TY - JOUR
AU - Vincent, Renaud
AB - Abstract We studied the impact of a catalyzed diesel particulate filter (DPF) on the toxicity of diesel exhaust. Rats inhaled exhaust from a Cummins ISM heavy-duty diesel engine, with and without DPF after-treatment, or HEPA-filtered air for 4h, on 1 day (single exposure) and 3 days (repeated exposures). Biological effects were assessed after 2h (single exposure) and 20h (single and repeated exposures) recovery in clean air. Concentrations of pollutants were (1) untreated exhaust (−DPF), nitric oxide (NO), 43 ppm; nitrogen dioxide (NO2), 4 ppm; carbon monoxide (CO), 6 ppm; hydrocarbons, 11 ppm; particles, 3.2×105/cm3, 60–70nm mode, 269 μg/m3; (2) treated exhaust (+DPF), NO, 20 ppm; NO2, 16 ppm; CO, 1 ppm; hydrocarbons, 3 ppm; and particles, 4.4×105/cm3, 7–8nm mode, 2 μg/m3. Single exposures to −DPF exhaust resulted in increased neutrophils, total protein and the cytokines, growth-related oncogene/keratinocyte chemoattractant, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1 in lung lavage fluid, as well as increased gene expression of interleukin-6, prostaglandin-endoperoxide synthase 2, metallothionein 2A, tumor necrosis factor-α, inducible nitric oxide synthase, glutathione S-transferase A1, heme oxygenase-1, superoxide dismutase 2, endothelin-1 (ET-1), and endothelin-converting enzyme-1 in the lung, and ET- 1 in the heart. Ratio of bigET-1 to ET-1 peptide increased in plasma in conjunction with a decrease in endothelial nitric oxide synthase gene expression in the lungs after exposure to diesel exhaust, suggesting endothelial dysfunction. Rather than reducing toxicity, +DPF exhaust resulted in heightened injury and inflammation, consistent with the 4-fold increase in NO2 concentration. The ratio of bigET-1 to ET-1 was similarly elevated after −DPF and +DPF exhaust exposures. Endothelial dysfunction, thus, appeared related to particle number deposited, rather than particle mass or NO2 concentration. The potential benefits of particulate matter reduction using a catalyzed DPF may be confounded by increase in NO2 emission and release of reactive ultrafine particles. diesel exhaust, diesel particulate filter, diesel emission particles, nitrogen dioxide, inhalation, inflammation. Epidemiological studies have shown a positive association between exposure to automotive combustion emissions and adverse cardiac (Auchincloss et al., 2008; Hoffmann et al., 2007; Lall et al., 2011; Raaschou-Nielsen et al., 2012; Tonne et al., 2007), pulmonary (Hazenkamp-von Arx et al., 2011), reproductive (Wilhelm and Ritz, 2003; Yorifuji et al., 2011) and neurological (Wang et al., 2009) outcomes, and cancer (Hung et al., 2012; Raaschou-Nielsen et al., 2011; Yorifuji et al., 2013). The effects are often attributed to the fine particulate component of traffic emissions (Auchincloss et al., 2008; Hoffmann et al., 2007; Hung et al., 2012; Lall et al., 2011). Biological responses to environmental air pollution exposure can be rapid. For example, changes in heart ventricular depolarization and repolarization that can potentially precipitate adverse cardiac outcomes have been shown to occur within a few hours after a rise in the level of fine particulate matter (He et al., 2010; Schneider et al., 2010). Controlled experimental investigations involving human and animal subjects generally support these findings and provide plausible mechanistic bases for the epidemiological observations. Diesel engines are a key source of fine and nanosized particulate matter in traffic dominated areas (Díaz-Robles et al., 2008), and therefore, control of diesel emission particles through technologies such as diesel particulate filters (DPFs) gains significance from a regulatory perspective. DPFs, also referred to as particle traps, have been broadly applied in both heavy-duty and light-duty diesel vehicles since 2007 to meet regulatory emission standards for particulate matter, with particle mass emission reduction efficiencies reaching >90% under most operating conditions. A drawback, however, is that the filters need to be regenerated to prevent clogging, and regeneration causes other inadvertent modifications to diesel exhaust. For example, nitrogen dioxide (NO2) level in the exhaust is increased when fuel-borne or coated oxidation catalysts are used to facilitate filter regeneration. These catalysts oxidize nitric oxide (NO) in the exhaust stream to NO2, a gas effective in the oxidative removal of trapped diesel soot, especially at low engine operation temperatures. The higher exhaust NO2/NOx ratio in the treated exhaust is a concern in spite of reductions in the levels of particulate matter, carbon monoxide (CO), and hydrocarbons accomplished by this process. Furthermore, regeneration of DPFs to remove deposited carbonaceous matter can generate a large number of newer and smaller (<10nm) nanoparticles (Khalek et al., 2011; Kittelson et al., 2008). It can be expected that these changes in the emission profile will impact the toxicity of resultant emissions. Indeed, there is mounting mechanistic evidence associating ambient ultrafine particles to adverse human outcomes (Tablin et al., 2012). A limited number of investigations have previously examined the impacts of DPF on the toxicity of exhaust emissions. Human exposures to dilute diesel exhaust with and without treatment of exhaust by a ceramic particle trap have shown that particle filtration did not significantly reduce airway inflammation associated with untreated diesel-exhaust exposure (Rudell et al., 1999). The noncatalyzed particle trap used in this work was able to reduce particle number concentrations by only 50%. A subsequent study (McDonald et al., 2004) showed that the use of a combination of low-sulfur fuel and a catalyzed particle trap could abolish inflammation, oxidative stress, and tendency to infection by respiratory syncytial virus in mice associated with a 7-day (6h/day) exposure to untreated, high-sulfur diesel exhaust by inhalation. But the study design did not allow clarification of effects attributable solely to diesel particle filtration. A recent study (Lucking et al., 2011) employed a catalyzed trap that resulted in a 90% reduction of diesel particles to demonstrate an abolition of cardiovascular effects (e.g., impaired vasodilatation and increased ex vivo thrombus formation) associated with a short-term (1h) untreated diesel-exhaust exposure in humans. This work provided important evidence that reduced particulate mass emission upon DPF treatment abrogates the vascular effects of a single, short-term inhalation exposure to diesel exhaust. However, additional investigations are required to fully assess the toxicological consequences of DPF-treated exhaust in relation to repeated exposures, range of biological endpoints that may be sensitive to exposures, and postexposure recovery periods relevant to manifestation of effects. In the present work, we exposed animals to dilute diesel exhaust from a heavy-duty diesel engine with and without filtration of diesel exhaust by a continuously regenerating catalyzed DPF to assess the biological impacts of acute exposure to untreated and post-DPF exhaust. Both immediate and delayed effects on a number of endpoints associated with cardiovascular toxicity, inflammation, and oxidative stress were assessed. MATERIALS AND METHODS Animals. Specific-pathogen-free Fischer-344 (F344/DuCrl) male rats (200–250g) obtained from Charles River (St Constant, QC, Canada) were housed in individual Plexiglass cages on wood-chip bedding under High Efficiency Particulate Air (HEPA)-filtered air and were held to a 12:12-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. Animals were received