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Published ahead of print on March 26, 2008, doi:10.1165/rcmb.2008-0057OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 198-207, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2008-0057OC

Soot Nanoparticles Promote Biotransformation, Oxidative Stress, and Inflammation in Murine Lungs

Rodney L. Rouse1, Gleeson Murphy1,*, Marc J. Boudreaux2, Daniel B. Paulsen3 and Arthur L. Penn1

1 Comparative Biomedical Sciences, 2 Division of Biotechnology and Molecular Medicine (BioMMED), and 3 Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana

Correspondence and requests for reprints should be addressed to Arthur Penn, PhD, Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Skip Bertman Drive, Baton Rouge, LA 70803. E-mail: apenn{at}vetmed.lsu.edu


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We previously described the physicochemical characteristics (particle size, adsorbed polynuclear aromatic hydrocarbons [PAHs], oxygen, and metal content) of butadiene soot (BDS) nanoparticles generated during incomplete combustion of the high-volume industrial petrochemical, 1,3-butadiene. We also demonstrated localization of BDS-delivered PAHs to lipid droplets of murine and human respiratory cells in vitro and up-regulation of biotransformation and oxidative stress responses in these cells. Here, the objective was to determine whether inhalation of BDS nanoparticles promotes up-regulation of Phase I biotransformation enzymes, oxidative stress responses, and inflammation in the lungs of mice. Female Balb/c mice exposed to BDS (5 mg/m3, 4 h/d, 4 d) were killed immediately or 1 day after final exposure; bronchoalveolar lavage fluid (BALF) was collected from the lungs; total RNA was extracted from one lung and histopathology performed on the other. Histopathology and BALF analysis revealed particle-laden macrophages in airways of BDS-treated mice, accompanied by neutrophilia and epithelial damage. Microarray and qRT-PCR analyses revealed up-regulation of (1) aryl hydrocarbon receptor (AhR)-responsive genes: AhR repressor (Ahrr) and cytochrome P450 IA1 and IB1(Cyp1a1, Cyp1b1); (2) oxidative stress response genes: heme oxygenase 1 (Hmox1), nuclear factor erythroid-derived 2–like 2 (Nfe2l2), NADPH dehydrogenase quinone 1 (Nqo1), and glutathione peroxidase 2 (Gpx2); and (3) pro-inflammatory genes: interleukin-6 (IL-6), C-X-C motif ligand 2 (Cxcl2; analog to human IL-8) and ligand 3 (Cxcl3), and granulocyte chemotactic protein (Cxcl6). Inhalation of PAH-rich, petrochemical combustion–derived nanoparticles causes airway inflammation and induces expression of AhR-associated and oxidative stress response genes, as seen in vitro, plus pro-inflammatory genes.

Key Words: inhalation • combustion nanoparticles • lung inflammation • biotransformation • cytokine genes



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
This research describes a novel nanoparticle source of human polynuclear aromatic hydrocarbon inhalation exposure and defines the early molecular events associated with, and in response to, this exposure.

 
Urban ambient pollution particles are composed of a complex array of organic and inorganic components, often adsorbed to a carbonaceous core. Fine particles (PM2.5; aerodynamic diameter 0.1–2.5 µm) contribute to ambient pollution and to cardiopulmonary morbidity (1). PM2.5 exposure correlates with increased cardiopulmonary and lung cancer mortality (2, 3), as well as increased risk of respiratory and cardiovascular disease (4, 5). Exposure to the coarse (PM10; aerodynamic diameter 2.5–10.0 µm) particulate component of air pollution generates an increase in the circulating inflammatory cytokines, IL-1β, IL-6, and IL-8 (6). After comparable exposure in vitro, alveolar macrophages and bronchial epithelial cells produced these same cytokines, suggesting that cytokines produced by respiratory cells can act locally and systemically (6, 7). To date, ultrafine particles (PM0.1; aerodynamic diameter < 0.1 µm) and nanoparticles (aerodynamic diameter < 0.150 µm) have received little attention from regulatory agencies (8), but in numerous experimental settings have been found to elicit a range of toxicologic effects (often more severe than those found with comparable exposures to fine particles), including inflammatory cell infiltration and impaired macrophage phagocytosis (9, 10).

The surface characteristics and crystalline structure of ambient particles can be major determinants of pulmonary inflammation and injury (11, 12). Individually or in concert, members of the complex chemical array adsorbed to particles also can promote disease processes. The aqueous, inorganic, transition metal–enriched fraction of residual oil fly ash (ROFA) induces edema, hemorrhage, and a profound inflammatory infiltrate in the lung (13). Diesel exhaust particles (DEPs) containing a variety of oxygen radical–generating quinones, polyaromatic hydrocarbons (PAHs), and metal species (14–16) produce pulmonary inflammatory cell infiltration and inflammatory cytokine production by bronchial epithelial cells and alveolar macrophages (17–19). Most of the effects have been attributed to the PAH and quinone components of DEPs. A body of literature, both in vitro and in vivo, has accumulated characterizing the effects of the parent particles and their isolated constituents in humans, as well as in animals (20, 21).

