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Published ahead of print on December 13, 2007, doi:10.1165/rcmb.2007-0204OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 532-540, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0204OC

Combustion-Derived Hydrocarbons Localize to Lipid Droplets in Respiratory Cells

Gleeson Murphy, Jr.1, Rodney L. Rouse1, William W. Polk1, William G. Henk1, Steven A. Barker1, Marc J. Boudreaux2, Z. Elizabeth Floyd3 and Arthur L. Penn1

1 Comparative Biomedical Sciences, School of Veterinary Medicine; 2 Division of Biotechnology & Molecular Medicine (BioMMED), School of Veterinary Medicine; and 3 Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana

Correspondence and requests for reprints should be addressed to Dr. Arthur Penn, 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
 
Combustion-generated radicals interact to form polynuclear aromatic hydrocarbons (PAHs), including carcinogens. PAHs aggregate into 20- to 50-nm particles, which extend into branched-chain structures (soots). Incomplete combustion yields black soot particles and black smoke. Many PAHs, including those in soots, fluoresce upon excitation. We have reported that butadiene soot (BDS), generated during combustion of the high-volume petrochemical 1,3-butadiene, serves as a reproducible example of combustion-derived fine and ultrafine particles, with the potential for acute or delayed health effects. Human bronchoepithelial cells (BEAS-2B) display time- and concentration-dependent responses to BDS exposure, culminating in concentration of fluorescent PAHs within discrete cytoplasmic bodies. Here we identify the cytoplasmic compartment(s) in which combustion-derived PAHs concentrate and assess the metabolic responses associated with this compartmentalization. BDS-associated fluorescence colocalized with a red fluorescent cholesterol analog and a transfected plasmid coding for a fluorescent lipid droplet surface protein within BEAS-2B cells. After BDS exposure, murine alveolar macrophages (MH-S) and adipocytes (3T3-L1) also develop fluorescence. These findings, especially within adipocytes, support the accumulation of PAHs within lipid droplets. Microarray data revealed up-regulation of aryl hydrocarbon receptor–induced Phase I biotransformation enzymes and nuclear erythroid-2 related factor 2–mediated oxidative stress responses in BEAS-2B cells. Quantitative RT-PCR results confirmed a time-dependent up-regulation of Phase I biotransformation enzymes (CYP1A1, CYP1B1, and ALDH3A1) in BDS-exposed BEAS-2B and MH-S cells. Thus, respiratory cell lipid droplets concentrate PAHs delivered by combustion-derived ultrafine particles. These PAHs, including several found in BDS and in cigarette smoke, activate xenobiotic metabolism pathways and thereby potentiate their toxicity.

Key Words: lipid droplets • fluorescence • soot • polynuclear aromatic hydrocarbons • xenobiotic metabolism



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Hydrocarbons, including carcinogens, on inhalable petrochemical combustion particles and present in cigarette smoke, localize to lipid droplets in respiratory cells and adipocytes. Lipid droplets may serve in storage/release of environmental toxicants.

 
Airborne particulates are of increasing concern for their contribution to ambient pollution and their toxic health effects. Health effects have been attributed both to the particles themselves, especially in the readily inhalable fine (<2.5 µm) and ultrafine (<0.1 µm) size ranges (1), and to inorganic and organic chemicals associated with the particles. Included among the organic chemicals are polynuclear aromatic hydrocarbons (PAHs) that can be adsorbed to the surface of the particles. Organic radicals generated during combustion of hydrocarbon substrates can interact to form PAHs, including carcinogens. These PAHs aggregate into bead-like 20- to 50-nm particles, which extend into branched-chain structures (soots). Soot particles that are incompletely combusted are visualized as black smoke (2). Many PAHs, including those in soots, fluoresce upon excitation.

