitivity to air pollutants (with humans being more responsive than rats), and the heightened sensitivity within a subset of the human population, such as the known increased adverse risk of individuals with congestive heart failure or atherosclerosis (Goldberg et al., 2001aGo).

Biological samples.
Rats were anaesthetized by administration of sodium pentobarbital (60 mg/kg, ip). Blood was collected from the abdominal aorta into Vacutainer tubes containing the sodium salt of ethylene diamine tetra acetic acid (EDTA) at 10 mg/ml and phenyl methyl sulfonyl fluoride (PMSF) at 1.7 mg/ml, mixed gently, and placed on ice (Kumarathasan et al., 2001Go). Plasma was isolated by centrifugation (2000 rpm for 10 min), aliquoted, and frozen at –80°C. The lungs were washed by bronchoalveolar lavage with warm saline (37°C) at 30 ml/kg body weight, then flash frozen in liquid nitrogen and stored at –80°C. The bronchoalveolar lavage fluid (BALF) was centrifuged (1500 rpm for 10 min at 4°C) to remove cells and frozen at –80°C.

Reverse transcription of lung RNA samples.
Frozen lung samples were homogenized in TRIzol reagent (Invitrogen Canada Inc., Burlington, ON, Canada), and total RNA was isolated according to the manufacturer's instructions. RNA was quantified using the RiboGreen RNA Quantitation Reagent and Kit (Molecular Probes, Eugene, OR), and quality was verified by electrophoresis on a formaldehyde-agarose gel. Total RNA was reverse transcribed using MuLV reverse transcriptase and random hexamers (Applied Biosystems, Mississauga, ON, Canada) according to the manufacturer's instructions. Briefly, 250 ng RNA was added to a reaction mixture of 5 mM MgCl2, 1x PCR Buffer II, 1 mM each dNTP, 1 U/µl RNase Inhibitor, 1 µM random hexamers, and water to produce a final volume of 50 µl. The mixture was incubated at 42°C for 1 h, MuLV reverse transcriptase was inactivated by heating to 99°C for 5 min, and the reaction was cooled to 5°C for 5 min followed by storage at –40°C until used.

Real-time PCR primers.
Primers for endothelin system genes (ET-1, ECE-1, ETA and ETB receptor), eNOS, and a reference gene (ß-actin) were designed using Vector NTI software (InforMax, Frederick, MD). The primer sequences for iNOS were from Ulrich et al. (2002)Go. Primers were designed to have 50 to 60% GC content, an optimal annealing temperature of 60–62°C, and yield PCR products 75–150 bp in length (Table 2). Primers and predicted amplicons were evaluated for any secondary structure that might inhibit primer annealing using m-fold software available online (http://www.bioinfo.rpi.edu/applications/mfold/old/dna/). Double-desalted primers were purchased from Invitrogen. High PCR reaction efficiency was verified and compared for all primer sets using a dilution series of rat cDNA. The ß-actin primer set was found to participate in high-efficiency reactions at both 60°C and 62°C. All other primer sets were validated at either 60°C or 62°C. Reaction products run on 1% agarose gels confirmed a unique band of the expected size for each amplicon. The identities of all amplicons were confirmed by TA cloning (Invitrogen) followed by automated fluorescence sequencing (3100 Genetic Analyser; Applied Biosystems Inc.) and sequence alignment against available nucleotide databases using the BLAST algorithm (http://www.ncbi.nih.gov/BLAST/) to verify uniqueness.


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TABLE 2 PCR primers designed to amplify a 75–150 bp expression product

 
Real-time PCR analysis of lung gene expression.
Master mixes of the reagents were prepared to minimize differences in reagent composition and pipetting errors. Twenty ng of cDNA were incubated with 25 µl iQ SYBR Green Supermix (Bio-Rad Laboratories (Canada) Ltd., Mississauga, ON, Canada) and 200 nM of each primer, and the reagent mixture was brought up to 50 µl with DNase/RNase-free water. All reactions were performed in duplicate on 96-well plates in a spectrofluorometric thermal cycler (iCycler iQ, Bio-Rad). PCR runs were initiated by incubation at 95°C for 3 min to activate the iTAQ polymerase, followed by 40 cycles of 95°C for 15 s, the appropriate annealing temperature for 15 s (see Table 2), and 30 s at 72°C. Fluorescence was monitored at every cycle during the elongation step. A melt curve was conducted following each run to verify product purity. Expression was calculated relative to ß-actin using the delta-delta Ct method (Livak and Schmittgen, 2001Go), and expressed as fold change relative to air control samples.