In urban areas, combustion of gasoline, diesel fuels, and industrial organics (simple aliphatics and/or fossil fuels) contributes significantly to the ambient PM2.5 fraction (22) and to the ultrafine particulate fraction (23). Incomplete combustion of low-molecular-weight hydrocarbons, as in the case of industrial flaring of fugitive volatiles, is also a real source of complex environmental particulate contamination. Furthermore, petrochemical-derived nanoparticles resulting, by accident or sabotage, from refinery or pipeline explosions and fires represent a real hazard both in the United States and abroad.

In the burning of hydrocarbons, radicals formed early in combustion interact, forming PAHs, including carcinogens, from less complex structures. PAHs will aggregate into nanoparticles, which can extend into branched-chain structures (soots); soot particles appear as black smoke. We have previously characterized the product of incomplete combustion of 1,3-butadiene (BD), a high-volume aliphatic hydrocarbon byproduct of petroleum refining that is used in the manufacture of synthetic rubber and other elastomers. This product, butadiene soot (BDS), is both a model mixture and a real-life example of PAH-laden, combustion-derived nanoparticles with potential for environmental contamination and for acute and/or chronic health effects (24).

BDS is an organic-rich mixture of 30- to 50-nm carbonaceous particles to which hundreds of PAH species, including benzo(a)pyrene [B(a)P] and other carcinogens, are adsorbed (24, 25). In contrast to ROFA and DEPs, BDS is relatively oxygen- and metals-poor (24, 25). Both human bronchoepithelial cells and mouse alveolar macrophages display a distinct punctate blue, PAH-associated cytoplasmic fluorescence after exposure to BDS in vitro. The fluorescence localizes to cytoplasmic lipid droplets even as Phase I biotransformation enzymes are induced (26). Fluorescence does not develop if PAHs are extracted from the particles before cell exposure. Fluorescent cells contain the same spectrum of PAHs present in the parent BDS (24). In vivo responses to BDS exposure have not been reported. We hypothesized that inhalation of PAH-containing BDS will result in activation of aryl hydrocarbon receptor (AhR)-associated genes (as is the case with other PAH-rich mixtures [DEPs, cigarette smoke]), will cause oxidative stress, and will result in inflammatory changes, including inflammatory infiltrate and up-regulation of inflammatory cytokines (as is the case with DEP). Although there is evidence associating inhalation of petrochemical combustion products to subsequent respiratory diseases, much less is known about lung-specific changes in gene expression during and after exposure to many of these compounds. In this study, we address that knowledge gap.

The questions addressed were:

(1) Does inhalation of freshly generated BDS nanoparticles induce pulmonary inflammatory responses in mice?
(a) Is there histologically demonstrable inflammatory pulmonary infiltrate?
(b) Is airway hyperresponsiveness increased?
(c) Are inflammatory changes seen in bronchoalveolar lavage (BAL) fluid (BALF) cytology?
(d) Are there increases in pro-inflammatory cytokines in BALF?
(2) Does inhalation exposure to BDS alter gene expression in the lungs?
(a) Phase I biotransformation enzyme genes?
(b) Oxidative stress response genes?
(c) Pro-inflammatory cytokine genes?


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals
Eighteen 6-week-old female Balb/c mice were obtained from Jackson Laboratories (Stock 000651; Bar Harbor, ME). After a 1-week quarantine period, animals were housed individually in suspended steel wire cages at the AAALAC-accredited Inhalation Research Facility at Louisiana State University. The mice were handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals (27); and all procedures were approved by the Louisiana State University Institutional Animal Care and Use Committee. Food and water were provided ad libitum between exposures, but were removed during the exposures to prevent their contamination with particles and/or chemical residues. All procedures included considerations for alleviation of animal discomfort or distress. Killing was by intraperitoneal injection of 0.2 ml Beuthanasia-D Special (Schering-Plough, Union, NJ).

BDS Exposures
BDS was generated as described previously (24). BD was burned in a 0.25 m3 stainless steel and Plexiglas chamber that was connected to an adjacent stainless steel and Plexiglas 0.25 m3 inhalation chamber in which mice were exposed to the resultant BDS. BDS was drawn from the generation chamber to the exposure chamber by static pressure differential. HEPA-filtered air was used to maintain a steady-state BDS particle concentration of 5.0 mg/m3 in the exposure chamber. Preliminary experiments at this concentration produced no overt toxicity. Particle concentration in the exposure chamber was monitored in real time with a DustTrak (Model 8520; TSI Inc., St. Paul, MN); and this concentration was calibrated daily by gravimetric filter comparison. Both the DustTrak probe and the filter holder were positioned in the airflow immediately above the cages housing the mice. Detailed descriptions of the PAH and metals content of BDS have been presented (25).