and housed in the animal care facilities at Health Canada in Ottawa. For inhalation exposures, the animals were transported in transit cages (Taconic, Hudson, NY) to a mobile exposure laboratory deployed at the Emissions Research and Measurement Section Laboratories (Environment Canada, Ottawa), which houses automotive emission testing facilities, and located 15 km from the Health Canada animal care facilities. After exposures, the animals were returned to Health Canada for postexposure holding and necropsies. Exhaust Generation. Exhausts were derived from a heavy-duty diesel engine (model year 2004 Cummins ISM 280, 10.8 l, inline 6 cylinder, 280 bhp at 2100rpm, 2004 emission standard) operated on an engine dynamometer. The engine was operated on commercial ultralow sulfur diesel. Experiments were conducted with and without exhaust after-treatment by a catalyzed, passively regenerating DPF (Engine Control Systems, Thornhill, ON, Canada). There were no other emission treatment systems installed. On each study day, the engine was preconditioned to ensure that the oil and cooling fluid temperatures, as well as the exhaust temperature (pre- and post-DPF, when applicable), were stabilized. During all tests, the engine was operated in a steady-state condition (1200rpm at 150 bhp, 650 ft lb torque), and exhaust was diluted using a constant volume sampling system with a dilution factor of about 15×. Inhalation Exposures. The study design is summarized in Table 1. All exposures were conducted from the exhaust of the same engine, with or without DPF. Direct diluted exhaust (−DPF) was first investigated. The single and repeated animal exposures for this configuration were conducted within a period of 1 week. The engine was then retrofitted with the DPF and validated over a period of 2 weeks. Single and repeated exposures to filtered exhaust (+DPF) were then conducted within a period of 1 week. Thus, two different cohorts of animals were ordered from the supplier for the −DPF and +DPF exposures, but each study contained air control animals, and diesel-exhaust-exposed animals transported to the test facility, as well as naive animals that remained at the animal care facilities for baseline. The exhaust was transferred from the engine test cell to a 1 m3 whole-body inhalation chamber (modified Hazelton) through a 100-ft-long, 2″ diameter stainless steel pipe. The flow rate through the delivery lines was 250 ft/min with a transit time of 15 s. Control animals were exposed to HEPA air in a 1 m3 chamber alongside the diesel-exhaust exposure chamber. The clean air and diesel-exhaust inhalation chambers were operated at 250 l/min (15 changes per hour) at a static pressure of −0.5″ H2O. Both chambers were tested prior to the study to ensure that there was no leakage of exposure atmospheres into the test facility or leakage of facility contaminants into the exposure atmospheres. The average temperatures during animal exposures in the chambers were clean air, 21±2°C, and diesel exhaust, 23±2°C. Table 1 Study Design and Deployment Cohort  Number of exposures  Exposure  Recovery after exposure  Group size  −DPF  1 day (4h/day)  Air  2 h  8  20 h  8  Diesel exhaust  2 h  8  20 h  8  3 days (4h/day)  Air  20 h  8  Diesel exhaust  20 h  8  +DPF  1 day (4h/day)  Air  2 h  8  20 h  8  Diesel exhaust  2 h  8  20 h  8  3 days (4h/day)  Air  20 h  8  Diesel exhaust  20 h  8  Cohort  Number of exposures  Exposure  Recovery after exposure  Group size  −DPF  1 day (4h/day)  Air  2 h  8  20 h  8  Diesel exhaust  2 h  8  20 h  8  3 days (4h/day)  Air  20 h  8  Diesel exhaust  20 h  8  +DPF  1 day (4h/day)  Air  2 h  8  20 h  8  Diesel exhaust  2 h  8  20 h  8  3 days (4h/day)  Air  20 h  8  Diesel exhaust  20 h  8  View Large Emission Characterization. Particle number and size distributions were measured using a TSI 3090 Engine Exhaust Particle Sizer (EEPS; TSI Inc., Shoreview, MN). Particle number concentrations were additionally analyzed by a set of Condensation Particle Counters (CPC 3007; TSI Inc.). Particle mass concentration was monitored using DustTrack II DRX (TSI Inc.). A number of Teflon filters were also sampled for gravimetric analysis. Dilute exhaust concentrations of carbon dioxide (CO2) and CO were measured continuously using Horiba analyzers (AIA 210 CO2 analyzer and AIA 210LE CO analyzer; Horiba, Burlington, ON, Canada). Total oxides of nitrogen and NO were monitored using a chemiluminescence analyzer (400-HCLD; California Analytical Instruments, Orange, CA). Nitrogen dioxide was calculated as the difference between NOX and NO levels. Total hydrocarbon levels were monitored by a flame ionization analyzer (300-HFID; California Analytical Instruments). The decrease of hydrated particulate mass during transport from dilution tunnel to the inhalation chamber based on filter gravimetry data was approximately 50% (Table 2). However, optical measurements indicated a 30% increase in particle count revealing secondary aerosol formation during transport. The DPF reduced inhalation chamber concentrations of most pollutants by 20–80%: CO (−79%), NO (−52%), NOx (−23%), and hydrocarbons (−75%). However, the levels of NO2 were increased in the +DPF atmosphere (+300%) from 4 to 16 ppm (Table 3). Particle mass was reduced in the +DPF atmosphere (−70% by gravimetry and −99% by optical measurements) but particle count increased (+38%) with the median size mode shifting from 70nm (Fig. 1A) down to 8nm (Fig. 1B). Exposure concentrations of volatile organic carbons and irritants such as formaldehyde, acetaldehyde, and acrolein were significantly decreased by DPF (−80 to −99%), in line with the mass concentration reduction (Supplementary Data). Table 2 Characteristics of Diesel-Exhaust Particles Condition  Location  Mass (gravimetry), µg/m3  Mass (opticala), µg/m3  Count (opticalb) × 105 particles/cm3  Size mode (electrical mobility) nm  −DPF  Dilution tunnel  546 (497,598)  212 (202,222)  2.5 (2.3,2.7)  n/a  Inhalation chamber  277 (260,310)  269 (244,307)  3.2 (2.5,3.8)  60–70  +DPF  Dilution tunnel  124 (113,138)  0.1 (0.07,0.13)  3.4 (1.7,4.2)  n/a  Inhalation chamber  82 (76,91)  1.7 (1.4,2.2)  4.4 (4.1,4.7)  7–8  Condition  Location  Mass (gravimetry), µg/m3  Mass (opticala), µg/m3  Count (opticalb) × 105 particles/cm3  Size mode (electrical mobility) nm  −DPF  Dilution tunnel  546 (497,598)  212 (202,222)  2.5 (2.3,2.7)  n/a  Inhalation chamber  277 (260,310)  269 (244,307)  3.2 (2.5,3.8)  60–70  +DPF  Dilution tunnel  124 (113,138)  0.1 (0.07,0.13)  3.4 (1.7,4.2)  n/a  Inhalation chamber  82 (76,91)  1.7 (1.4,2.2)  4.4 (4.1,4.7)  7–8  Note. Particle mass and counts are averages (minimum and maximum) for 3–4 separate engine runs under each of the two conditions: −DPF and +DPF. n/a, not available. aMeasured using DustTrak DRX. bMeasured using a Condensation Particle Counter. View Large Table 3 Gaseous Pollutant Concentrations During Animal Exposures Condition  Location  CO (ppm)  CO2 (%)  NOX (ppm)  NO (ppm)  NO2 (ppm)  HC (ppm)  −DPF  Dilution tunnel  6.5 (6.4,6.7)  0.96 (0.94,0.98)  47.8 (45.1,48.9)  43.8 (41.2,44.9)  4.0 (3.7,4.2)  10.7 (10.1,11.0)  Inhalation chamber  6.3 (6.2,6.5)  n/a  47.0 (44.4,48.1)  42.66 (40.1,43.8)  4.2 (4.0,4.5)  n/a  +DPF  Dilution tunnel  1.3 (1.2,1.4)  0.92 (0.91,0.93)  37.9 (36.9,39.7)  21.5 (20.5,22.8)  16.3 (15.6,16.9)  2.7 (2.6,2.9)  Inhalation chamber  1.3 (1.2,1.4)  n/a  36.1 (35.1,37.9)  20.3 (19.3,21.5)  15.7 (15.0,16.3)  n/a  Condition  Location  CO (ppm)  CO2 (%)  NOX (ppm)  NO (ppm)  NO2 (ppm)  HC (ppm)  −DPF  Dilution tunnel  6.5 (6.4,6.7)  0.96 (0.94,0.98)  47.8 (45.1,48.9)  43.8 (41.2,44.9)  4.0 (3.7,4.2)  10.7 (10.1,11.0)  Inhalation chamber  6.3 (6.2,6.5)  n/a  47.0 (44.4,48.1)  42.66 (40.1,43.8)  4.2 (4.0,4.5)  n/a  +DPF  Dilution tunnel  1.3 (1.2,1.4)  0.92 (0.91,0.93)  37.9 (36.9,39.7)  21.5 (20.5,22.8)  16.3 (15.6,16.9)  2.7 (2.6,2.9)  Inhalation chamber  1.3 (1.2,1.4)  n/a  36.1 (35.1,37.9)  20.3 (19.3,21.5)  15.7 (15.0,16.3)  n/a  Note. Values are averages (minimum and maximum) for four separate engine runs under each of the two conditions: −DPF and +DPF. n/a, not available. View Large Fig. 1. View largeDownload slide Particle size distributions in diesel-exhaust exposure atmospheres. Dilute diesel exhaust without any emission treatment (−DPF) produced a particle size distribution with a 70nm size mode (A), whereas the treatment of exhaust by DPF (+DPF) removed this size mode and generated larger number concentrations of much smaller ultrafine particles with a size mode of 8nm (B). Fig. 1. View largeDownload slide Particle size distributions in diesel-exhaust exposure atmospheres. Dilute diesel exhaust without any emission treatment (−DPF) produced a particle size distribution with a 70nm size mode (A), whereas the treatment of exhaust by DPF (+DPF) removed this size mode and generated larger number concentrations of much smaller ultrafine particles with a size mode of 8nm (B). Biological Samples. Following inhalation exposures and recovery in clean air, animals were anaesthetized by inhaled isofluorane. The trachea was exposed and cannulated, blood was withdrawn from the abdominal aorta into vacutainer tubes containing the sodium salt of EDTA at 10mg/ml and PMSF at 1.7mg/ml, mixed gently, and placed on ice. The diaphragm was then punctured. The lungs were filled by intratracheal delivery of warm saline (37°C) at 30ml/kg body weight (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) was collected in a centrifuge tube kept on ice. Secondary lavages were obtained with additional volumes of saline (5ml/animal), three times, to increase the yield of lavage cells. The lavage fluids were centrifuged (1500rpm for 10min at 4°C) to separate cells from the supernatants. The cell pellets from both primary and secondary lavages were combined to recover the total BAL cells. Primary lavage supernatants were used to analyze biochemical endpoints. Secondary lavage supernatants were discarded. Whole blood samples were centrifuged at 2000rpm for 10min at 4°C to obtain plasma. Plasma aliquots were frozen at −80°C. Lung and heart tissues were collected, flash frozen in liquid nitrogen, and stored at −80°C for reverse transcriptase-PCR analyses. Cytology. Lung BAL cells were counted using a Coulter Multisizer II (Coulter Canada, Ville St-Laurent, QC, Canada), and differential cell counts were obtained from cytospin preparations, using Wright stain and following standard procedures (Poon et al., 2002). Counts of white blood cells (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, platelets, as well as hemoglobin content, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and platelet volume were analyzed in EDTA-containing whole blood with the ADVIA 120 hematology analyzer (Siemens Healthcare Diagnostics, Deerfield, IL). Clinical Chemistry. Protein concentration in the lavage samples were measured, after appropriate dilution of the primary lavage supernatant in deionized water, using Pierce BCA protein assay kit (Thermo Scientific Inc., Rockford, IL) according to the manufacturer’s instructions. Levels of blood urea nitrogen (BUN), inorganic phosphate, albumin, cholesterol, glucose, total protein, creatinine, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), as well as the enzymes alkaline phosphatase (ALP), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), lipase, and creatine kinase-NAC (CK-NAC) were quantitated in plasma using an ABX Pentra 400 clinical chemistry analyzer employing reagents and analyses protocols supplied by the instrument manufacturer (Horiba ABX, Montpellier, France). Cytokines. Levels of interleukin (IL)-1α, IL-1β, IL-5, IL-6, IL-10, growth-related oncogene/keratinocyte chemoattractant (GRO/KC), granulocyte macrophage colony-stimulating factor, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP-1α), and regulated on activation and normal T cell expressed and secreted chemokine in the BAL fluid were analyzed using Milliplex cytokine assay kits (Millipore Corporation, Billerica, MA). Plasma levels of brain natriuretic peptide, myeloperoxidase, MCP-1, troponin I, troponin T, IL-6, von Willebrand factor (vWF), TNF-α, tissue inhibitor of metalloproteinase-1 (TIMP-1), vascular endothelial growth factor (VEGF), tissue plasminogen inhibitor, serum intracellular adhesion molecule (sICAM), s-selectin, fibrinogen, adiponectin, and C-reactive protein (CRP) were analyzed using the Milliplex multiplex cardiovascular assay kits (Millipore Corporation). Analyses were conducted according to the kit manufacturer’s instructions using a Bio-Plex 200 multiplex luminescence assay system (Bio-Rad Laboratories Canada Ltd., Mississauga, ON, Canada). Plasma 8-iso-PGF2α (8-isoprostane). Plasma samples were deproteinized and affinity purified for 8-iso-PGF2α as described by Bielecki et al. (2012), and the reconstituted samples were analyzed by a competitive enzyme immunoassay (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer’s instructions. Plasma Endothelins. Plasma levels of big ET-1, ET-1[1–21], ET-2[1–21], and ET-3[1–21] peptides were analyzed by a high-performance liquid chromatography method using fluorescence detection, as previously described (Kumarathasan et al., 2001). Gene Expression Analyses. Lung and heart 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 total RNA was reverse transcribed using MuLV reverse transcriptase and random hexamers (Applied Biosystems, Mississauga, Canada), according to the manufacturer’s instructions. Primers for peptidylprolyl isomerase A (cyclophilin A), IL-6, IL-1β, prostaglandin-endoperoxide synthase 2 (PTGS2), TNF-α, metallothionein 2A (MT2A), cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), ET-1, endothelin-converting enzyme-1 (ECE-1), endothelin receptor type A (ETAR), endothelin receptor type B (ETBR), heme oxygenase-1 (HO-1), glutathione S-reductase (GSR), glutathione peroxidase-1 (GPX-1), glutathione S-transferase A1 (GSTA1), glutathione S-transferase M1 (GSTM1), superoxide dismutase 2 (SOD2), and superoxide dismutase 3 (SOD3) were obtained from Thomson et al. (2013), or designed and validated to produce amplicons with an optimal annealing temperature of 60°C (Supplementary Data), as described in Thomson et al. (2013). Real-time PCR was performed on 96-well plates in a spectrofluorometric thermal cycler (Lightcycler 480, Roche Diagnostics Canada, Laval, QC, Canada) using iQ SYBR Green Supermix (Bio-Rad Laboratories [Canada] Ltd.). A melt curve was conducted following each run to verify product purity. Expression was calculated relative to peptidylprolyl isomerase A expression using the delta-delta Ct method and expressed relative to time-matched controls. Statistical Analyses. The data from single (1-day) exposures were analyzed by three-way ANOVA with Exposure (air, diesel), Treatment (−DPF, +DPF), and Recovery (2 and 20h) as factors. The data from repeated (3-day) exposures were analyzed by 2-way ANOVA with Exposure (air, diesel) and Treatment (−DPF, +DPF) as factors. Data sets not meeting the assumptions of normality and equal variance for ANOVA were subjected to log10, natural log (ln), inverse or square root transformations (in the order given) until the assumptions were met, or else rank transformed prior to analyses. Pairwise multiple comparisons were carried out using Tukey’s procedure to elucidate the pattern of significant effects (α = 0.05). The analyses were