Analysis of plasma endothelin-1.
Plasma big ET-1 and ET-1[1-21] were analysed by HPLC-fluorescence in a subset of the animals immediately after exposure as previously described (Kumarathasan et al., 2001Go).

Gelatin zymography.
BALF samples were evaluated for MMP activity by gelatin zymography in a subset of the animals immediately after exposure. Equal volumes of BALF (20 µl) were loaded on 10% SDS-acrylamide gels containing 1 mg/ml gelatin (Sigma) and run for 1 h at 200 mV. In addition to the samples, each gel also contained prestained molecular weight markers (Bio-Rad) and a dilution series of a human MMP-2 standard (Calbiochem, La Jolla, CA). Gels were incubated in Zymogram Renaturation Buffer (Bio-Rad) for 30 min, then incubated overnight at 37°C in Zymogram Development Buffer (Bio-Rad). Following incubation, gels were stained in 0.25% Coomassie Blue R-250 staining solution (in 40% methanol/10% acetic acid) for 1 h, and then destained in a solution of 40% methanol/10% acetic acid. Clear bands were assessed by densitometric analysis using NIH shareware. To verify MMP activity, control gels were incubated under the same conditions in buffer containing 25 mM EDTA.

Statistical analyses.
Data are expressed as means ± SEM. The effects of EHC-93 and ozone were tested for statistical significance by multi-way ANOVA (OZONE, EHC, and TIME as factors), followed by Tukey's multiple comparison procedure to elucidate the pattern of significant effects ({alpha} = 0.05) using Sigma-Stat (Sigma-Stat 2.0, Chicago, IL). The systematic description of the statistically significant effects determined from multi-way ANOVAs and post-hoc comparisons in studies involving three factors can be cumbersome. For the purpose of clarity and brevity, we have adhered to the following guidelines. Significant factor interactions are indicated in the text of the Results section. Significant main effects are described in text only if they were not part of a significant factor interaction. Statistical significance reported in the figure legends refers to the Tukey's post-hoc comparisons, as directed by significant main effects or significant factor interactions in the ANOVAs. Statistical analysis of data by two-way and three-way ANOVAs and post-hoc comparisons are summarized in Tables 3 and 4, respectively.


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TABLE 3 Summary of Statistical Analyses of the Dose-Response Data Immediately After Exposure to the Pollutants

 

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TABLE 4 Summary of Statistical Analyses of the Time-Course Data Immediately and 24 h after Exposure to the Pollutants

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of ozone and particles on pulmonary endothelin system genes were investigated for the individual pollutants as well as for the combined pollutants. Factoring doses of ozone and particles provided insight into the dose-dependent changes associated with each pollutant, as well as the potential toxicological interactions at the early stages of lung response. Effects in the high-exposure groups were also analyzed 24 h after exposure in order to characterize the dynamics of changes in relation to the toxicokinetics of the pollutants and the inflammation and repair processes in the lungs. PreproET-1 mRNA levels were significantly increased immediately following exposure to EHC-93 (two-way ANOVA; EHC main effect, p = 0.010; Fig. 1A) or ozone (OZONE main effect, p < 0.001; Fig. 1A). Although both pollutants could regulate preproET-1 mRNA, there was no statistical interaction between EHC-93 and ozone with respect to the modulation of preproET-1 mRNA levels immediately after exposure (Table 3). The effects of each pollutant on preproET-1 mRNA were additive. After 24 h recovery in clean air, preproET-1 mRNA expression remained elevated in the lungs of rats exposed to EHC-93 alone (three-way ANOVA; OZONE x TIME, p < 0.001; Tukey, 0 versus 24 h within 0 ppm O3, p < 0.05; Fig. 1B). However, preproET-1 mRNA returned to control levels at 24 h in the lungs of rats exposed to ozone or to the combined pollutants (three-way ANOVA; OZONE x TIME, p < 0.001; Tukey, 0 versus 24 h within 0.8 ppm O3, p < 0.05; Fig. 1B). The elevation of preproET-1 mRNA levels immediately after exposure coincided with a significant increase in ECE-1 mRNA expression (two-way ANOVA; EHC main effect, p < 0.001; OZONE main effect, p < 0.001; Fig. 1C). There was no statistically significant EHC-93 and ozone factor interaction with respect to ECE-1 mRNA expression immediately after exposure, and the independent effects of particles and ozone were additive. After 24 h recovery of the animals in clean air, ECE-1 mRNA levels decreased in all exposure groups (three-way ANOVA; EHC x TIME, p = 0.004; OZONE x TIME, p < 0.001; Fig. 1D).



