90-Day Repeated Inhalation Exposure of Surfactant Protein-C/Tumor Necrosis Factor- α (SP-C/TNF- α ) Transgenic Mice to Air Pollutants

Abstract

Tumor necrosis factor (TNF)-α, a cytokine present in inflammed lungs, is known to mediate some of the adverse effects of ozone and inhaled particles. The authors evaluated transgenic mice with constitutive pulmonary expression of TNF-αunder transcriptional regulation of the surfactant protein-C promoter as an animal model of biological susceptibility to air pollutants. To simulate a repeated, episodic exposure to air pollutants, wild-type and TNF mice inhaled air or a mixture of ozone (0.4 ppm) and urban particles (EHC-93, 4.8 mg/m³) for 4 h, once per week, for 12 consecutive weeks and were sacrificed 20 h after last exposure. TNF mice exhibited chronic lung inflammation with septal thickening, alveolar enlargement, and elevated protein and cellularity in bronchoalveolar lavage fluid (genotype main effect, p <.001). Repeated exposure to pollutants did not result in measurable inflammatory changes in wild-type mice and did not exacerbate the inflammation in TNF mice. The pollutants decreased recovery of alveolar macrophages in lavage fluid of both wild-type and TNF mice (exposure main effect, p < .001). Exacerbation of the rate of protein nitration reactions specifically in the lungs of TNF mice was revealed by the high ratio of 3-nitrotyrosine to _L-DOPA after exposure to the air pollutants (Genotype × Exposure factor interaction, p = .014). Serum creatine kinase-MM isoform increased in TNF mice exposed to pollutants (Genotype × Exposure factor interaction, p = .043). The marked pollutant-related nitration in the lungs of the TNF mice reveals basic differences in free radical generation and scavenging in the inflamed lungs in response to pollutants. Furthermore, elevation of circulating creatine kinase-MM isoform specifically in TNF mice exposed to pollutants suggests systemic adverse impacts from lung inflamma-tory mediators, possibly on muscles and the cardiovascular system.

Episodic variations of the concentration of ambiant air pollutants are associated with increased cardiopulmonary morbidity and mortality (Burnett et al. 1994; Burnett, Cakmak, and Brook 1998; US Environmental Protection Agency [EPA] 1996). Susceptible subgroups are persons with cancer, acute lower respiratory diseases, any form of cardiovascular disease, chronic coronary artery diseases, or congestive heart failure (Goldberg et al. 2000). Animal models are essential for the investigation of the biological bases for such epidemiological associations between air pollution and adverse effects in sensitive subpopulations.

Inflammation of the lungs as well as fibrotic and obstructive lung diseases are associated with increased levels of tumor necrosis factor (TNF)-α (Pan et al. 1996; Piguet, Grau, and Vassalli 1990; Nelson et al. 1989). Furthermore, adverse effects of ozone and particles in the lungs are associated with, and to some extent are dependent on the production of TNF-α (Driscoll et al. 1997; Fakhrzadeh, Laskin, and Laskin 2004; Ulrich et al. 2002; Yang et al. 2004). In transgenic surfactant protein (SP)-C/TNF-α mice (Miyazaki et al. 1995; Kanehiro et al. 2001), the murine TNF-α gene is expressed under transcriptional regulation of the human SP-C gene promoter. The TNF mice constitutively express TNF-α in the lungs, which produces chronic inflammation, alveolar disruption, enlarged lung volumes, and right ventricular hypertrophy (Fujita et al. 2001, 2002). The use of transgenic animal models to study the interaction of chemicals with the development of chronic disorders offers promising possibilities (Chhabra et al. 2003; Costa and Kodavanti 2003). In particular, long-term exposures to air pollutants are technically challenging and costly, and there is little guarantee that chronic exposure of animals to pollutants over several months will result in lesions that are directly relevant to the progression of a human disease over several decades. Our objective was to assess the suitability of TNF mice with chronic lung inflammation as a sensitivity model for investigation of air pollution–induced biological effects.