conducted using SigmaPlot, version 12 (Systat Software, Inc., San Jose, CA). RESULTS Blood Clinical Chemistry and Cytology Blood inorganic phosphate, ALP, CK-NAC, and LDL were slightly lower (3–18%) in experimental animals (moved across facilities, transferred to the inhalation chambers, and exposed to air) compared with naive animals that remained at the animal facility (data not shown). Otherwise, there were no changes in the levels of all clinical chemistry endpoints analyzed (BUN, inorganic phosphate, albumin, cholesterol, glucose, total protein, creatinine, ALP, CK-NAC, LDH, ALT, AST, lipase, triglycerides, LDL, and HDL), which were attributable to Exposure (air, diesel), Treatment (−DPF, +DPF), or Recovery (2 and 20h). All values remained within normal range for healthy rats (Sharp and LaRegina, 1998). Single exposure to diesel exhaust resulted in a 25% increase in blood lymphocytes by comparison with exposure to clean air (Exposure, air vs. diesel, p = 0.01; data not shown). Red-blood-cell numbers were increased by 3% after diesel-exhaust exposure (Exposure, air vs. diesel, p = 0.027; data not shown). For both endpoints, there were no Treatment or Recovery main effects or interactions. There were no changes in lymphocytes and red-blood-cell counts after three repeated exposures to diesel exhaust. Platelet count increased by 7% in animals exposed to −DPF exhaust (Exposure × Treatment, air vs. diesel within −DPF, p = 0.051, −DPF vs. +DPF within diesel, p = 0.053; data not shown). Cell counts were within normal range for healthy rats (Sharp and LaRegina, 1998). Lung Lavage Protein and Cytology Single exposure of animals to diesel exhaust resulted in an increase in the level of total proteins in the lung lavage fluid indicative of lung injury (Exposure, air vs. diesel, p = 0.002; Fig. 2A). The effect size was larger for +DPF diesel-exhaust exposure (Exposure × Treatment, p = 0.052). Lavage total protein levels were not significantly elevated after three repeated exposures to diesel exhaust (Fig. 2B). Yield of neutrophils in the lung lavage fluid was enhanced by a single exposure to diesel exhaust, with larger changes in animals exposed to +DPF exhaust by comparison with −DPF exhaust (Exposure, air vs. diesel, p < 0.001, Treatment, −DPF vs. +DPF, p < 0.001; Fig. 3A). However, there were no significant exposure-related changes in neutrophils after repeated diesel-exhaust exposures (Fig. 3B). Single exposure to diesel-exhaust did not alter the yield of macrophages in the lung lavage fluid in a statistically significant manner. The yield of macrophages was higher in the +DPF exhaust exposure experiment (Treatment, −DPF vs. +DPF, p = 0.022); effect was seen in air- and diesel-exhaust-exposed animals and at 2 and 20h recovery time points (Fig. 3C). Repeated (3-day) exposure to diesel resulted in enhanced yield of macrophages, an effect that was higher in +DPF configuration (Exposure, air vs. diesel, p = 0.038, Treatment, −DPF vs. +DPF, p = 0.005; Fig. 3D). Fig. 2. View largeDownload slide Changes in BAL total protein after exposure to diesel exhaust with (+DPF) or without (−DPF) treatment by DPF. Values are mean fold changes relative to time matched controls ± SEM, n = 8. (A) Single exposure. Three-way ANOVA, Exposure, *air versus diesel, p = 0.002. (B) Repeated exposures. Two-way ANOVA, not statistically significant. Fig. 2. View largeDownload slide Changes in BAL total protein after exposure to diesel exhaust with (+DPF) or without (−DPF) treatment by DPF. Values are mean fold changes relative to time matched controls ± SEM, n = 8. (A) Single exposure. Three-way ANOVA, Exposure, *air versus diesel, p = 0.002. (B) Repeated exposures. Two-way ANOVA, not statistically significant. Fig. 3. View largeDownload slide Recovery of neutrophils and macrophages following single and repeated exposures to diesel exhaust. Values are mean ± SEM, n = 8. (A) BAL neutrophils, single exposure. Three-way ANOVA, Exposure, *air versus diesel, p < 0.001; Treatment, **−DPF versus +DPF, p < 0.001. (B) BAL neutrophils, repeated exposures. Two-way ANOVA, Treatment, *−DPF versus +DPF, p < 0.001. (C) BAL macrophages, single exposure. Three-way ANOVA, Treatment, *−DPF versus +DPF, p = 0.022. (D) BAL macrophages, repeated exposures. Two-way ANOVA, Exposure, *air versus diesel, p = 0.038; Treatment, **−DPF versus +DPF, p = 0.005. Fig. 3. View largeDownload slide Recovery of neutrophils and macrophages following single and repeated exposures to diesel exhaust. Values are mean ± SEM, n = 8. (A) BAL neutrophils, single exposure. Three-way ANOVA, Exposure, *air versus diesel, p < 0.001; Treatment, **−DPF versus +DPF, p < 0.001. (B) BAL neutrophils, repeated exposures. Two-way ANOVA, Treatment, *−DPF versus +DPF, p < 0.001. (C) BAL macrophages, single exposure. Three-way ANOVA, Treatment, *−DPF versus +DPF, p = 0.022. (D) BAL macrophages, repeated exposures. Two-way ANOVA, Exposure, *air versus diesel, p = 0.038; Treatment, **−DPF versus +DPF, p = 0.005. BAL Cytokines Consistent with an inflammatory response, the levels of GRO/KC in the lung lavage were significantly elevated following single exposure to diesel exhaust (Fig. 4A). The effects were significantly higher 2h after exposure to diesel exhaust and returned to air control levels after 20h recovery in clean air (Exposure × Recovery, 2h vs. 20h within diesel, p < 0.001, air vs. diesel within 2h recovery, p < 0.001). Exposure to +DPF exhaust significantly increased the levels of GRO/KC beyond the effect observed in −DPF animals (Treatment × Recovery, −DPF vs. +DPF within 2h recovery, p = 0.002; 2h vs. 20h within +DPF, p < 0.001). Levels of GRO/KC were not significantly altered after three repeated exposures to diesel exhaust (Fig. 4B). Levels of MIP-1α were significantly elevated 20h post +DPF diesel-exhaust exposure (Exposure × Treatment × Recovery, −DPF vs. +DPF within diesel at 20h recovery, p = 0.004, air vs. diesel within +DPF at 20h recovery, p = 0.005; Fig. 4C). However, the diesel-exhaust effects were not statistically different from air exposure effects after three repeated exposures (Fig. 4D). Levels of MCP-1 were also significantly elevated by single exposure to +DPF diesel exhaust (Exposure × Treatment, air vs. diesel within +DPF, p < 0.001, −DPF vs. +DPF within diesel, p < 0.001; Fig. 4E) but were below detection limit after repeated exposures to clean air or diesel exhaust (Fig. 4F). Fig. 4. View largeDownload slide Inflammatory cytokines in BAL fluid following diesel-exhaust exposures. Values are mean ± SEM, n = 8. (A) GRO/KC, single exposure. Three-way ANOVA, Exposure × Recovery. *2h versus 20h within diesel, p < 0.001; **air versus diesel within 2h recovery, p < 0.001; Treatment × Recovery, †−DPF versus +DPF within 2h recovery, p = 0.002; ††2h versus 20h recovery within +DPF, p < 0.001. (B) GRO/KC, repeated exposures. Two-way ANOVA, no significant effects. (C) MIP-1α, single exposure. Three-way ANOVA, Exposure × Treatment × Recovery, *−DPF versus +DPF within diesel at 20h recovery, p = 0.004; **air versus diesel within +DPF at 20h recovery, p = 0.005. (D) MIP-1α, repeated exposures. Two-way ANOVA, no significant effects. (E) MCP-1, single exposure. Three-way ANOVA, Exposure × Treatment, *air versus diesel within +DPF, p < 0.001; **−DPF versus +DPF within diesel, p < 0.001. (F) MCP-1, repeated exposures, values below detection limit. Fig. 4. View largeDownload slide Inflammatory cytokines in BAL fluid following diesel-exhaust exposures. Values are mean ± SEM, n = 8. (A) GRO/KC, single exposure. Three-way ANOVA, Exposure × Recovery. *2h versus 20h within diesel, p < 0.001; **air versus diesel within 2h recovery, p < 0.001; Treatment × Recovery, †−DPF