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FIG. 1. Particulate matter and ozone increase expression of ET-1 and ECE-1 mRNA. Rats were exposed by inhalation for 4 h to the indicated doses of particulate matter and ozone and euthanized immediately after exposure or following 24 h recovery in filtered air. Lung ET-1 and ECE-1 mRNA expression was determined by real-time PCR. The results are expressed as mean ± SEM (n = 4–12 animals/treatment). Letters over bars indicate statistical significance (Tukey, p < 0.05). (A) PreproET-1 mRNA immediately after exposure. a5 versus 50 mg/m3 within EHC; b0 versus 0.4 ppm and 0 versus 0.8 ppm within OZONE. (B) PreproET-1 mRNA after 24 h recovery. a0 versus 24 h within 0 ppm O3; b0 versus 0.8 ppm O3 within 0 h; c0 versus 0.8 ppm OZONE within 24 h; d0 versus 24 h within 0.8 ppm O3. (C) ECE-1 mRNA immediately after exposure. a0 versus 5 mg/m3 within EHC; b0 versus 50 mg/m3 and 5 versus 50 mg/m3 within EHC; c0 versus 0.8 ppm and 0.4 versus 0.8 ppm within OZONE. (D) ECE-1 mRNA after 24 h recovery. a0 versus 50 mg/m3 EHC within 0 h; b0 versus 24 h within 50 mg/m3 EHC; c0 versus 0.8 ppm O3 within 0 h; d0 versus 24 h within 0.8 ppm O3.

 
Endothelin-1[1-21] was measured in plasma to assess the potential immediate systemic impacts of the lung responses to the inhaled pollutants. HPLC-fluorescence analyses of ET-1[1-21] peptide levels revealed an interaction of particles and ozone, with both pollutants independently causing elevation of ET-1[1-21] immediately after exposure, but not when inhaled in combination (two-way ANOVA; EHC x OZONE factor interaction, p < 0.001; Tukey, 0 versus 50 mg/m3 EHC-93 within 0 ppm O3, p < 0.05; 0 versus 0.8 ppm O3 within 0 mg/m3 EHC, p < 0.05; Fig. 2). Plasma bigET-1 levels followed a similar pattern of response (two-way ANOVA; EHC x OZONE, p = 0.003; Fig. 2).



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FIG. 2. Immediate effects of acute exposure to particulate matter and ozone on plasma ET-1 and bigET-1 peptides. Plasma was collected from rats immediately after 4 h inhalation exposure to the indicated doses of particulate matter and ozone, and analyzed by HPLC. The results are expressed as mean ± SEM (n = 4 animals/treatment). Letters over bars indicate statistical significance (Tukey, p < 0.05). a0 versus 50 mg/m3 EHC within 0 ppm O3; b0 versus 0.8 ppm O3 within 0 mg/m3 EHC; c0 versus 0.8 ppm O3 within 50 mg/m3 EHC; d0 versus 50 mg/m3 EHC within 0.8 ppm O3; e0 versus 0.8 ppm O3 within 50 mg/m3 EHC; f0 versus 50 mg/m3 EHC within 0.8 ppm O3.