Diesel exhaust and cigarette smoke are among the most frequently studied "real-world" examples of complex combustion-derived particulate mixtures. In both cases, there is a growing body of literature that emphasizes the distinction between the toxicity of the particles versus the toxicity of chemicals adsorbed to the particles (3, 4). Another environmental source of complex particulates is flaring of fugitive volatile compounds by industry. In these settings, volatiles that escape the processing stream or that remain unused are combusted, as strict regulations are in place limiting the amounts of highly reactive volatile organic compounds, such as 1,3-butadiene (BD), that can be released to the atmosphere (5). BD is a high-volume, aliphatic hydrocarbon byproduct of petroleum refining and is used in the manufacture of synthetic rubber and other elastomers. The United States' production of BD is 3 x 109 lb/year (6). Butadiene soot (BDS), generated during the combustion of BD, is both a model mixture and a real-life example of a petrochemical product of incomplete combustion with the potential for environmental contamination and for contributing to health problems (7). Free BDS particles have been found apposed to the luminal surface of lung epithelium in mice exposed to BDS by inhalation, while alveolar macrophages filled with BDS particles have been identified in the lung parenchyma even 4 weeks after BDS exposures end (Murphy et al., manuscript in preparation).

We have previously characterized BDS as a metals-poor, organic-rich mixture of ultrafine (30–50 nm) carbonaceous particles to which hundreds of PAH species are adsorbed (7, 8). Sixteen percent of the total weight of fresh BDS is comprised of PAHs, including benzo(a)pyrene [B(a)P] and other carcinogens, many of which display a characteristic blue or blue-green fluorescence in organic solutions. Human bronchoepithelial cells exposed to BDS develop blue fluorescence, which over time becomes localized in discrete cytoplasmic bodies. Following BDS exposure, these cells display a profile of extractable PAHs similar to that of the parent BDS (10). The fluorescence does not develop if the cells are exposed to carbon black instead of BDS, or if the BDS is extracted with organic solvents before the soot particles are presented to the cells (7). To this point, the cellular sites of BDS fluorescence localization have not been identified.

Lipid droplets are spherical organelles ranging in diameter from 50 nm at formation up to 200 µm in mature adipocytes, with the majority being approximately 1 µm in most mammalian cells (9). Initially, lipid droplets were regarded as repositories of intracellular lipids used for energy production and membrane maintenance (10). Recent studies on the dynamic behavior of lipid droplets have led to the elucidation of their role in other processes, including fatty acid oxidation and inflammatory eicosanoid production in leukocytes, where lipoxygenases and cyclo-oxygenases interact with arachidonic acid within these droplets (11, 12). During their formation, probably in the membrane of the endoplasmic reticulum (13), and through their association with the plasma membrane (14), lipid droplets become armed with an array of proteins that are responsible for the organelle's structure, function, and signaling activities (9, 15). Three of the most extensively characterized of these lipid droplet surface proteins are perilipin, adipose differentiation-related protein (ADFP or adipophilin), and TIP47, collectively referred to as the PAT family of proteins (16). As the molecular characteristics of lipid droplets have expanded, links to various disease processes, including atherosclerosis, have been identified. Expression of ADFP is increased significantly in atherosclerotic plaques, and increased ADFP expression in macrophages alters lipid transport by promoting storage of triglycerides and cholesterol while reducing cholesterol efflux (17).

The compounds that concentrate in lipid droplets are not restricted to lipids. Indeed, a number of proteins, including caveolin-1, caveolin-2 (13, 18, 19), and the core protein of the hepatitis C virus (20), have been localized to lipid droplets. Proteomic characterization has revealed that proteins responsible for lipid transport and metabolism localize to lipid droplets (21). Hydrophobic environmental chemicals, including PAHs, are another group of compounds that might concentrate within lipid droplets. Verdin and colleagues (22) have demonstrated that fluorescent PAHs, including B(a)P, concentrate in the lipid droplets of fungi, which sequester these noxious compounds (pollutant dissipation) and perhaps metabolize them to less toxic derivatives.

The experiments described here demonstrate that combustion-derived PAHs adsorbed onto inhalable ambient particles are concentrated in lipid droplets of respiratory system cells and that these PAHs concomitantly activate xenobiotic metabolism pathways known to potentiate the toxicity of certain PAHs, including several found in BDS and in cigarette smoke.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Culture
BEAS-2B cells (1.5 x 106), a human bronchoepithelial cell line (23), were seeded into T-25 flasks (Corning, Corning, NY) containing bronchial-epithelial growth medium (BEGM), before expansion in T-150 flasks. BEGM is a basal medium (BEBM; Cambrex, Walkersville, MD) supplemented (per 500 ml) with 2 ml of 13 mg/ml bovine pituitary extract and 0.5 ml each of 0.5 mg/ml hydrocortisone, 0.5 µg/ml human recombinant epidermal growth factor, 0.5 mg/ml epinephrine, 10 mg/ml transferrin, 5 mg/ml insulin, 0.1 µg/ml retinoic acid, 6.5 µg/ml triiodothyronine, and 50 mg/ml gentamicin.