versus +DPF within 2h recovery, p = 0.002; ††2h versus 20h recovery within +DPF, p < 0.001. (B) GRO/KC, repeated exposures. Two-way ANOVA, no significant effects. (C) MIP-1α, single exposure. Three-way ANOVA, Exposure × Treatment × Recovery, *−DPF versus +DPF within diesel at 20h recovery, p = 0.004; **air versus diesel within +DPF at 20h recovery, p = 0.005. (D) MIP-1α, repeated exposures. Two-way ANOVA, no significant effects. (E) MCP-1, single exposure. Three-way ANOVA, Exposure × Treatment, *air versus diesel within +DPF, p < 0.001; **−DPF versus +DPF within diesel, p < 0.001. (F) MCP-1, repeated exposures, values below detection limit. Plasma Cytokines Plasma levels of MCP-1, vWF, TIMP-1, VEGF, s-selectin, fibrinogen, adiponectin, and CRP were not significantly altered by exposure to diesel exhaust (Supplementary Data), whereas the levels of sICAM were significantly reduced after repeated exposures to diesel exhaust (Exposure, air vs. diesel, p = 0.007; Supplementary Data). Gene Expression Single exposure to diesel exhaust under +DPF configuration resulted in a reduction in the expression of CYP1A1 gene in the lungs. The reduction was seen both after 2 and 20h recovery in clean air (Exposure × Treatment, −DPF vs. +DPF within diesel, p < 0.001, air vs. diesel with +DPF, p = 0.002; Fig. 5A). Repeated exposures to diesel exhaust also caused a reduction in CYP1A1 expression under both −DPF and +DPF configurations (Treatment, air vs. diesel, p = 0.050; Fig. 5B). Lung expression of GSTA1 gene was significantly enhanced after a single exposure to both −DPF and +DPF diesel exhaust (Exposure × Treatment × Recovery, air vs. diesel within −DPF at 20h recovery, p = 0.021; −DPF vs. +DPF within Diesel at 20h recovery, p < 0.001; Exposure × Recovery, air vs. diesel within 2h recovery, p < 0.001; Fig. 5C). Repeated exposures to diesel exhaust caused a reduction in the expression of GSTA1, but the effect was not statistically significant (Fig. 5D). The levels of MT2A mRNA were enhanced by a single exposure to diesel exhaust, with the effect greater after exposure to +DPF exhaust. The levels returned to air control levels after 20h recovery in clean air (Exposure × Treatment × Recovery, air vs. diesel within −DPF at 2h recovery, p = 0.002, air vs. diesel, within +DPF at 2h recovery, p < 0.001, −DPF vs. +DPF, within diesel at 2h recovery, p < 0.001; Fig. 5E). Repeated exposures to diesel exhaust did not cause any statistically significant changes to MT2A expression (Fig. 5F). Fig. 5. View largeDownload slide Alteration of gene expression of CYP1A1, GSTA1, and MT2A in the lung tissue following diesel-exhaust exposures. Values are mean fold change relative to time matched controls ± geometric standard deviation; n = 8. (A) CYP1A1, single exposure. Three-way ANOVA, Exposure × Treatment, *−DPF versus +DPF within diesel, p < 0.001; **air versus diesel within +DPF, p = 0.002. (B) CYP1A1, repeated exposures. Two-way ANOVA, Exposure, p = 0.050. (C) GSTA1, single exposure, Exposure × Treatment × Recovery, *air versus diesel within −DPF at 20h recovery, p = 0.021; **−DPF vs. +DPF within diesel at 20h recovery, p < 0.001; Exposure × Recovery, †air versus diesel within 2h recovery, p < 0.001. (D) GSTA1, repeated exposures. Two-way ANOVA, no significant effects. (E) MT2A, single exposure, Exposure × Treatment × Recovery, *air versus diesel within −DPF at 2h recovery, p = 0.002; **air versus diesel, within +DPF at 2h recovery, p < 0.001; †−DPF versus +DPF, within diesel at 2h recovery, p < 0.001. (F) MT2A, repeated exposures. Two-way ANOVA, no significant effects. Fig. 5. View largeDownload slide Alteration of gene expression of CYP1A1, GSTA1, and MT2A in the lung tissue following diesel-exhaust exposures. Values are mean fold change relative to time matched controls ± geometric standard deviation; n = 8. (A) CYP1A1, single exposure. Three-way ANOVA, Exposure × Treatment, *−DPF versus +DPF within diesel, p < 0.001; **air versus diesel within +DPF, p = 0.002. (B) CYP1A1, repeated exposures. Two-way ANOVA, Exposure, p = 0.050. (C) GSTA1, single exposure, Exposure × Treatment × Recovery, *air versus diesel within −DPF at 20h recovery, p = 0.021; **−DPF vs. +DPF within diesel at 20h recovery, p < 0.001; Exposure × Recovery, †air versus diesel within 2h recovery, p < 0.001. (D) GSTA1, repeated exposures. Two-way ANOVA, no significant effects. (E) MT2A, single exposure, Exposure × Treatment × Recovery, *air versus diesel within −DPF at 2h recovery, p = 0.002; **air versus diesel, within +DPF at 2h recovery, p < 0.001; †−DPF versus +DPF, within diesel at 2h recovery, p < 0.001. (F) MT2A, repeated exposures. Two-way ANOVA, no significant effects. Single exposure to diesel exhaust caused statistically significant increases to IL-6 gene expression both in −DPF and +DPF configurations, with the largest increases observed 2h after diesel-exhaust exposure (Exposure, air vs. diesel, p < 0.001, Recovery, 2h vs. 20h, p = 0.025; Fig. 6A). Repeated exposures to diesel exhaust did not cause any statistically significant changes to IL-6 gene expression (Fig. 6B). Expression of TNF-α gene was significantly elevated 2h after a single exposure to diesel exhaust. However, the levels were not significantly different 20h after recovery in clean air (Exposure × Recovery, air vs. diesel at 2h recovery, p < 0.001, 2h vs. 20h within Diesel, p = 0.002; Fig. 6C). Repeated exposures to diesel exhaust did not cause any statistically significant changes to TNF-α gene expression (Fig. 6D). Gene expression of PTGS2 was significantly elevated 2h after a single exposure to diesel exhaust both in −DPF and +DPF atmospheres. The levels returned to control levels 20h after recovery of animals in clean air (Exposure × Recovery, air vs. diesel at 2h recovery, p < 0.001, 2h vs. 20h within diesel, p < 0.001; Fig. 6E). Expression of PTGS2 gene was not significantly altered by repeated exposures (Fig. 6F). Fig. 6. View largeDownload slide Changes in the expression of IL-6, TNF-α, and PTGS2 in the lung following diesel-exhaust exposures. Values are mean fold change relative to time-matched controls ± geometric standard deviation; n = 8. (A) IL-6, single exposure. Three-way ANOVA, Exposure, *air versus diesel, p < 0.001; Recovery, **2h versus 20h, p = 0.025. (B) IL-6, repeated exposures. Two-way ANOVA, no significant effects. (C) TNF-α, single exposure. Three-way ANOVA. Exposure × Recovery, *air versus diesel at 2h recovery, p < 0.001; **2h versus 20h within diesel, p = 0.002. (D) TNF-α, repeated exposures. Two-way ANOVA, no significant effects. (E) PTGS2, single exposure. Three-way ANOVA, Exposure × Recovery, *air versus diesel at 2h recovery, p < 0.001; **2h versus 20h, within diesel, p < 0.001. (F) PTGS2, repeated exposures. Two-way ANOVA, no significant effects. Fig. 6. View largeDownload slide Changes in the expression of IL-6, TNF-α, and PTGS2 in the lung following diesel-exhaust exposures. Values are mean fold change relative to time-matched controls ± geometric standard deviation; n = 8. (A) IL-6, single exposure. Three-way ANOVA, Exposure, *air versus diesel, p < 0.001; Recovery, **2h versus 20h, p = 0.025. (B) IL-6, repeated exposures. Two-way ANOVA, no significant effects. (C) TNF-α, single exposure. Three-way ANOVA. Exposure × Recovery, *air versus diesel at 2h recovery, p < 0.001; **2h versus 20h within diesel, p = 0.002. (D) TNF-α, repeated exposures. Two-way ANOVA, no significant effects. (E) PTGS2, single exposure. Three-way ANOVA, Exposure × Recovery, *air versus diesel at 2h recovery, p < 0.001; **2h versus 20h, within diesel, p < 0.001. (F) PTGS2, repeated exposures. Two-way ANOVA, no significant effects. Gene expression of HO-1 was significantly