Ten mice inhaled BDS mixed with HEPA-filtered air (4 h/d, 4 d), and eight were exposed only to HEPA-filtered air for the same time. Six BDS-exposed mice and four control mice were killed and sampled immediately after the fourth day of exposure. The remaining four mice from each group were killed and sampled the following day.

Airway Hyperresponsiveness
We assessed airway hyperresponsiveness in unrestrained mice by whole body plethysmography (Buxco, Troy, NY) immediately after the final BDS exposure and 1 day before killing. Data were expressed as a dimensionless value, Penh, or "enhanced pause." After acclimation and aerosol saline exposure, we challenged mice with graded doses (1.56–50.0 mg/ml) of nebulized methacholine (Sigma, St. Louis, MO) to assess airway response. We recorded post-exposure Penh values over 5 minutes for each dose.

BALF Collection and Analysis
After mice were killed, we lavaged lungs twice with 0.5 ml phosphate-buffered saline passed through a 19-gauge cannula anchored in the trachea. We immediately placed the pooled BALF on ice. We performed 300 cell leukocyte differential counts on modified Wright's-stained cytocentrifuge slide preparations of 400-µl aliquots of raw BALF. The leukocytes were categorized by type (macrophage, neutrophil, eosinophil), and macrophages were graded by particle burden. Group 1 macrophages (M{Phi}1) had less than 10% cytoplasmic particle burden. Group 2 macrophages (M{Phi}2) had 10 to 50% of the cytoplasm occupied by particles. Group 3 macrophages (M{Phi}3) had more than 50% of their cytoplasm occupied by particles. BALF was analyzed by enzyme-linked immunosorbent assay (ELISA) for the presence of selected cytokines (TNF-{alpha}, IL-6, and the murine homolog of interleukin-8 [MIP2/ Cxcl2]). ELISA kits (BD-Pharmingen, San Diego, CA) were used according to manufacturer's guidelines. The lower limit of detection for all of the ELISA kits was 7.8 ng/ml.

Histopathology
We perfused the right lung with 0.4 ml of freshly prepared 0.02 M periodate/0.1 M lysine/0.25% paraformaldehyde (PLP) fixative in phosphate buffer (pH 7.4), then excised and stored the lungs in PLP for 24 to 48 hours before standard histologic sectioning, processing for hematoxylin and eosin staining, and evaluation by a board-certified veterinary pathologist. Lung histopathology was scored according to six parameters: particles found within (1) alveolar macrophages, (2) interstitial macrophages, and (3) bronchial epithelium; numbers of (4) peribronchial neutrophils and (5) transmigratory neutrophils; and by (6) epithelial damage. Scoring was 0/1 (present or absent) for particulate matter in macrophages and epithelium. All other parameters were scored 0 to 3 (0, none; 1, mild; 2, moderate; 3, severe).

RNA Isolation
We isolated and excised the left lung for storage in RNAlater (Applied Biosystems, Foster City, CA). Each lung was removed from RNAlater 24 to 48 hours after collection, gently blotted of excess solution, and placed into a clean 2-ml microcentrifuge tube with 1 ml TRIzol Reagent (Invitrogen, Carlsbad, CA) and a 4.5 mm copper-coated bead. We homogenized the lung tissue with two 2-minute 25-Hz passages on a Mixer Mill MM300 (Qiagen, Valencia, CA). We purified RNA from the aqueous phase of the lung homogenate with the Qiagen RNeasy Mini Kit, including RNase-free DNase treatment. We used an additional Buffer RPE wash to remove residual salts, followed by an additional 2-minute spin to evaporate residual ethanol. We measured RNA concentrations with an ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE), and we assessed RNA quality and integrity with the Agilent RNA 6,000 Nano Assay Kit and the Model2100 BioAnalyzer (Agilent, Santa Clara, CA). We converted total RNA to cDNA with a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol.

Gene Microarray Assay
Global gene expression was assessed from total RNA on Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA). The arrays were processed at the Research Core Facility of Louisiana State University Health Science Center-Shreveport. Double-stranded cDNA synthesized from total RNA was used to create cRNA, which was biotinylated, fragmented, and added to a hybridization cocktail that included probe array controls, bovine serum albumin, and herring sperm DNA. This cocktail was hybridized (16 h; 45°C) to oligonucleotide probes on microarrays in a GeneChip Hybridization Oven 640. Immediately after hybridization, the array underwent an automated washing and staining protocol on a GeneChip Fluidics Station and was scanned with a GeneChip Scanner 3000. Collection and processing of initial raw data were performed by a GeneChip Workstation. All gene chips and instrumentation were from Affymetrix (Santa Clara, CA). All data collected and analyzed here adhere to the guidelines for Minimal Information About a Microarray Experiment (MIAME).

