* Healthy Environments and Consumer Safety Branch, and Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada, K1A 0K9, and
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
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ABSTRACT |
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Key Words: lung; pollution; particles; ozone; endothelin; cardiovascular.
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INTRODUCTION |
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Endothelin-1 is a potent vasoconstrictor peptide involved in the homeostatic control of vascular smooth muscle tone (Haynes et al., 1995). Circulating and tissue ET-1 levels are elevated in many cardiovascular diseases, including atherosclerosis, congestive heart failure, and hypertension (Luscher and Barton, 2000
). 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., 2000
) and in tissue through degradation by neutral endopeptidases (D'Orléans-Juste et al., 2003
). 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., 1989
), 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., 2000
). 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., 2003
), and matrix metalloproteinase-2 (MMP-2) to form the vasoactive ET-1[1-32] peptide (Fernandez-Patron et al., 1999
). This alternate processing pathway may be notably significant in tissue injury (Fernandez-Patron et al., 2001
).
We have reported that inhaled urban particles, while not directly injurious to normal lungs (Adamson et al., 1999; Vincent et al., 1997a
), nevertheless increased the circulating levels of ET-1[1-21] (Bouthillier et al., 1998
; Vincent et al., 2001
). 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., 2001
). 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., 1998
). 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., 2004
), 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].
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MATERIALS AND METHODS |
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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 min1 over the entire day (20.2 m3 air inhaled/d), oronasal breathing, and an alveolar surface area of 54 m2. Deposition rates for the 0.0510 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, 1989). Using these parameters, and assuming a 24-h exposure to an average PM10 concentration of 175 µg/m3 (Tellez-Rojo et al., 2000
), 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 106 µg O3/cm2/h per µg ambient O3/m3 (Miller et al., 1988
). 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., 1997a
).
Model assumptions for rats were a tidal volume of 2.1 ml, a breathing frequency of 102 min1 (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 106 µg O3/cm2/h per µg ambient O3/m3 (Miller et al., 1988). 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., 2001a).
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., 2001). 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). Primers were designed to have 50 to 60% GC content, an optimal annealing temperature of 6062°C, and yield PCR products 75150 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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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., 2001).
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 ( = 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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RESULTS |
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DISCUSSION |
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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., 1997a), 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., 2005
) and in endothelial cells (Jornot and Junod, 1989
). Regulation of ET-1 is thought to be predominantly at the transcriptional level (Fagan et al., 2001
), but translational regulation of ET-1 has been reported in endothelial cells exposed to high-density lipoprotein (Hu et al., 1994
). Furthermore, atrial natriuretic peptide has been shown to inhibit ET-1 synthesis while, at the same time, stabilizing preproET-1 mRNA (Hu et al., 1992
). 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., 2000), 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., 2000
), 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, 2000
). 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 1520% increase in immunoreactive ET-1 reported previously (Bouthillier et al., 1998
). 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., 2000). Endothelins are substrates for a variety of metallopeptidases that can be induced or activated in the injured lungs (D'Orléans-Juste et al., 2003
). 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., 2001
). 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., 1997a
) and thickening (Bouthillier et al., 1998
) that results from coexposure to EHC-93 and ozone, by comparison to the changes induced by the individual pollutants. The alveolar airblood 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., 1999). 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., 2001). In agreement with this observation, human subjects exposed to ozone and urban particulate matter exhibit a constriction of the brachial artery (Brook et al., 2002
). 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., 2003
). Such an increase of ET-1 is associated with an unfavorable prognosis in congestive heart failure patients (Galatius-Jensen et al., 1996
) or after myocardial infarction (Omland et al., 1994
). Furthermore, heart rate variability is reduced in humans within an hour of a peak ozone episode (Gold et al., 2000
), and high circulating ET-1 levels have been shown to correlate with decreased heart rate variability (Aronson et al., 2001
; Pekdemir et al., 2004
). 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., 1998
; Lepailleur-Enouf et al., 2001
) and has been shown to predispose to pulmonary edema (Carpenter et al., 2003
). Transcriptional activation of preproET-1 and ECE-1 is implicated in atherosclerosis progression (Rossi et al., 1999
), and repeated exposure of hyperlipidemic rabbits to EHC-93 has indeed been shown to accelerate plaque formation (Suwa et al., 2002
). 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., 2000
; Peters et al., 2001
), 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., 2001), hypertension (Jin et al., 2003
), and idiopathic dilated cardiomyopathy (Charron et al., 1999
), 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.
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ACKNOWLEDGMENTS |
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