 
The plasma levels of the mature peptide and its precursor did not strictly correlate with the mRNA levels of preproET-1 and ECE-1 in the lungs, since the combination of ozone plus particles increased lung preproET-1 and ECE-1 mRNA, but not the circulating levels of ET-1[1-21] and bigET-1. Matrix metalloproteinase-2, known to be activated in injured lungs, can process bigET-1 to ET-1[1-32], a peptide distinct from the ET-1[1-21] form monitored in the HPLC assay. Analysis of bronchoalveolar lavage by gelatin zymography revealed a band in all samples that migrated with the 72 kD MMP-2 standard (Fig. 3). The intensity of this band increased immediately after exposure only in animals coexposed to both particulate matter and ozone (one-way ANOVA; p = 0.03), but not in animals exposed to air or the individual pollutants (Fig. 3). No gelatinolytic activity was observed in gels incubated with EDTA (data not shown). Based on its comigration with the MMP-2 standard and the inhibition of activity by EDTA, this band likely corresponds to the latent 72 kD form of MMP-2.



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FIG. 3. Gelatin zymography of bronchoalveolar lavage fluid (BALF) immediately after exposure to the pollutants. BALF was collected from rats exposed by inhalation to the indicated doses of particulate matter (EHC) and ozone and analyzed for MMP activity as described in Materials and Methods. Cleared bands indicating gelatinase activity migrated with the 72 kDa MMP-2 standard. The results are expressed as mean ± SEM (n = 3 animals/treatment). *p < 0.05, (Holm-Sidak multiple comparison), 0 mg/m3 EHC/0 ppm O3 versus 50 mg/m3 EHC/0.8 ppm O3. AU, arbitrary units.

 
Changes in expression of the specific endothelin receptors have the potential to impact on the physiological significance of higher ET-1 peptide levels as well as on clearance of the peptide. Expression of ETA receptor mRNA was reduced by ozone immediately after exposure (two-way ANOVA; OZONE main effect, p < 0.001; Fig. 4A) and after 24 h recovery in clean air (three-way ANOVA; OZONE main effect, p = 0.003; Fig. 4B), and was not affected by the urban particles. In contrast, expression of ETB receptor mRNA increased immediately after exposure to EHC-93 or ozone. There was no evidence of pollutant interactions except for an apparent additive effect of ozone and particles (two-way ANOVA; EHC main effect, p = 0.023; OZONE main effect, p = 0.004; Fig. 4C). After 24 h recovery in clean air, ETB receptor mRNA expression decreased about 20% below air control level in all exposure groups (three-way ANOVA; EHC x TIME, p = 0.012; OZONE x TIME, p = 0.011; Fig. 4D).



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FIG. 4. Particulate matter and ozone differentially modulate endothelin receptor mRNA expression. Lung ETA and ETB receptor mRNA levels were determined by real-time PCR after 4 h inhalation exposure to the indicated pollutant doses immediately after exposure and after 24 h recovery. The results are expressed as mean ± SEM (n = 4–12 animals/treatment). Letters over bars indicate statistical significance (Tukey, p < 0.05). (A) ETA receptor mRNA immediately after exposure. a0 versus 0.8 ppm within OZONE. (B) ETA receptor mRNA after 24 h recovery. a0 versus 0.8 ppm within OZONE. (C) ETB receptor mRNA immediately after exposure. a0 versus 50 mg/m3 within EHC; b0 versus 0.8 ppm and 0.4 versus 0.8 ppm within OZONE. (D) ETB receptor mRNA after 24 h recovery. a0 versus 50 mg/m3 EHC within 0 hr; b0 versus 24 h within 50 mg/m3 EHC; c0 versus 0.8 ppm O3 within 0 h; d0 versus 24 h within 0.8 ppm O3.

 
Higher expression of ET-1 in endothelial cells is usually counterbalanced by elevation of nitric oxide production. Both ozone and the urban particles independently increased eNOS mRNA expression immediately after exposure, with additive effects after exposure to both pollutants in combination (two-way ANOVA; EHC main effect, p = 0.027; OZONE main effect, p = 0.017; Fig. 5A). Overall, eNOS mRNA levels decreased to control levels after 24 h recovery (three-way ANOVA; TIME main effect, p < 0.046; Fig. 5B). There were no significant changes of iNOS mRNA (data not shown).