Gene Expression Analysis
GeneChip Workstation data were sent to Expression Analysis Systems (Durham, NC). Initially, a principle component analysis was used to determine clustering of experimental units. The analysis revealed clustering by treatment group with greater variation between groups than within groups (data not presented), thus validating subsequent comparisons between treatment groups. Before making these comparisons, data were subjected to reduction of invariant probes (REDI) to remove previously determined mal-performing probes from the data set. Treatment group data underwent permutation analysis for differential expression (PADE); this accounts for false positives by tabulating a false discovery rate (FDR) based on a permutation-generated reference curve (technical information on REDI and PADE analyses available at www.expressionanalysis.com). After REDI and PADE, expression data were tabulated for each remaining transcript, including individual transcript P value, FDR, fold change, Affymetrix probe ID, gene symbol, and functional summary. All transcripts included in this study had a fold change of at least 1.5 (up or down), and both an individual P value and FDR <= 0.05.

Pathway Analysis
We analyzed gene expression data with the network- and pathway-building software, Ingenuity Pathways Analysis 4.0 (Ingenuity Systems, Redwood City, CA). We examined gene networks and canonical pathways using the Ingenuity Analysis Knowledge Database (Ingenuity Systems) and identified networks and pathways for phenotypic relevance. We identified select genes from the literature for confirmation by quantitative real-time PCR analysis. We created custom networks to demonstrate the connections between the genes identified in our expression analyses.

Quantitative Real-Time RT-PCR
We performed quantitative RT-PCR (qRT-PCR) for selected genes on cDNA from lung homogenates with inventoried TaqMan Gene Expression Assays primer-probe sets (Applied Biosystems, Foster City, CA). Reaction volumes were 25 µl, and 40 reaction cycles were performed for each gene in an Applied Biosystems 7300 Real Time PCR System. We determined relative gene expression by the comparative cycle threshold ({Delta}{Delta}CT) method, with each gene normalized to hypoxanthine guanine phosphoribosyl transferase (Hprt1) expression (48). Results are reported as fold change over control ± standard error of the mean [(2–{Delta}{Delta}CT) ± SEM].

Statistical Analysis
We used the UNIVARIATE and TTEST procedures of the SAS statistical package (version 9.1.3; SAS Institute, Inc., Cary, NC) to compare qRT-PCR data from lungs. We used a folded F test for each dataset to determine if the variance across the set was statistically "equal," in which case the variances could be pooled for determining statistical differences. For the occasional cases of unequal variance across a dataset, we used Satterthwaite's approximation of degrees of freedom to determine statistical significance. We used SigmaStat 3.1 software (Systat Software Inc., San Jose, CA) to analyze cytokine data via Mann-Whitney Rank Sum t tests and to compare histopathology scores with a Kruskal-Willis one-way ANOVA on ranks with post hoc pairwise evaluation by the Holm-Sidak method at {alpha} = 0.05.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Airway Hyperresponsiveness
No significant differences between BDS-exposed mice and controls were noted with unrestrained whole body plethysmography (data not shown).

BALF Differentials
The differential cytology of BALF from air- and BDS-exposed mice, as well as the grouping of macrophages by particle burden, is presented in Figure 1. In air control mice, essentially all the BALF cells were M{Phi}1 macrophages with fewer than five particles per cell. Those particles may be incidental room air particles that escaped the HEPA filter, or food/litter particles. In the BDS-exposed mice, neutrophil concentration was profoundly increased (> 10x); less than 1% of neutrophils contained particles. The particle burden of alveolar macrophages also was increased (> 50% of the macrophages present were M{phi}2 or M{phi}3); with 1 day of recovery after BDS exposure, BALF neutrophilia increased in BDS-exposed mice (> 2x; P = 0.002). Thus, BALF differential results support BDS-induced airway inflammation.


Figure 1
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  		Figure 1. Differential leukocyte counts of bronchoalveolar lavage fluid (BALF) from mice exposed to filtered air or butadiene soot (BDS). Inhalation exposure to 5 mg/m3 BDS for four consecutive days leads to neutrophilic infiltration into the bronchoalveolar space. Alveolar macrophages (M{phi}), the major cell-type found in BALF of normal mice, ingest the BDS particles. One day after exposure, neutrophilia is augmented and macrophages containing particulate material predominate (> 50% M{phi}2 or M{phi}3). Representative particle-laden macrophages are shown in the lower portion of the figure.