MATERIALS AND METHODS

Chemicals

Dulbecco’s phosphate-buffered saline (PBS; calcium and magnesium free), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DETPA), phenyl methyl sulfonyl fluoride (PMSF), sodium acetate, trisodium salt of citric acid, Trizma hydrochloride and base, molecular weight cut-off filters (10 kDa and 30 kDa), l-DOPA, p-tyrosine, 3-nitrotyrosine, CK Isotrol, CK-MB/LD-1 Isotrol, trifluoracetic acid (TFA), 3,4-dichloroisocoumarin, NADPH, sodium hydroxide, nitrate reductase, sodium nitrite, potassium nitrate, endothelin-1 (human, porcine), endothelin-2 (human), and endothelin-3 (human, rat) were purchased from Sigma Chemical (St. Louis, MO). The standard big endothelin-1 (human) was obtained from BACHEM California, (Torrance, CA). 2,3-Diaminonaphthalene was obtained from Molecular Probes (Eugene, OR). Creatine kinase isoenzyme reagents came from Beckman (Fullerton, CA). Analytical reagent grade sodium chloride came from BDH (Toronto, Ontario). Acetonitrile, acetone, methanol, and hydrochloric acid of reagent grade purity were obtained from common commercial suppliers. Amber glass vials and screw caps with septa were purchased from Chromatographic Specialities (Brockville, Ontario). Butylated hydroxytoluene (BHT) was from United States Biochemical Corporation (Cleveland, OH). Deionized water was obtained from a Super-Q Plus High Purity Water System (Millipore Corporation, Bedford, MA). Compressed gaseous nitrogen of UHP grade quality was supplied by Matheson Gas Products (Whitby, Ontario). The enzyme-linked immunosorbent assay (ELISA) for TNF-α was obtained from Biosource (Camarillo, CA).

Animals

All experimental protocols were reviewed by the Animal Care Committee of Health Canada, and carried as set forth in the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. Male SP-C/TNF-α transgenic hemizygous mice from the colony of the National Jewish Medical and Research Center in Denver (Fujita et al. 2001, 2002; Kanehiro et al. 2001) were crossed in Ottawa with C57BL/6 female mice (Charles River Laboratories, St. Constant, Qüebec) to generate F1 hybrid transgenic mice. At birth, male and female offsprings were separated, with males individually caged. The animals were housed in Plexiglass cages on wood chip bedding, under charcoal-filtered and 0.2 μm–filtered air stream, in a 12-h dark and 12-h light cycle. Food and water were provided ad libitum. Six weeks after weaning all pups were genotyped. Ear notches were sampled under anesthesia and frozen at -20°C until analysis as described earlier (Fujita et al. 2001). Wild-type and liter-mate TNF hemizygous transgenic male mice and female mice were used in the inhalation studies. Animals were 12 to 19 weeks of age and 15 to 25 g at start of exposures. Overall, clinical end points indicative of general physiological and health status did not differ between wild-type and TNF mice. With the exception of a somewhat lower white blood cell count in TNF mice (wild-type, 2.8 ± 1.4 × 10⁹/L; TNF, 1.9 ± 0.3 × 10⁹/L), which could be due to sequestration in the lungs because of a chronic inflammation, other end points such as cholesterol (114–160 mg/dl), glucose (133–196 mg/dl), lactate dehydrogenase (135–236 U/L), creatine (0.47–0.63 mg/dl), blood urea nitrogen (13–21 mg/dl), red blood cells (8–9 × 10⁹/L), hemoglobin (10–14 g/L), hematocrit (32–41%), platelets (1097–1535 × 10⁹/L), and mean platelet volume (3.4-4.4 fl) were all within normal range (Wolford et al. 1986).

Inhalation Exposure to Air Pollutants

The ambient urban particles EHC-93 were recovered in a dry form from bag-house videlon filters of the single-pass air-purification system of the Environmental Health Centre in Ottawa (100% outdoor air). The material was mechanically sieved (300 μm, 100 μm, 80 μm, 56 μm, 36 μm) and the sieved dust recovered after the 36-μm-mesh filter is referred to as EHC-93 bulk particles. The toxicity of EHC-93 was studied in cell culture models (Vincent et al. 1997b) and in vivo models (Adamson, Vinvent, and Bjarnason 1999; Bouthillier et al. 1998; Vincent et al. 1997a), establishing its relevance as an environmental material.