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FIG. 5. Endothelial nitric oxide synthase (eNOS) mRNA expression after pollutant inhalation. Lung eNOS mRNA levels were assessed by real-time PCR in rats exposed to the indicated pollutant doses immediately after exposure and after 24 h recovery. The results are expressed as mean ± SEM (n = 4–12 animals/treatment). Letters over bars indicate statistical significance (Tukey, p < 0.05). (A) eNOS mRNA immediately after exposure. a0 versus 5 mg/m3 within EHC; b0.4 versus 0.8 ppm within OZONE. (B) eNOS mRNA after 24 h recovery. a0 versus 24 h within TIME.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study we demonstrate for the first time that inhaled particulate matter and ozone independently regulate pulmonary endothelin system genes, and that these changes coincide with increased circulating levels of the potent vasoconstrictor ET-1[1-21] in plasma. Unexpectedly, we found that, while coexposure to both particulate matter and ozone resulted in similar activation of lung endothelin system genes, the response immediately after exposure was not associated with increased spill-over of bigET-1 or ET-1[1-21] peptides into circulation (Table 5).


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TABLE 5 Summary of Pollutant Effects on the Rat Lung Endothelin System and Plasma Endothelins Relative to Air Controls

 
To clarify the early impacts of the pollutants on the regulation of ET-1[1-21], we measured gene expression in the lungs by real-time RT-PCR, and plasma bigET-1 and ET-1[1-21] levels by HPLC-fluorescence immediately after exposure. Our data reveal a rapid increase of circulating ET-1[1-21] after a 4 h exposure to either particulate matter or ozone. The lungs are the principal source of circulating ET-1 (Dupuis et al., 1996Go), and concurrent increase of preproET-1 and ECE-1 mRNA in the lungs confirms that both particulate matter and ozone can regulate the pulmonary endothelin system in rats. Indeed, the impact of inhaled pollutants on pulmonary preproET-1 mRNA levels measured here is similar in magnitude to that measured in rat lungs after acute inhalation exposure to cigarette smoke (Adachi et al., 2000Go), which is also known to rapidly increase plasma ET-1[1-21] levels (Haak et al., 1994Go). Changes in the precursor peptide bigET-1 correlated with levels of the mature ET-1[1-21] peptide, confirming that elevation of circulating ET-1[1-21] in our animal model was due, at least in part, to increased de novo synthesis of ET-1.

It is possible that the extent of oxidative stress and tissue injury produced from coexposure to particles plus ozone, in excess of what is observed with ozone or particles alone (Vincent et al., 1997aGo), inhibited translation of preproET-1 mRNA in the affected central acinus in these animals. Oxidative stress is known to inhibit translation of a number of proteins in the lungs (Shenberger et al., 2005Go) and in endothelial cells (Jornot and Junod, 1989Go). Regulation of ET-1 is thought to be predominantly at the transcriptional level (Fagan et al., 2001Go), but translational regulation of ET-1 has been reported in endothelial cells exposed to high-density lipoprotein (Hu et al., 1994Go). Furthermore, atrial natriuretic peptide has been shown to inhibit ET-1 synthesis while, at the same time, stabilizing preproET-1 mRNA (Hu et al., 1992Go). Since translational regulation of ET-1 has not been studied to a significant extent, additional work is required to assess its relevance to ET-1 production by the lungs in normal and disease states.