increased by a single exposure to both −DPF and +DPF diesel exhaust, with the effects of +DPF exhaust significantly greater than −DPF effects (Exposure × Treatment, air vs. diesel within −DPF, p = 0.001; air vs. diesel within +DPF, p < 0.001; −DPF vs. +DPF within diesel, p < 0.001; Recovery, 2h vs. 20h, p = 0.041; Fig. 7A). Although repeated exposures to −DPF diesel exhaust reduced the expression of HO-1 gene, +DPF exhaust enhanced HO-1 expression (Exposure × Treatment, air vs. diesel within −DPF, p = 0.011; air vs. diesel within +DPF, p = 0.024; −DPF vs. +DPF within diesel, p < 0.001; Fig. 7B). Single exposure to diesel exhaust did not cause any statistically significant changes to iNOS gene expression (Fig. 7C), whereas repeated exposures to diesel exhaust under the +DPF configuration caused a statistically significant increase (Exposure × Treatment, −DPF vs. +DPF within diesel, p < 0.001, air vs. diesel within +DPF, p = 0.002; Fig. 7D). Single exposure to both −DPF and +DPF diesel exhaust caused a significant increase in the expression of SOD2 gene after 2h recovery of animals in clean air, but the expression returned to control levels after 20h recovery (Exposure × Recovery, air vs. diesel within 2h, p < 0.001; 2h vs. 20h within diesel, p < 0.001; Fig. 7E). Repeated exposures to diesel exhaust caused a significant reduction in lung SOD2 gene expression (Exposure, air vs. diesel, p = 0.003; Fig. 7F). Fig. 7. View largeDownload slide Alteration in the gene expression of HO-1, iNOS, and SOD2 in the lung following diesel-exhaust exposures. Values are mean fold change relative to time matched controls ± geometric standard deviation; n = 8. (A) HO-1, single exposure. Recovery, *2h versus 20h, p = 0.041; Exposure × Treatment, **air versus diesel within −DPF, p = 0.001; †air versus diesel within +DPF, p < 0.001; ††−DPF versus +DPF within diesel, p < 0.001. (B) HO-1, repeated exposures. Two-way ANOVA, Exposure × Treatment, *air versus diesel within −DPF, p = 0.011; **air versus diesel within +DPF, p = 0.024; †−DPF versus +DPF within diesel, p < 0.001. (C) iNOS, single exposure. Three-way ANOVA, no significant effects. (D) iNOS, repeated exposures. Two-way ANOVA, Exposure × Treatment, *−DPF versus +DPF within diesel, p < 0.001; **air versus diesel within +DPF, p = 0.002. (E) SOD2, single exposure. Three-way ANOVA, Exposure × Recovery, *2h versus 20h within diesel, p < 0.001; **air versus diesel within 2h, p < 0.001. (F) SOD2, repeated exposures. Two-way ANOVA, Exposure, *air versus diesel, p = 0.003. Fig. 7. View largeDownload slide Alteration in the gene expression of HO-1, iNOS, and SOD2 in the lung following diesel-exhaust exposures. Values are mean fold change relative to time matched controls ± geometric standard deviation; n = 8. (A) HO-1, single exposure. Recovery, *2h versus 20h, p = 0.041; Exposure × Treatment, **air versus diesel within −DPF, p = 0.001; †air versus diesel within +DPF, p < 0.001; ††−DPF versus +DPF within diesel, p < 0.001. (B) HO-1, repeated exposures. Two-way ANOVA, Exposure × Treatment, *air versus diesel within −DPF, p = 0.011; **air versus diesel within +DPF, p = 0.024; †−DPF versus +DPF within diesel, p < 0.001. (C) iNOS, single exposure. Three-way ANOVA, no significant effects. (D) iNOS, repeated exposures. Two-way ANOVA, Exposure × Treatment, *−DPF versus +DPF within diesel, p < 0.001; **air versus diesel within +DPF, p = 0.002. (E) SOD2, single exposure. Three-way ANOVA, Exposure × Recovery, *2h versus 20h within diesel, p < 0.001; **air versus diesel within 2h, p < 0.001. (F) SOD2, repeated exposures. Two-way ANOVA, Exposure, *air versus diesel, p = 0.003. Lung gene expression of pre-proET-1 was elevated following +DPF diesel-exhaust exposure. The expression was significantly increased 2h after exposure to diesel exhaust but returned to control levels after 20h recovery in clean air (Exposure × Treatment × Recovery, −DPF vs. +DPF within diesel at 2h recovery, p < 0.001, air vs. diesel within +DPF at 2h Recovery, p = 0.015; Fig. 8A). There were no statistically significant changes in pre-proET-1 expression following repeated exposures (Fig. 8B). A single exposure to −DPF diesel exhaust caused a statistically significant increase in lung expression of ECE-1 after 20h recovery of animals in clean air (Exposure × Treatment × Recovery, −DPF vs. +DPF within diesel at 20h recovery, p < 0.001; air vs. diesel within −DPF at 20h recovery, p < 0.001; Fig. 8C), whereas repeated exposures to +DPF diesel exhaust caused a significant reduction in the gene expression of ECE-1 (Exposure × Treatment, −DPF vs. +DPF within diesel, p < 0.001; air vs. diesel within +DPF, p = 0.003; Fig. 8D). Single exposure to diesel exhaust caused a statistically significant decrease in the gene expression of eNOS in the lung (Exposure, air vs. diesel, p < 0.001; Fig. 8E), an effect seen across all treatment groups (−DPF and +DPF) and recovery time points (2 and 20h); changes were not statistically significant in the repeated exposure animals (Fig. 8F). Expression of other genes analyzed in the lung (namely IL-1β, ETAR, ETBR, GSR, GPX-1, GSTM1, and SOD3; Supplementary Data) and heart (namely eNOS; Supplementary Data) was also significantly affected by either single or repeated exposures to diesel exhaust. Gene expression of IL-6, IL-1β, PTGS2, TNF-α, MT2A, CYP1A1,iNOS, ET-1, ECE-1, ETAR, ETBR, HO-1, GSR, GPX-1, GSTA1, GSTM1, SOD2, and SOD3 in the heart were not significantly altered by exposure to diesel exhaust (Supplementary Data). Fig. 8. View largeDownload slide Changes in the gene expression of pre-proET-1, ECE-1, and eNOS in the lung, and the ratio of bigET-1 to ET-1 peptides in the plasma of animals following exposures to diesel exhaust. Gene expression data are mean fold change relative to time-matched controls ± geometric standard deviation; plasma endothelin ratios are mean fold changes relative to time matched controls ± SEM; n = 8. (A) Lung pre-proET-1, single exposure. Three-way ANOVA, Exposure × Treatment × Recovery, *−DPF versus +DPF, within diesel at 2h recovery, p < 0.001; **air versus diesel within +DPF at 2h recovery, p = 0.015. (B) Lung pre-proET-1, repeated exposures. Two-way ANOVA, no significant effects. (C) Lung ECE-1, single exposure, Exposure × Treatment × Recovery, *−DPF versus +DPF within diesel at 20h recovery, p < 0.001; **air versus diesel within −DPF at 20h recovery, p < 0.001. (D) Lung ECE-1, repeated exposures. Exposure × Treatment, *−DPF versus +DPF within diesel, p < 0.001; **air versus diesel within +DPF, p = 0.003. (E) Lung eNOS, single exposure, three-way ANOVA, Exposure, *air versus diesel, p < 0.001. (F) Lung eNOS, repeated exposures. Two-way ANOVA, no significant effects. G) Plasma bigET-1/ET1, single exposure. Three-way ANOVA, Exposure × Recovery, *2h versus 20h, within diesel, p = 0.004; **air versus diesel at 2h recovery, p = 0.037. (H) Plasma bigET-1/ET1, repeated exposures. Two-way ANOVA, no significant effects. Fig. 8. View largeDownload slide Changes in the gene expression of pre-proET-1, ECE-1, and eNOS in the lung, and the ratio of bigET-1 to ET-1 peptides in the plasma of animals following exposures to diesel exhaust. Gene expression data are mean fold change relative to time-matched controls ± geometric standard deviation; plasma endothelin ratios are mean fold changes relative to time matched controls ± SEM; n = 8. (A) Lung pre-proET-1, single exposure. Three-way ANOVA, Exposure × Treatment × Recovery, *−DPF versus +DPF, within diesel at 2h recovery, p < 0.001; **air versus diesel within +DPF at 2h recovery, p = 0.015. (B) Lung pre-proET-1, repeated exposures. Two-way ANOVA, no significant effects. (C) Lung ECE-1, single exposure, Exposure × Treatment × Recovery, *−DPF versus +DPF within diesel at 20h recovery, p < 0.001; **air versus diesel within −DPF at 20h recovery, p < 0.001. (D) Lung ECE-1, repeated exposures. Exposure × Treatment, *−DPF versus +DPF within diesel, p < 0.001; **air versus diesel within +DPF, p = 0.003. (E) Lung eNOS, single exposure, three-way ANOVA, Exposure, *air versus diesel, p < 0.001. (F) Lung eNOS, repeated exposures. Two-way ANOVA, no significant effects. G) Plasma bigET-1/ET1, single exposure. Three-way ANOVA, Exposure × Recovery, *2h versus 20h, within diesel, p = 0.004; **air versus diesel at 2h recovery, p = 0.037. (H) Plasma bigET-1/ET1, repeated exposures. Two-way ANOVA, no significant effects. Plasma Endothelins Inhalation of diesel exhaust resulted in consistent, yet nonstatistically significant elevation of plasma bigET-1, ET-1, and ET-2 (Supplementary Data). Alteration of the endothelinergic system was nevertheless confirmed by the statistically significant elevation of the ratio of bigET-1 to ET-1 after acute exposure to both –DPF and +DPF exhausts (Exposure × Recovery, 2h vs. 20h within diesel, p = 0.004, air vs. diesel at 2h recovery, p = 0.037; Fig. 8G). No significant effects of diesel-exhaust exposure were noted after repeated exposures (Fig. 8H). Plasma 8-Isoprostane Single exposures to diesel exhaust did not cause any statistically significant changes in plasma levels of 8-isoprostane (Fig. 9A), although marginally significant increases in the levels of 8-isoprostane were noted in response to repeated exposures to diesel exhaust under both −DPF and +DPF atmospheres (Exposure, air vs. diesel, p = 0.059; Fig. 9B). Fig. 9. View largeDownload slide Changes in plasma levels of 8-isoprostane following diesel-exhaust exposures. Values are mean fold changes relative to time matched controls ± SEM, n = 8. (A) Single exposure. Three-way ANOVA, no significant effects. (B) Repeated exposures. Two-way ANOVA, Exposure, air versus diesel, p = 0.059. Fig. 9. View largeDownload slide Changes in plasma levels of 8-isoprostane following diesel-exhaust exposures. Values are mean fold changes relative to time matched controls ± SEM, n = 8. (A) Single exposure. Three-way ANOVA, no significant effects. (B) Repeated exposures. Two-way ANOVA, Exposure, air versus diesel, p = 0.059. DISCUSSION The following key observations were made. (1) Treatment of exhaust using a catalyzed DPF not only reduced particle mass emissions but also reduced levels of hydrocarbons including carbonyls and volatile organic compounds measured in the gas phase, CO, NO, and NOx. (2) In contrast, the +DPF treatment led to a 300% increase in the levels of NO2 in the exhaust and 38% increase in particle count, with a shift in particle size mode from 70nm down to 8nm measured in the inhalation chambers. (3) Inhaled diesel exhaust affected markers of acute inflammation, oxidative stress, and cardiovascular effects in lung, heart and plasma. Inflammation was significantly enhanced after single exposure to diesel exhaust but disappeared after repeated exposures. (4) Biological effects of diesel exhaust were generally enhanced after treatment of exhaust with a catalyzed DPF, consistent with the toxicology of nitrogen dioxide, a strong oxidant gas. (5) Nevertheless, changes of several endpoints did not correlate with the levels of NO2, indicating that different components of the inflammation response may be driven by different agents within the exhaust. (6) Vascular oxidative stress and endothelial dysfunction, reflected in the activation of the endothelinergic system and decreased expression of lung eNOS appeared to relate more directly to the ultrafine particle count rather than with inhaled particle mass or NO2 concentration. Diesel exhaust is a complex mixture consisting of carbonaceous soot particles coated with organic species including aldehydes, alkanes and alkenes, polyaromatic hydrocarbons (PAH), PAH derivatives and inorganic ions, as well as a gas phase consisting of CO, CO2, oxides of nitrogen, oxides of sulfur, volatile and semivolatile organic compounds, and water vapor (Ris, 2007; Wichmann, 2007). In the present study, substantial reductions were observed in particle mass concentration, as well as in the levels of CO and gas-phase total hydrocarbons after treatment of diesel exhaust with a catalyzed DPF, and these emission changes are in line with previous reports (Herner et al., 2009; Shah et al., 2007). However, a large increase in exhaust levels of NO2 is also expected from catalyzed DPFs (Herner et al., 2009; Lucking et al., 2011), which is intended to facilitate the oxidative removal of diesel soot trapped in the filter. Notwithstanding the large reductions in particle mass, the formation of a larger number of ultrafine particles of 8nm size mode in the DPF-treated exhaust in the present study points to potential formation of new nucleation mode particles during, as well as immediately post, filter regeneration, as has been shown previously for particulate filters (Holmén and Ayala, 2002; Khalek et al., 2011). Evidently, these changes in the emission profile led to biological responses that appear sensitive to elevated NO2 concentration of the +DPF exhaust and those impacted by changes in the particulate phase, as discussed below. We attribute the enhanced inflammatory responses in animals exposed to +DPF exhaust, largely to the elevated levels of NO2 in the exhaust as the pattern of inflammatory changes observed in our study are in agreement with the known toxicology of NO2, an oxidant gas with strong inflammatory potential. The inflammation response to untreated diesel exhaust was typified by lung neutrophilia and was accompanied by increases in BAL total protein, indicative of mild lung injury. The levels of neutrophil chemoattractant and activator GRO/KC in BAL fluid anticipated the inflammatory cascade after exposure to diesel exhaust. The inflammation response was corroborated by enhanced gene expression of MT2A, IL-6, and PTGS2 in the lung tissue 2h post diesel-exhaust exposure. When animals were exposed to exhaust treated by DPF, the levels of most inflammatory endpoints were 2- to 4-fold higher, consistent with the 4-fold increase in NO2 concentration. For example, the magnitude of changes in lavage MCP-1 and lung MT2A gene expression after exposure to +DPF exhaust containing 16 ppm NO2 in our study were comparable with previous reports for BALB/c mice exposed to 15 ppm NO2 (Johnston et al., 2000; Johnston et al., 2001). Channell et al. (2012) showed that blood plasma from human subjects exposed to 0.5 ppm NO2, or to dilute diesel exhaust containing 100 µg/m3 particles and 0.8 ppm NO2, was able to activate expression of intracellular cell adhesion molecule when fed to primary cultures of coronary endothelial cells. Interestingly, the expression of CYP1A1 decreased with +DPF exhaust exposure, potentially in response to the heightened inflammation status. Transcriptional suppression of P450 enzymes in response to inflammation has previously been demonstrated (Wright and Morgan, 1990). Although NO2 may have largely contributed to the inflammatory changes observed in our study, it is possible that the smaller ultrafine mode generated by +DPF treatment also contributed to the inflammatory response. For example, gene expression of the inflammatory mediators TNF-α and IL-1β in the lungs did not correlate well with NO2 concentration. Although we used ultralow sulfur diesel, the ultrafine mode is known to contain sulfuric acid particles. Catalyzed DPF oxidizes sulfur dioxide contained in the exhaust to SO3, which combines with water vapor to form sulfuric acid aerosols. These aerosols nucleate to sulfuric