Wild-type and TNF mice were exposed simultaneously by the nose-only route in a flow-pass system to clean air or to a pollutant mixture containing ozone at 0.4 ppm and urban air particles EHC-93 at 4.8 mg/m³, for 4 h per day, 1 day per week, over 12 consecutive weeks. Air and pollutant exposures were conducted simultaneously in parallel nose-only exposure systems. Animals for biochemical endpoints were exposed as two cohorts, and the two inhalation studies were conducted 7 months apart. The total numbers of animals exposed were wild-type mice exposed to air, six males and three females; wild-type mice exposed to pollutants, five males and two females; TNF mice exposed to air, six males and three females; TNF mice exposed to pollutants, seven males and six females. Animals were returned to normal housing conditions immediately after exposure, and were euthanised 20 h after the last exposure.

Ozone and EHC-93 particle atmospheres were generated and characterized as described previously (Bouthillier et al. 1998; Guénette, Hayes, and Vincent 1997; Vincent et al. 2001). The ozone concentration throughout the study was stable within 10 ppb of the target 400 ppb concentration; there were no concentration excursions during the exposures. The time-weighted average EHC-93 particle concentration was 4.8 mg/m³. The aerosol concentration was evaluated at the inhalation ports by isokinetic sampling using 0.2 μm Teflon filters (TF-200, 47 mm; Gelman Sciences, Ann Arbor, MI) during two 2-h samplings; filter weight change divided by the sampling volume provided a direct estimate of the time-weighted average particulate concentration. Real-time particle counts and size measurements at the inhalation ports (optical size range of 0.3 to 10 μm; Lasair Model 301; Particle Measuring Systems, Boulder, CO) were also obtained continuously throughout each exposure and provided measurements of count median diameter for confirmation of concentration stability. The aerodynamic size characteristics of the particulate atmospheres were determined by gravimetric cascade impactor analyses on isokinetic samples at the inhalation ports (seven-stage Mercer cascade impactor, 1 L/min, 0.2 to 4.6 μm effective cut-off diameter; Intox, Albuquerque, NM) or the chamber exhaust (seven-stage Mercer cascade impactor, 10 L/min, 0.2 to 9.5 μm effective cut-off diameter; Intox, Albuquerque, NM). Multimodal particle size distribution analyses of the cascade impactor gravimetric data revealed that the resuspended EHC-93 particles contained a respirable fraction with an aerodynamic diameter (Dae) mode at 1.3 μm Dae (mode 1, 20% of aerosol mass) and a respirable mode at 3.6 μm Dae (mode 2, 35% of aerosol mass). These two respirable modes accounted for about 55% of the total aerosol mass. An additional coarse mode at 15 μm Dae (mode 3) accounted for the remaining 45% of the aerosol mass (Vincent et al. 2001).