 
BALF Cytokines
IL-8 was significantly higher in BDS-exposed mice with or without a day of recovery (Figure 2). However, after a day of recovery, IL-8 was further elevated in BDS-exposed mice. This finding is consistent with the increased BALF neutrophilia (described above) in these mice. No significant differences were detected in TNF-{alpha} and IL-6 levels between BDS-exposed and control mice. Mice from both groups that were allowed 1 day of recovery had significantly lower IL-6 levels than their immediately killed counterparts (data not presented).


Figure 2
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  		Figure 2. IL-8 Concentrations are elevated in BALF of BDS-exposed mice. IL-8 enzyme-linked immunosorbent assay reveals significantly higher IL-8 in BALF of mice exposed to BDS relative to control cohorts regardless of the timing of killing and sampling. Samples presented in ng/ml, mean ± SE. BDS-exposed, immediately killed mice n = 6; other groups n = 4. *Significant difference (P < 0.001) between BDS-exposed and control mice; {dagger}significant difference (P = 0.029) between BDS-exposed and control mice.

 
Lung Histopathology
Figure 3 shows examples of histopathologic changes in the lungs of control (Figure 3A), BDS-exposed mice killed immediately after their final BDS exposure (Figure 3B), and BDS-exposed mice killed 1 day after final exposure (Figure 3C). Lung histopathology was assessed according to whether particles were found within alveolar macrophages or interstitial macrophages, by particles apposed to or within cells of the bronchial epithelium, and by location of neutrophils (Table 1). Control mice show no signs of pulmonary inflammation (Figure 3A). An inflammatory response in BDS-exposed mice killed immediately after final exposure (Figure 3B) was evidenced by peribronchial neutrophilia and migration of neutrophils into the bronchoalveolar space. BDS-exposed mice killed 1 day after exposure exhibited increasing neutrophilia, as well as bronchiolar epithelial damage, including basement membrane disruption accompanied by the neutrophilic infiltration (Figure 3C). The BALF and histopathology results indicate that inhalation of these combustion-derived nanoparticles elicits a time-dependent increase in particle accumulation by alveolar macrophages, an associated deep lung neutrophilia, and persistence of particles in the pulmonary interstitium.


Figure 3
Figure 3
Figure 3
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  		Figure 3. BDS-exposed mice experience a neutrophilic inflammatory pulmonary infiltrate. (A) Photomicrograph of lungs from a mouse exposed to HEPA-filtered air. Notice the scattered resident population of macrophages, in the interstitium and alveolar spaces (black arrowheads). Hematoxylin and eosin (H&E) stain. (B) Photomicrograph lung from a mouse exposed to 5 mg/m3 of BDS for four consecutive days and killed immediately. Soot particles are within macrophages (red arrowhead) and bronchiolar epithelial cells (red arrow). Neutrophils are migrating into the bronchiolar interstitium (black arrows) but not through the epithelium. H&E stain. (C) Photomicrograph of lungs from a mouse exposed to 5 mg/m3 of BDS for four consecutive days then allowed rest for one day. Soot particles are within alveolar (red arrowheads) and interstitial macrophages. There is a focus of moderate neutrophilic infiltration and transmucosal exocytosis (black arrowheads) with mild disruption of the continuity of the bronchiolar epithelium (black arrows). An interstitial lymphatic vessel is filled with neutrophils (green arrowhead), most of which contain one to three individual soot particles (not apparent at this magnification). H&E stain.

 

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  		TABLE 1. HISTOPATHOLOGY SCORES DEMONSTRATE BUTADIENE SOOT–INDUCED INFLAMMATORY INFILTRATION

 
Microarray
Expression Analysis filtering of raw Affymetrix data generated from mouse lung RNA yielded 261 transcripts (73 down-regulated and 188 up-regulated) that passed all criteria (>= 1.5-fold-change, P <= 0.05, FDR <= 0.05) in mice killed immediately after 4 days of BDS exposure. Ingenuity Systems identified 248 of these transcripts, including duplicates. These transcripts represented 154 individual genes (33 down-regulated and 121 up-regulated) for which literature-based network relationships exist (see Table E1 in the online supplement). Table 2 lists some of the significantly up-regulated genes in the lungs of BDS-exposed mice. Aryl hydrocarbon receptor (AhR)-responsive genes were prominently up-regulated, including the aryl hydrocarbon receptor repressor (Ahrr), cytochrome P450 IA1 (Cyp1a1) and IB1 (Cyp1b1), aldehyde 3 dehydrogenase (aldh3a1), and TCDD-inducible poly (ADP-ribose) polymerase (Tiparp). Oxidative stress response genes, including heme oxygenase 1 (Hmox1), nuclear factor erythroid-derived 2 like 2 (Nfe2l2), NADPH dehydrogenase quinine 1 (Nqo1), thioredoxin reductase 1 (Txnrd1), and glutathione peroxidase 2 (Gpx2), were also significantly up-regulated, as were genes associated with inflammation, including the mouse homolog of IL8 (Cxcl2; Mip2), granulocyte chemotactic protein (Cxcl6), and prostaglandin-endoperoxide synthase 2 (Ptgs2; Cox2). These results show that uptake of BDS nanoparticles by alveolar macrophages and airway epithelium is accompanied by up-regulation of biotransformation enzymes, oxidative stress, and pro-inflammatory cytokines.