On the other hand, there is ample evidence that the relative abundance of ETA and ETB receptors determines the effects of endothelin on target cells and impacts clearance of ET from the systemic circulation. Since the ETA receptor is recycled back to the cell surface after binding its ligands and internalization (Bremnes et al., 2000Go), the early decrease of ETA receptor mRNA in the lungs after inhalation of ozone may not immediately affect ETA receptor density. In contrast, because the ETB receptor is not recycled (Bremnes et al., 2000Go), the increased ET-1 peptide levels should accelerate turnover of the ETB receptor, which can be compensated only by increased synthesis of the ETB protein, and hence higher mRNA levels. Binding of ET-1[1-21] to the ETB receptor of endothelial cells stimulates the release of the vasodilators prostacyclin and nitric oxide (Luscher and Barton, 2000Go). Such a response of endothelial cells to the elevated ET-1[1-21] is substantiated here by the up-regulation of eNOS mRNA immediately after inhalation of particles or ozone. The later 20% decrease in ETB receptor mRNA levels in the lungs 24 h after exposure to the pollutants should result in lower receptor density and slower ET-1 clearance, which seems in agreement with the 15–20% increase in immunoreactive ET-1 reported previously (Bouthillier et al., 1998Go). In short, our data suggest that the observed increase in circulating levels of mature ET-1[1-21] in rats following inhalation of pollutants may be due to a combination of primary effects in the lungs, namely elevated expression of preproET-1 and ECE-1 mRNA in endothelial cells resulting in a higher rate of production, basolateral secretion, and luminal spill-over of ET-1[1-21], combined with a lower expression of ETB mRNA in the endothelium resulting in lower receptor density and slower clearance of ET-1[1-21].

The changes in bigET-1 in plasma tracked those of ET-1, but in contrast to clearance of ET-1 by the ETB receptor in the pulmonary endothelium, bigET-1 is cleared from blood mainly by the liver and the kidneys through a mechanism that is not receptor mediated (Burkhardt et al., 2000Go). Endothelins are substrates for a variety of metallopeptidases that can be induced or activated in the injured lungs (D'Orléans-Juste et al., 2003Go). For example, cleavage of bigET-1 by MMP-2 to produce ET-1[1-32] may be significant in tissue injury (Fernandez-Patron et al., 2001Go). We found that combined exposure to particulate matter and ozone, but not the individual pollutants, caused an immediate increase of MMP-2 in the alveoli. The presence of MMP-2 is in line with the enhanced septal remodeling (Vincent et al., 1997aGo) and thickening (Bouthillier et al., 1998Go) that results from coexposure to EHC-93 and ozone, by comparison to the changes induced by the individual pollutants. The alveolar air–blood barrier has a thickness of less than 1 µm, and since bigET-1 is secreted basolaterally by endothelial cells, MMP-2 produced within the septum will colocate with the secreted peptide. Furthermore, the volume of extracellular lining fluid where alveolar macrophages distribute is small, and the cells are in effect juxtaposed to type 1 epithelial cells. Consequently, any MMP-2 secreted by alveolar macrophages will immediately access the alveolar interstitium through the permeable epithelial barrier in the injured lungs of the animals coexposed to particles and ozone.

A shift in the processing of bigET-1 in the affected areas of the lungs from the ECE-dependent production of ET-1[1-21] to alternate pathways would explain the lack of measurable excess spill-over of ET-1[1-21] despite increases of preproET-1 and ECE-1 mRNAs in the coexposure group. Endothelin-1[1-32] is a potent vasoconstrictor (Fernandez-Patron et al., 1999Go). If our interpretation is correct, that coexposure to particulate matter plus ozone increased production of ET-1[1-32], this alternate pathway could play a role in mediating the acute cardiovascular effects of inhaled pollutants, particularly in lungs with existing inflammation. We did not monitor alternate endothelin peptides such as ET-1[1-31] and ET-1[1-32] in our study, and we are not aware of studies that have actually documented ET-1[1-32] in blood or tissues of animals, aside from simpler systems such as perfused arterial segments or in silico. Confirmation of the extent and relevance of the various alternate endothelin processing pathways will require detection of those species in the plasma, lungs, or BAL.