Dosimetric Modelling

Using an established deposition model, the deposition efficiency (percent of inhaled mass deposited in the stated compartment) of the 1.3 μm Dae mode (mode 1) was estimated at 13% for the pulmonary compartment and 12% for the tracheobronchial compartment of the mouse lungs (Regional Deposited Dose Ratio RDDR2 modelling software; US EPA). Similarly, the deposition efficiency of the 3.6 μm Dae mode (mode 2) was estimated at 5% for the pulmonary compartment and 7% for the tracheobronchial compartment. Note that the dosimetric compartments are defined functionally based on clearance kinetics but correspond nevertheless to anatomical compartments. The pulmonary compartment is associated functionally with a slow clearance and corresponds anatomically to the alveolar space, the central acinus, and small bronchioles. The tracheobronchial compartment is associated with a fast clearance (≤24 h) and corresponds to the trachea, bronchi, and large bronchioles. The default assumptions used in the calculations of particle deposition in the mice are a minute ventilation of 23 ml/min, a pulmonary compartment surface area of 500 cm², and a tracheobronchial compartment surface area of 3.5 cm². Thus, the mass of particle inhaled during each 4-h exposure session (4.8 μg particle/L air × 0.023 L air inhaled/min × 240 min per session) was estimated at about 26 μg, of which 5.3 μg was associated with mode 1 (20% of aerosol mass), and 9.3 μg with mode 2 (35% of aerosol mass). Consequently, the particle depositions were estimated in the pulmonary compartment at 1.15 μg (mode 1: 5.3 μg inhaled × 0.13 deposition efficiency = 0.69 μg deposition; mode 2: 9.3 μg inhaled × 0.05 deposition efficiency = 0.47 μg deposition), or 2.3 ng/cm², and in the tracheobronchial compartment at 1.29 μg (mode 1: 5.3 μg inhaled × 0.12 deposition efficiency = 0.64 μg deposition; mode 2: 9.3 μg inhaled × 0.07 deposition efficiency = 0.65 μg deposition) or 368 ng/cm². Note that deposition of the coarse mode (mode 3) in the pulmonary or tracheobronchial compartments should be insignificant. For comparison, the estimated particle deposition values for rats exposed under the above conditions (4.8 mg/m³, 4 h) would be 2.3 ng/cm² for the pulmonary compartment and 251 ng/cm² for the tracheobronchial compartment (Vincent et al. 2001). The rate of deposition of ozone in the central acinus of rats, 68 × 10⁻⁶ μg O ₃/cm²/h per μg O₃/m³ can be used to estimate deposition in mouse lungs (Miller et al. 1988). Thus, the centriacinar dose of ozone in the mice of the present experiments (0.4 ppm O₃ or 785 μg O₃/m³, 4 h) was estimated at 214 ng O₃/cm².

Considering a plausible human exposure scenario of 175 μg particles/m³ over 24 h (Tellez-Rojo et al. 2000), and a deposition efficiency of the fine particles in the pulmonary compartment of 20% (Schlesinger 1989), the surface relative dose in the pulmonary compartment would be 1.3 ng/cm². Assuming 12 h exposure at 0.12 ppm O₃ and 12 h exposure at 0.06 ppm O₃, the centriacinar dose of ozone in a human subject would be about 127 ng O₃/cm² (Thomson et al. 2004). There are evidently large uncertainties with respect to the deposition of particles and ozone in the experimental animals as well as under the human exposure scenarios. Nevertheless, the internal doses of the pollutants estimated for each exposure episode in our experiments (2.3 ng particles/cm² alveolar surface; 214 ng O₃/cm² centriacinar surface area) are directly relevant to estimated human internal doses from realistic exposure scenarios (1.3 ng particles/cm² alveolar surface; 127 ng O₃/cm² centriacinar surface).

Biological Samples

Animals were anaesthetized with sodium pentobarbital (65 mg/kg, intraperitoneal [i.p.]). The trachea was exposed and cannulated, blood was withdrawn from the abdominal aorta and transferred into vacutainer tubes containing the sodium salt of EDTA (10 mg/ml) and PMSF (1.7 mg/ml), mixed gently, and placed on ice. The diaphragm was then punctured, the lungs were filled by intratracheal instillation of warm saline (37°C) at 30 ml/kg body weight (Hatch et al. 1994; Vincent et al. 1996). Lungs were massaged gently by rubbing the thoracic cage. Saline was aspirated and reinjected twice more, and the primary bronchoalveolar lavage (BAL) fluid was collected in a cold centrifuge tube. Secondary lavages were obtained with additional volumes of saline (3 ml/mouse), three times, to increase the yield of lavage cells. The lavage fluids were centrifuged (1500 rpm for 10 min at 4°C) to separate cells from the supernatants. Primary lavage supernatants were used to analyze biochemical endpoints. Secondary lavage supernatants were discarded. The cell pellets from both primary and secondary lavages were combined to recover the total BAL cells. BAL cells were counted in a Coulter Multisizer II (Coulter Canada, Ville St. Laurent, PQ, Canada), and differential cell counts were obtained from cytospin preparations using Wright stain and with the use of Ames-Haematic slide stainer following standard procedures (Poon et al. 2002).