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  		TABLE 2. MICROARRAY GENE EXPRESSION DATA DEMONSTRATE UP-REGULATION OF ARYL HYDROCARBON RECEPTOR–RESPONSIVE BIOTRANSFORMATION, OXIDATIVE STRESS RESPONSE, AND INFLAMMATORY CYTOKINE GENES AFTER 4 d (4 h/d) INHALATION EXPOSURE TO BUTADIENE SOOT

 
Pathway Analysis
Filtered microarray data were entered into the Ingenuity Analysis Knowledge Database and canonical pathways described. Our three pathways of interest (Ahr-associated, oxidative stress response, pro-inflammatory) were each identified as significant within differentially expressed gene networks (groups of interrelated genes). A merging of these networks is depicted in Figure 4. Relationships associating the three pathways of interest can be defined or inferred from the merged network.


Figure 4
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  		Figure 4. Network demonstrates the interaction of gene pathways. This gene network constructed using experimental expression data with the Ingenuity Analysis Knowledge Database (Ingenuity Systems, Redwood City, CA) shows the interaction of the three gene pathways investigated in this study: (1) Ahr-related genes (Cyp1a1, Cyp1b1, Ahrr); (2) Nrf2-mediated oxidative stress response genes (Hmox1, Nfe2l2, Nqo1, Gpx2); (3) inflammatory genes (IL-6, Cxcl2, Cxcl3, Cxcl6, Cox2). Other genes unassociated with these pathways were significantly up-regulated but were not further investigated in the present study. Red indicates up-regulation and green represents down-regulation, with the intensity of color directly related to the degree of up-regulation or down-regulation of the gene transcripts.

 
Quantitative RT-PCR
We performed qRT-PCR on lung tissue to confirm that BDS inhalation elicited up-regulation of biotransformation enzyme and inflammatory cytokine genes. In vitro exposure of human and mouse respiratory cells to BDS results in up-regulation of these genes (26). These qRT-PCR results from mouse lung cDNA are presented in Table 3. In the lungs of mice killed immediately after BDS exposure, significant increases in expression were confirmed for selected AhR-responsive biotransformation enzymes: aldehyde dehydrogenase 3A1 (Aldh3a1), cytochrome P450 IA1 (Cyp1a1), and IB1 (Cyp1b1), as well as the AhR repressor (Ahrr) and TCDD-inducible poly(ADP-ribose) polymerase (Tiparp), while expression of AhR itself was unchanged. In the lungs of BDS-exposed mice allowed 1 day of recovery before killing, the expression of these AhR-responsive genes had decreased; but, with the exception of Aldh3a1 and Tiparp, each was still significantly up-regulated relative to control mice.


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  		TABLE 3. QUANTITATIVE RT-PCR OF LUNG TISSUE REVEALS UP-REGULATION OF ARYL HYDROCARBON RECEPTOR–RESPONSIVE BIOTRANSFORMATION AND INFLAMMATORY CYTOKINE GENES AFTER INHALATION EXPOSURE TO BUTADIENE SOOT

 
Chemokine (C-X-C motif) ligand 2 (Cxcl2; MIP-2; human IL-8 analog) up-regulation was confirmed by qRT-PCR. IL-6 up-regulation was demonstrated by qRT-PCR but was not seen on microarray, a phenomenon that we have previously reported for other BALF-derived interleukins (28). Although up-regulation of IL-6 and Cxcl2 was decreased in animals allowed to recover for 1 day after BDS exposure, their expression of these genes still was significantly elevated relative to that of control mice. With the exception of IL-1β, whose expression was elevated after 1 day of recovery, the expression levels of the other cytokines that we examined were not significantly different from controls.


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Previously, particulates in ambient air have been linked to increases in cardiopulmonary-related morbidity and mortality (1–3). A recent study reported the relatively minimal impact of acute inhalation (4 or 24 consecutive hours) of inert ultrafine carbon particles on pulmonary function and inflammation (29). No significant changes in histopathology were seen in BALB/c mice as a result of ultrafine carbon particle exposure. Significant gene expression changes identified in the lung were primarily representative of cellular protective mechanisms, including up-regulation of heat shock protein, homeostatic, and immunomodulatory genes. In contrast, we report that inhalation of combustion-derived BDS nanoparticles causes acute pulmonary inflammation in the bronchoalveolar space of mice accompanied by up-regulation of biotransformation, oxidative stress, and pro-inflammatory genes. The different results in these two studies reflect the differing responses to highly purified (>= 96%) carbon particles versus carbon particles carrying adsorbed PAHs.