In summary, we propose that regulation of the pulmonary endothelin system by air pollutants may have profound human health impacts. Based on the responses of ECE-1 and eNOS mRNAs, the lowest-observed-effect level (LOEL) for inhaled urban particles EHC-93 with respect to changes in the endothelin system in the lungs of rats in our study corresponds to an internal effective pulmonary dose of 2.5 ng/cm2. Based on the response of preproET-1 mRNA, the LOEL for ozone here corresponds to an internal dose of 214 ng/cm2. These values are only two-fold higher than the reference values for a plausible human exposure scenario (fine particles, 1.3 ng/cm2; ozone, 127 ng/cm2). Elevation of plasma ET-1[1-21] and ET-3 in rats after inhalation of EHC-93 is accompanied by increased systemic blood pressure (Vincent et al., 2001Go). In agreement with this observation, human subjects exposed to ozone and urban particulate matter exhibit a constriction of the brachial artery (Brook et al., 2002Go). Higher plasma ET-1 levels (+25%) have been detected in children from southwest metropolitan Mexico City by comparison to children from low-pollution areas (Calderon-Garciduenas et al., 2003Go). Such an increase of ET-1 is associated with an unfavorable prognosis in congestive heart failure patients (Galatius-Jensen et al., 1996Go) or after myocardial infarction (Omland et al., 1994Go). Furthermore, heart rate variability is reduced in humans within an hour of a peak ozone episode (Gold et al., 2000Go), and high circulating ET-1 levels have been shown to correlate with decreased heart rate variability (Aronson et al., 2001Go; Pekdemir et al., 2004Go). Reduced ETB receptor expression in the lungs, resulting in slower clearance of ET-1 and, hence, elevated steady-state levels of circulating ET-1, has been proposed as a fundamental change in congestive heart failure (Kobayashi et al., 1998Go; Lepailleur-Enouf et al., 2001Go) and has been shown to predispose to pulmonary edema (Carpenter et al., 2003Go). Transcriptional activation of preproET-1 and ECE-1 is implicated in atherosclerosis progression (Rossi et al., 1999Go), and repeated exposure of hyperlipidemic rabbits to EHC-93 has indeed been shown to accelerate plaque formation (Suwa et al., 2002Go). Finally, acute cardiac effects in humans have now been documented within 1 to 3 h after exposure to occupational and ambient air pollutants (Gold et al., 2000Go; Peters et al., 2001Go), and the rapid response of the pulmonary endothelin system in animals exposed to ozone and urban particles is consistent with these observations.

Perspectives
Our animal data suggest several verifiable theoretical implications for human health. For one, the extent of the changes to the pulmonary endothelin system induced by ambient pollutants may well depend on the pollutant mix, since ozone and particulate matter in our study appeared to display some basic differences in their toxicodynamics, as well as some level of toxicological interaction. In turn, the pathophysiological impacts and health significance of the activation of the pulmonary endothelin system should depend on host factors, such as health status or genetic predisposition. Individuals with a compromised cardiovascular system and ineffective compensation for the vasopressor effect of ET-1 may respond adversely to an acute surge of circulating ET-1. Some of the documented effects associated with higher circulating ET-1 are hypertension, decreased heart rate variability, myocardial ischemia, and arrhythmia. In individuals with underlying pulmonary inflammation, such as a lung infection, chronic obstructive pulmonary disorder, or asthma, elevation of endothelin production may enhance the pulmonary inflammation cascade and tissue hyperplasia and hypertrophy. In individuals with no apparent health conditions but nevertheless with some ET-1 and ETA receptor polymorphisms that are associated with higher risk for asthma (Immervoll et al., 2001Go), hypertension (Jin et al., 2003Go), and idiopathic dilated cardiomyopathy (Charron et al., 1999Go), it remains possible that recurring activation of the pulmonary endothelin system by air pollutants will interact with these genetic determinants of susceptibility and precipitate disease development. Molecular epidemiology tools are available to investigate such outcomes.


    ACKNOWLEDGMENTS
 
Errol Thomson is the recipient of a scholarship from the Natural Sciences and Engineering Research Council of Canada and a Strategic Areas of Development scholarship from the University of Ottawa. The authors are grateful to Josée Guénette, Stephen Bjarnason, D. J. MacIntyre, and Erica Blais for conducting the inhalation exposures, Dr. Anushuyadevi Saravanamuthu for performing the HPLC analyses of endothelins, and Marc-André Joly for sequencing the PCR amplicons. This work was supported by the Toxic Substances Research Initiative (TSRI-60) and Health Canada (SEP 4320010). Conflict of interest: none declared.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
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