Lavage supernatant (ca. 1 ml) were vortexed with 50 μl of aqueous 0.1 M DETPA solution and 50 μl of 0.3 M BHT solution in isopropanol to prevent autoxidation. Each supernatant was aliquotted for total protein analysis by the Coomassie brilliant blue dye binding assay (Vincent and Nadeau 1983), TNF-α measurements by ELISA assay (Biosource International, Camarillo, CA), and for analysis of oxidative stress markers p-tyrosine and 3-nitrotyrosine by high-performance liquid chromatography–(HPLC-EC) (Kumarathasan and Vincent 2003). Whole blood samples were centrifuged at 2000 rpm for 10 min to obtain plasma. A set of aliquots (250 μl ) of plasma were analyzed for endothelins, and whole blood was analyzed for nitrite (Kumarathasan, Goegan, and Vincent 2001). Serum creatine kinase measurements were carried out by the procedure of Szasz, Gruber, and Berndt (1976). Total serum creatine kinase isozymes were 120 units/L in wild-type mice (MM, 34%; MB, 20%; BB, 47%) and 162 units/L in TNF mice (MM, 25%; MB, 16%, BB, 60%).

Lung morphology was examined on a limited number of animals. Lungs were fixed by intratracheal instillation of 2% glutaraldehyde in 0.085 M sodium cacodylate, 0.05% (w/v) calcium chloride, pH 7.4, at a pressure of 25 cm H₂O above the chest for 10 to 15 min. The lungs and trachea were excised, immersed in fixative, and stored at 4°C. Tissue blocks were postfixed in formalin for 24 h, dehydrated in ethanol, and embedded in glycol methacrylate. Sections (0.75 μm) were stained with basic fuchsin.

Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR)

Frozen lung samples were homogenized in Trizol (Invitrogen Life Technologies, Burlington, Ont) at a ratio of 50 to 100 mg tissue per ml. Chloroform (2 ml) was added, the samples were vortexed and spun, and the upper phase was recovered. The RNA was precipitated at 4°C after addition of one half volume of isopropanol, and then washed in 75% ethanol. The resulting RNA pellet was air-dried, resuspended in RNase-free Tris-EDTA buffer, and frozen at -80°C. RNA purity was determined by measuring absorbance at 260 and 280 nm, ensuring that the A₂₆₀/A₂₈₀ ratio was 1.85 to 2.0. RT-PCR as- says for preproendothelin-1 (preproET-1), preproendothelin-3 (preproET-3), endothelin-converting enzyme-1 (ECE-1), and endothelin-A receptor (ET[A]R) were carried out using GeneAmp reagents (Perkin Elmer, Foster City, CA). The reverse transcription reaction mix consisted of 5 mM MgCl₂, 1× PCR buffer II, 1 mM each dNTP, 1 unit/μl RNase inhibitor, 2.5 units/ μl MuLV reverse transcriptase, and 2.5 μM random hexamers. Total RNA was consistent in all reactions (∼1 μg). Reverse transcription was carried out at 42°C for 45 min, 99°C for 5 min, and 5°C for 5 min. The PCR reaction mix consisted of 2 mM MgCl₂, 1× PCR buffer II, 1.25 units/reaction AmpliTAQ gold polymerase, and 0.5 μM of each primer. PCR cycling conditions were as follows: hot start, 95°C for 10 min; denaturation, 95°C for 1 min; annealing, 50°C for 1 min; extension, 72°C for 1 min for 32 cycles. The following primers were used:

ET-1	forward	5′-GCTCCTGCTCCTCCTTGATG-3′
		reverse	5′-CTCGCTCTATGTAAGTCATGG-3′
ET-3	forward	5′-GCACTTGCTTCACTTATAAGG-3′
		reverse	5′-CAGAAGCAAGAAGCATCAGTT G-3′
ECE-1	forward	5′-CGTAGCGATAGTCTTAGCAC-3′
		reverse	5′-GTGCCACACCAAAACTACAG-3′
ET[A]R	forward	5′-TTCGTCATGGTACCCTTCGA-3′
		reverse	5′-GATACTCGTTCCATTCATGG-3′
β-Actin	forward	5′-CTGATCCACATCTGCTGGAAGG TGG-3′
		reverse	5′-ACCTTCAACACCCCAGCCATGT ACG-3′