Metals-rich ROFA, oxygen-rich DEPs, and tobacco smoke particles also produce inflammatory lung infiltrate. The bulk of effects from these particles are attributed to metals (13), hydroquinones, that are oxidized in air to semiquinone radicals that ultimately produce superoxide (30) and other PAH oxidation products present upon initial cellular exposure. Combustion and storage conditions of these complex mixtures will affect the in vivo and in vitro responses to them. In contrast, BDS is an oxygen- and metals-poor mixture of PAHs and nanoparticles that remains stable for many months (24) and that serves as a unique model and real-life example of particulate exposure arising from petrochemical combustion. Although oxidation of PAHs in BDS clearly occurs within the cell (see next paragraph), the low oxygen content of BDS indicates that, unlike DEP and cigarette smoke, the initial chemical presentation of BDS to the cell is not primarily in the form of quinones, hydroquinones, and semiquinones.

The numerous PAHs, including extensively investigated carcinogens, e.g., B(a)P, found in DEPs, cigarette smoke, and to a lesser extent in ROFA, are metabolized for detoxification and excretion (31, 32). The first step in this process is Phase I biotransformation, most often performed by microsomal oxidases, including CYP1A1 and CYP1B1. The results here reveal up-regulation of these genes in response to BDS inhalation. We found no change in expression of the AhR subsequent to BDS exposure, in contrast to a prior report on in vivo responses to DEP exposure (33). Our results are consistent with a more recent study describing the constitutive and PAH-induced expression of AhR, ARNT, AHRR, and CYP1A1 in various human adult and fetal tissues (34). There, all four genes were constitutively expressed in adult lung tissue; however, after PAH exposure of isolated pulmonary mononuclear cells (monocytes and lymphocytes), AhR expression did not change, while expression of ARNT, AHRR, and CYP1A1 increased. Our present study shows increases in Ahrr, Cyp1a1, and Cyp1b1 and is consistent with our reported in vitro findings in both murine and human lung cell lines exposed to BDS (26).

Mice killed immediately after the final BDS exposure had higher expression of Cyp1a1, Cyp1b1, Aldh3a1, and Tiparp than was observed in lungs of BDS-exposed mice allowed 1 day of recovery. However, the expression of these genes after recovery was still significantly elevated when compared with values from air control mice (Table 2). These results are consistent both with (1) absence of additional BDS exposure during the recovery day and (2) retention of previously inhaled BDS within the lung.

The oxidized products of select PAHs metabolized by the cytochrome P450 system are capable of forming carcinogenic DNA adducts and/or eliciting oxidative stress responses in cells. These effects may be exacerbated by the presence of particles, as in the case of DEPs, where cellular oxidative stress has been linked directly to inflammatory endpoints (17). We have demonstrated up-regulation both of Nrf2 oxidative stress response genes and of pro-inflammatory genes in the lungs of mice after BDS exposure. NRF2 protein is encoded by the gene NFE2L2. The murine homolog of this gene was up-regulated in BDS-exposed mice. NRF2 is constitutively bound in the cytoplasm through association with the cytoskeletal protein, kelch-like ECH-associated protein 1 (KEAP1 [35]). Under conditions of oxidative stress, NRF2 dissociates from KEAP1 and moves to the nucleus, where it functions as a transcription factor. NRF2 binds to the antioxidant response element (ARE) of targeted genes that code proteins with cytoprotective and detoxifying functions (36). Through the ARE, NRF2 induces expression of HMOX1, NQO1, GPX2, and TXNRD1 (33). Here, expression of each of these genes was elevated in BDS-exposed mice (Table 2; Figure 4). The protein products of these genes metabolize xenobiotics and provide cellular defense against oxidative damage. Relative to wild-type mice, Nrf2 knockout mice accumulate increased levels of oxidative DNA adducts in airway epithelium (37). With increasing oxidative stress, inflammatory responses also escalate. In support of this, we demonstrate oxidative stress responses and inflammatory responses in the lungs of mice that have inhaled BDS.

The up-regulation of IL-6, Cxcl2 (Il8), Cxcl3, and Cxcl6 (Table 2) is consistent with the neutrophilic inflammatory response observed histopathologically in lung sections. Subsequent to BDS exposure, a significant influx of neutrophils to the bronchoalveolar space also was observed via BALF cytology. A number of molecules (including Cxcl2, Cxcl3, and Cxcl6) are capable of recruiting neutrophils and macrophages to the lungs; these inflammatory effectors may be produced by pulmonary epithelial cells (38) or fibroblasts (39). The inflammatory infiltrate intensified after 1 day of rest subsequent to 4 days of BDS exposure. In support of the neutrophilia, BALF IL-8 levels were elevated in BDS-exposed mice and were increased after a day of recovery. Increasing neutrophilia was accompanied by the emergence of M{Phi}2s and M{Phi}3s in the BALF and pulmonary interstitium (Figure 3C). Retention of particles in alveolar macrophages has been hypothesized to potentiate long-term health effects through either persistent physical irritation or inflammation after an acute particulate inhalation exposure (23).