acid particles (Kittelson et al., 2008). DPFs do not remove these aerosol nanoparticles. Sulfuric acid is a known irritant with a potential to cause an inflammatory response (Amdur and Chen, 1989). Whereas single exposures to diesel exhaust caused inflammation, inflammatory changes were generally absent after repeated exposures, possibly due to an adaptive response after repeated exposures. Involvement of adaptive defense mechanisms has previously been proposed in pulmonary and cardiovascular effects of long-term exposure to traffic-related particulate matter (Gerlofs-Nijland et al., 2010). Significantly increased macrophage counts in the repeated exposure scenario measured at the end of 3 days of exposure followed the particle burden and initial inflammation response (peaked at 2 and 20h postexposure), consistent with known biological role of macrophages in the cleanup of apoptotic neutrophils from sites of inflammation (Bratton and Henson, 2011). Accordingly, enhanced neutrophilia was accompanied by increased production of the macrophage chemoattractant proteins, MIP-1α and MCP-1. Most oxidative stress effects were sensitive to heightened NO2 levels of +DPF exhaust, whereas some effects appeared responsive to changes in the particulate phase of diesel exhaust. Significant induction of MT2A and HO-1 (single and repeated exposures) and iNOS (repeated exposures) gene expression in the lungs in response to diesel-exhaust exposure may relate to pulmonary oxidative stress driven by NO2. It has been previously shown that NO2 exposures in Wistar rats can result in marked oxidative stress as indicated by increased activity of Mn-superoxide dismutase and glutathione peroxidase, and levels of malondialdehyde and protein carbonyls in the heart (Li et al., 2011). Early increase of SOD2 and GSTA1 gene expression in the lungs 2h after exposure and increase in circulating levels of 8-isoprostane, as well as decrease of SOD2 gene expression after repeated exposures, were not related to NO2 concentration. It is possible that changes in these particular oxidative stress endpoints may be attributable to effects of the ultrafine particles penetrating the septum and potentially reaching the endothelium. Induction of SOD2, GSTA1, and GPX-1 is a classical response to oxidative stress and may account for a transient adaptive response. However, the decrease of SOD2 after repeated exposure to diesel exhaust is of concern as this may be due to mitochondrial injury with consequent elevation of superoxide flux, evidenced here by the elevation of 8-isoprostane. Therefore, damage from diesel exhaust may persist despite a transient adaptive response. Endothelin-1 is expressed in endothelial cells and macrophages (Khimji and Rockey, 2010). Because of the strong inflammatory response after exposure of the animals to diesel exhaust, the changes in pulmonary pre-proET-1 gene expression observed in our model cannot be attributed specifically to changes within the pulmonary endothelium. The lungs are known to be a major site of ETB receptor-mediated clearance of ET-1, as well as a source of circulating ET-1 and bigET-1 (Fagan et al, 2001). In our study, we have observed a consistent elevation of circulating bigET-1, ET-1, and ET-2 peptides after exposure to diesel exhaust, although changes of individual markers were not statistically significant. Inhalation of urban particulate matter increases plasma ET-1 and blood pressure in rats (Bouthillier et al., 1998; Vincent et al., 2001), and elevation of plasma ET-2 has been observed following inhalation of ultrafine particles (Elder et al., 2004). Activation of the endothelinergic system in the present study is supported by the statistically significant early elevation of the bigET-1 to ET-1 ratio in plasma. The bigET-1 to ET-1 ratio reflects the relative abundance of the precursor peptide and the mature peptide from the balance between the rate of de novo synthesis of bigET-1, the rate of conversion of bigET-1 to ET-1 by endothelin-converting enzymes and chymase, and the rate of clearance from circulation. In general terms, an increase of the bigET-1 to ET-1 ratio suggests an activation of endothelin production due to transcriptional activation, and a decrease of the ratio suggests a higher rate of conversion by ECE-1 (Télémaque et al., 1998; Battistini and Kingma, 2000). Increased production of the vasoconstrictor ET-1 and decreased release of the vasodilator NO are hallmarks of endothelial dysfunction in cardiovascular disease and diabetes (Fagan et al., 2001; Farhangkhoee et al., 2006; Shah, 2007). Increased expression of pre-proET-1, indicated here by the increase of plasma bigET-1 to ET-1 peptides ratio in animals exposed to diesel exhaust, coupled with alteration of pulmonary endothelial cell NO pathway, indicated by the decrease of endothelial cell eNOS gene expression, argues for an alteration of the physiology of pulmonary capillary endothelial cells. Cardiovascular effects of diesel exhaust can be mediated by the gas phase, as shown by Campen et al., (2005) when bradycardia and T-wave depression in ApoE−/+ mice were not abolished by removal of the particulate phase. However, the enhanced ratio of plasma bigET-1 to ET-1 peptides (p = 0.037), the elevation of plasma isoprostane (p = 0.059), and the decreased eNOS gene expression in the lungs (p < 0.001) under both −DPF and +DPF configurations suggest that deposition of primary and secondary ultrafine particles is an important driver of vascular oxidative stress and endothelial dysfunction. In this respect, particulate mass deposition may not be a useful metric of ultrafine particle dose. We aimed in our study to assess the impact of a catalyzed diesel particle filter on the toxicity of whole diesel-exhaust emissions. The challenge in the toxicological investigation of whole diesel exhaust is to disentangle the biological responses to a very complex mixture and reveal the relative effects of the gas and particulate phases, and their interaction. We have identified effects clearly attributable to the potent oxidant NO2, notably the elevation of indices of acute lung injury and pulmonary inflammation. Nevertheless, changes in several indicators of oxidative stress and endothelial dysfunction did not correlate with NO2 levels and suggest effects attributable to the ultrafine particles; exposure of animals to HEPA-filtered diesel exhaust should allow the validation or refutation of our interpretation. With the continued use of catalyzed DPFs, concerns remain on large NO2/NOx ratios in the exhaust and formation of smaller nanoparticles including sulfuric acid aerosols. Biological effects relating to potential nitration and chlorination of primary exhaust constituents by oxidation catalysts and release of catalytically active metals from fuel additives warrant continued investigations. Integration of DPF with new engine technologies, fuels and fuel blends, and other emission treatment systems will only increase the need for further toxicological assessment. FUNDING Natural Resources Canada through the Program of Energy Research and Development (Advanced Fuels and Technologies for Emission Reduction Project C24.002 and Particles and Related Emissions Project C14.003). 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TI - Nitrogen Dioxide and Ultrafine Particles Dominate the Biological Effects of Inhaled Diesel Exhaust Treated by a Catalyzed Diesel Particulate Filter
JF - Toxicological Sciences
DO - 10.1093/toxsci/kft162
DA - 2013-07-27
UR - https://www.deepdyve.com/lp/oxford-university-press/nitrogen-dioxide-and-ultrafine-particles-dominate-the-biological-FNTwAVFUOg
SP - 437
EP - 450
VL - 135
IS - 2
DP - DeepDyve
ER -