The amplified products were quantitated on agarose gels. Equal amounts of PCR products were separated by electrophoresis on 1% agarose gels containing 1:10000 dilution of Syber Green-1 (Molecular Probes, Eugene, OR). Gels were run for 1 h at an applied voltage of 80 volts. After electrophoresis, gels were scanned with a Storm 840 (Amersham BioSciences, Qüebec, Canada) and the band intensities were quantitated using the ImageQuant software package (Molecular Devices Corporation, Sunnyvale, CA).

Statistical Analyses

Results are expressed as mean ± standard error (sample size, N = 3–13). Plasma volumes did not allow for analyses of all end points in all animals. The data from the exposure studies were analyzed initially by two-way analysis of variance (ANOVA) with genotype (wild-type, TNF) and exposure (air, pollutants) as factors, using the general linear model to observe any main effects and interaction between treatment groups. All pairwise multiple comparisons were carried out using the Holm-Sidak procedure. All statistical analyses were conducted using Sigma Stat (Jandel, San Rafael, CA).

RESULTS

The histologic assessment confirmed that the TNF mice had large alveolar volumes with thick cellular septa as compared to wild type mice (Figure 1). More inflammatory cells could be observed in the alveoli as well as the interstitium of the TNF mice. This was confirmed by the cell counts in BAL fluid (genotype main effect, p < .001; Figure 2A ). The differential cell counts showed that there was an increase in inflammatory cell types in the TNF mice, in particular macrophages, neutrophils, multinucleated giant cells and lymphocytes (genotype main effect, p < .05; Figure 2B ). Repeated exposure to the pollutants did not result in measurable inflammatory changes in wild-type mice as defined by increased neutrophils, lymphocytes, monocytes, and eosinophil. Likewise, exposure to the pollutants did not amplify the cellular changes in the BAL of TNF mice. Nevertheless, exposure to the pollutants caused a decrease in total cell counts in BAL of both TNF mice and wild-type mice compared to air-exposed animals (exposure main effect, p = 0.007; Figure 2A ), attributable to lower recovery of macrophages from BAL (exposure main effect, p < .001; Figure 2B ). Total BAL protein increased in TNF mice compared to wild-type mice (genotype main effect, p = .051; Figure 3), but there were no statistically significant effects of the pollutants. There were no statistically significant changes of TNF-α levels in BAL of wild-type and TNF mice after exposure to the pollutants (data not shown). The levels of p-tyrosine, a product of hydroxylation of phenylalanine and indicative of free radicals formation namely, reactive oxygen species (ROS) generation, was not affected significantly in BAL by the genotype of the animals or by exposure to pollutants (data not shown). In contrast, 3-nitrotyrosine, a product of nitration of the amino acid tyrosine in protein and indicative of higher reactive nitrogen species (RNS), was increased in BAL of TNF mice. The ratio of 3-nitrotyrosine/l-DOPA in BAL, which provides a ratio of the relative activity of the nitration pathway over that of the hydroxylation pathway, was increased specifically in TNF mice exposed to the pollutants (genotype × exposure factor interaction, p = .014; Figure 4).

Expression of preproET-1 mRNA did not appear to be affected (Figure 5A ), but expression of ECE-1 (Figure 5B ), preproET-3 (Figure 5C ), and ET[A] receptor (Figure 5D ) mRNAs were decreased in the lungs of TNF mice (one-way ANOVA, p < .05). Although preproET-1 gene expression did not appear to be affected by the lung remodeling, the TNF mice exhibited a decrease in plasma ET-1 (genotype main effect, p = .044; Figure 6B ) and ET-2 (genotype main effect, p = .052; Figure 6C ) compared to the wild-type mice. Although not statistically significant, plasma bigET-1 (Figure 6A ) and plasma ET-3 (Figure 6D ) were reduced in TNF mice. None of the changes in the peptides after exposure to the pollutants were statistically significant, but overall repeated exposure to the pollutants apparently reduced spillover of the peptides from the lungs. Blood nitrite levels were not statistically different between experimental groups (Figure 7A ). The ratio of the sum of ET-1 and bigET-1 (pressor component) to the nitrite level (dilator component) was decreased in animals exposed to the pollutants (exposure main effect, p = .033; Figure 7B ), suggesting a compensatory down regulation of ET-1 in the lungs with repeated exposure to the pollutants. Serum creatine kinase isoenzymes (CK-MM, CK-MB, CK-BB) analyses indicated a relative increase of CK-MM (genotype × exposure interaction, p = .043) in TNF mice exposed to pollutants (Figure 8).