The merged network of genes depicted in Figure 4 demonstrates direct and indirect relationships between differentially expressed genes in the three pathways that we examined (Ahr-associated, oxidative stress response, pro-inflammatory). Within this network, IL-6 was not differentially expressed according to microarray but was up-regulated by qRT-PCR in BDS-exposed mice. Other genes within the network were not differentially regulated according to microarray and were not examined via qRT-PCR. Genes having known relationships with genes from Ahr-associated, oxidative stress response, and pro-inflammatory pathways comprise a large percentage of the differentially expressed genes in the merged network. The role that these genes might play in the described processes (biotransformation, oxidative stress and stress response, and inflammation) is apparent in some cases but obscure in others. A relationship between PAH exposure, Cyp1a1 up-regulation, and increased pro-inflammatory cytokines has been described previously (30). The protective induction of Nrf2-mediated oxidative stress responsive genes by PAH exposure also has been described (34). We report here the BDS-mediated induction of AhR-associated genes, Nrf2-mediated oxidative stress response genes, and pro-inflammatory cytokines and chemokines. We note the phase I enzyme response to PAH cellular insult, link this with subsequent attempts to moderate injury via phase II enzymes, and ultimately with inflammation induced by PAH-mediated cellular damage and death.

Inhalation of particles may elicit not only local effects in the respiratory tract, but systemic effects as well. An increase in circulating levels of inflammatory cytokines subsequent to PM10 exposure has been associated with progression of atherosclerosis (40). Direct translocation of particles from the respiratory tree to the systemic circulation has been described as influencing the subsequent development of cardiovascular disease conditions (41), although this conclusion has been actively challenged by more recent human exposure studies (42, 43). Ultrafine particles deposited in the nasopharyngeal area access the central nervous system via olfactory neuronal transport (44). Our study deals only with effects of BDS in the lung parenchyma, though we recognize the potential for BDS nanoparticles to reach other organ systems.

Studies in rats and dogs have demonstrated that pulmonary epithelial cells have a saturation point with respect to PAHs (45). Combustion-derived ultrafine particles have the potential to translocate to extrapulmonary sites with their PAH payload intact, if this PAH saturation point is exceeded. This is unlikely through exposure to ambient environmental particles, which usually are present in relatively low concentration and have a low overall PAH burden (46). The saturating concentration might be exceeded, however, during an acute exposure to high levels of such particles: among firefighters, rescue workers, and local residents after a terrorist attack (as in September 11th), petrochemical industrial accidents, or soldiers and civilian populations exposed to petroleum fires (as in the Gulf Wars).

Although studies in dogs demonstrated acute loss in less than 60 seconds of approximately one-third of adsorbed PAHs from inhaled nanparticles, follow-up experiments found the remaining PAHs adsorbed to particles within the lung and regional lymph nodes up to 5 months later (47). This supports the possibility of retained soot nanoparticles delivering PAHs to pulmonary and nonpulmonary tissue long after particle inhalation has ceased. Our in vitro finding that BDS-derived PAHs concentrate in lipid droplets of respiratory cells further supports this possibility (26).

In summary, we have reported that brief inhalation exposure to a moderate dose of PAH-rich, metals-poor combustion-derived ultrafine BDS particles initiates (1) AhR-responsive biotransformation responses capable of transforming PAHs into more toxic metabolites, as evidenced by increased expression of AhR-responsive genes; (2) cellular oxidative stress, as reflected by up-regulation of Nrf2-mediated oxidative stress response genes; and (3) acute pulmonary inflammation, as demonstrated by inflammatory cell infiltration and up-regulation of pro-inflammatory cytokine genes. Finally, our histopathologic analyses indicate that freshly generated inhaled BDS nanoparticles reach the deepest bronchoalveolar spaces in lungs of exposed mice.


   Acknowledgments
 
The authors thank Terry Ahlert, Brandon LaGroue, and Daniel Lundquist for their technical assistance and Michael Kearney for help with the statistical analyses.


   Footnotes
 
* Present affiliation: U.S. Army Veterinary Medical Corps, Analytical Toxicology Division, USAMRICD, Aberdeen, Maryland Back

This work was supported by the Louisiana Governor's Biotechnology Initiative.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2008-0057OC on March 26, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 31, 2008

Accepted in final form March 8, 2008


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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