DISCUSSION

In this study, we have assessed the potential use of the SP-C/TNF-α mouse model in investigations of air pollution-induced health effects in susceptible individuals with chronic lung inflammation. Analysis of lung morphology confirmed that over-expression of TNF-α led to chronic inflammation, alveolar septal destruction with air space enlargement, increased septal cellularity, and remodeling of the pulmonary compartment compared to the wild-type mice. This is similar to an emphysematous condition as reported previously (Miyazaki et al. 1995; Fujita et al. 2001, 2002). Significantly increased macrophages and neutrophils in BAL of unexposed TNF mice indicated a progressive inflammation process in line with previous findings (Fujita et al. 2001). In addition, we have also seen increased multinucleated giant cells in the BAL of unexposed TNF mice. Multi-nucleated giant cells are a hallmark of granulomatous reactions, and are formed by alveolar macrophage proliferation and fusion (Prieditis and Adamson 1996). Repeated coexposure to ozone and urban particles did not induce overall changes in BAL cellularity counts. Recovery of alveolar macrophages was decreased after exposure to the pollutants in both the wild-type mice and the TNF mice. Decreased macrophage recovery from BAL fluid has been previously reported in conditions of lung injury (Hatch et al. 1994; Hudak, Zhang, and Kleeberger 1993; Kumarathasan et al. 2002). This decrease in alveolar macrophage counts may be due to adhesion to lung epithelial cells or sequestration or altered lung structure. However, the absence of measurable inflammation in the wild-type mice after repeated exposure to the pollutants, and the absence of exacerbation of the cellular inflammatory changes in TNF mice beyond what was observed after exposure to clean air, is consistent with adaptation of the mice to some of the primary effects of the pollutants (Wiester et al. 2000).

Likewise, total BAL protein was significantly elevated in the TNF mice, but repeated exposure to the pollutants did not statistically affect BAL protein in wild-type or TNF mice. TNF-α levels in BAL were slightly increased in TNF mice compared to wild-type mice, although this effect seemed more prominent in younger mice (data not shown). The elevated cellularity, protein, and TNF-α in the TNF mice was accompanied by increased gene expressions for caspase-3, bcl2, and I B in the lungs (data not shown), in line with TNF-α inducing some epithelial apoptosis reported under inflammatory conditions (Hamzaoui et al. 1999; Aggarwal, Gollapudi, and Gupta 1999). However, our data did not reveal clear interactions between genotype and pollutants with respect to the primary endpoints of lung inflammation and injury. Any potential exacerbation by the pollutants of the lung remodelling induced by the chronic lung inflammation could not be detected with those endpoints.

Hydroxylation reactions mediated by ROS were monitored here from the levels of p-tyrosine and l-DOPA levels. Nitration reactions facilitated by RNS were monitored by tracking 3-nitrotyrosine formation. p-Tyrosine is formed by hydroxylation of phenylalanine. The hydroxylation of p-tyrosine leads to the formation of l-DOPA, whereas nitration of p-tyrosine produces 3-nitrotyrosine. The ratio of 3-nitrotyrosine to l-DOPA therefore reflects the competition between RNS and ROS pathways. The TNF mice exhibited some increase (not statistically significant) in BAL p-tyrosine compared to wild-type mice, but with no changes in 3-nitrotyrosine, suggesting increased oxidative stress caused by a higher background rate of ROS generation. Treatment of guinea pigs with TNF-α was previously reported to result in ROS formation in lung effluents, but not nitric oxide (RNS) production (Serfilippi, Ferro, and Johnson 1994). Exposure to the pollutants elevated slightly (not statistically significant) the levels of p-tyrosine in the wild-type mice. In contrast, exposure to the pollutants caused an exacerbation of RNS specifically in the TNF mice, but not in the wild-type mice. Acceleration of RNS pathways can be relevant to tissue damage through nitration of key cellular proteins. Nitration and oxidation of various crucial alveolar proteins have been associated with diminished function in vitro, and also ex vivo in proteins sampled from acute lung injury patients (Lang et al. 2002). Our data clearly indicate an interaction between genotype and pollutants with respect to nitration reactions in the lungs. Therefore, in individuals with pulmonary inflammation, pollutants may exacerbate biochemical and cellular lesions promoted by protein nitration.

We have shown before that exposure to pollutants increases expression of endothelin system genes in the lungs (Thomson et al. 2004) and results in elevated circulating ET-1 peptide in plasma of rats (Bouthillier et al. 1998; Vincent et al. 2001). The TNF transgenic mice were also shown to exhibit pulmonary hypertension and right ventricular hypertrophy with no inflammatory cell infiltration or fibrosis in the heart (Fujita et al. 2001). We were interested in evaluating the impact of TNF-α overexpression on the lung endothelinergic system, and in particular the potential interaction between genotype and pollutants in the modulation of plasma ET-1. The reduction of ET-1 mRNA in the lungs of TNF mice measured by RT-PCR is in line with the previous observation of Fujita et al. (2002) by Northern hybridization. In general, our results indicated that overexpression of TNF-α and repeated inhalation exposure to the pollutants decreased expression of endothelin system genes, and reduced circulating peptides. The genotype effect may possibly be associated with a net loss of endothelial cells in the parenchyma, and thus a lower spill-over rate of the peptides in the systemic circulation, but this interpretation is speculative. The pollutant effect may represent a compensatory response to an earlier induction of the endothelinergic system (Vincent et al. 2001; Thomson et al. 2004). The circulating endothelins bigET-1 and ET-1 play a role in vasoconstriction as opposed to the function of nitric oxide as vasodilator. In particular, NO is known to down-regulate ET-1. The shift of total ET-1 (bigET-1 plus ET-1) over NO due to pollutant exposure suggests a compensatory decrease of ET-1 production with repeated inhalation exposures to the pollutants. In brief, our data do not reveal a sustained overactivation of the endothelinergic system in the lungs of the TNF mice after repeated exposure to the pollutants.

Total serum creatine kinase (CK) activity values were similar in both unexposed TNF mice and the wild type mice, with similar relative abundance of the three isoforms BB > MM > MB. Interestingly, exposure to the pollutants produced shifts in the CK isoforms only in the TNF mice. Serum CK-MM isoenzyme is a muscular injury marker (Saito et al. 2001), whereas changes in both serum CK-MM and CK-MB have been detected in car-diomyopathy (Apple, 1989; Hossein-Nia et al. 1997). TNF-α has been associated with the pathophysiology of muscle damage (Carrol et al. 2002; Saito et al. 2001). It is interesting to note the similarity in the patterns of effects of pollutants and genotype on RNS/ROS ratios and CK isozymes. Further investigations will be necessary to assess the relationship between RNS/ROS pathways and CK-MM changes, and their relevance to cardiac effects in the TNF mice.

CONCLUSIONS

It remains difficult to identify subtle air pollutant effects against progressing and severe pathological changes induced by the chronic lung inflammation caused by constitutive overexpression of TNF-α in the lungs. Nevertheless, we have identified two distinct sets of biomarkers in the mouse model that are sensitive to an interaction between genotype and pollutants. First, the data indicate that the pollutants exacerbated oxidative stress in the lungs of TNF mice, specifically enhancing the nitration pathway. Second, TNF mice exhibited elevation of CK-MM in serum after coexposure to ozone and urban particles, suggesting a potential systemic impact following primary pulmonary effects of the pollutants. These observations provide critical leads for the investigation of the interaction of repeated exposure to air pollutants with progression of chronic disorders.