MH-S cells (1 x 106), a murine alveolar macrophage cell line (24), were propagated in T-150 flasks containing RPMI 1640 medium supplemented with 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/liter glucose, 1.5 g/liter bicarbonate, 0.05 mM 2-mercaptoethanol, and 10% FBS. BEAS-2B and MH-S cells were grown to 80 to 90% confluence (37°C, 5% CO2/95% air), split into 60-mm dishes (2.5 x 105 cells/dish; grown on 25 x 25 mm glass coverslips) or 6-well plates (1 x 105 cells/well), and expanded until approximately 90% confluent.

Murine 3T3-L1 preadipocytes (25) were plated in 6-well plates and grown to 2 days postconfluence in DMEM containing 4.5 g/liter glucose with 10% calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were induced to differentiate by changing the medium to DMEM containing 4.5 g/liter glucose, 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.5 mM 3-isobutyl-methylxanthine, 1 µM dexamethasone, and 1.7 µM insulin. After 48 hours, this medium was replaced with DMEM containing 4.5 g/liter glucose supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, and the cells were maintained in this medium until used (26).

BDS Generation and Collection
The process of BDS generation and collection has been described in detail (7). Briefly, room temperature BD gas (>=99% purity) (Sigma, St. Louis, MO) was passed through a back-flash-protected stainless steel two-stage regulator to a stainless steel Bunsen burner at flow rates of 5 to 7 ml/second under normal atmosphere and ignited. Soot particles passing through the feed pipe were collected on acetone-washed Whatman cellulose filters placed in a porcelain Buchner funnel connected to a vacuum pump. The BDS was scraped gently off the filters and stored in aluminum-foil-wrapped glass vials capped with foil-lined lids.

BDS Exposures
In each exposure, the culture medium was changed immediately before addition of BDS or staining solutions. A stock solution was prepared by suspending 10 mg BDS in 50 ml of culture medium and sonicating the suspension with three 15-second pulses of a Branson Sonifier (Model 450; Constant Duty Cycle, Danbury, CT). BEAS-2B cells, MH-S cells, and adipocytes were exposed to 20 µg/ml sonicated BDS for 24 hours before fluorescence imaging. For microarray and/or quantitative real-time (qRT)-PCR experiments, BEAS-2B and MH-S cells were exposed to BDS for 0, 1, 3, or 24 hours before RNA extraction.

Fluorescent Dye Colocalization
Organelle-specific fluorescent probes were used to investigate the subcellular localization of BDS-associated fluorescence. The probes were prepared in BEGM, and BEAS-2B cells were exposed as described in Table 1. LysoTracker Red DND-99, to label lysosomes; dextran-tetramethylrhodamine (D-TMR), to label endosomes, Cholesteryl BODIPY 542/563 C11, to label lipid droplets and ER-Tracker Red, to label endoplasmic reticulum (ER), were obtained from Molecular Probes (Invitrogen, Carlsbad, CA). To investigate peroxisomes as candidate organelles for sequestration of the PAH fluorescence, we transfected BEAS-2B cells with a plasmid whose protein product is a fusion of the red fluorescent protein DsRed2 with the peroxisomal targeting sequence 1 (27). The characteristics of the optical filter set for each test agent are listed in Table 1.


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  		TABLE 1. FLUORESCENT PROBE SPECIFICATIONS, EXPOSURE PARAMETERS, AND FILTER WAVELENGTH BOUNDARIES FOR BUTADIENE SOOT (BDS)-ASSOCIATED FLUORESCENCE COLOCALIZATION

 
Fluorescence Microscopy
We collected images (Figures 1A–1D, 2A–2B, and 4D–4E)–from a Leica DM RXA2 upright microscope, through 40x (HCX PL APO CS, 1.25 NA), 63x (HCX PL APO CS, 1.32 NA), or 100x (HCX APO U-V-I, 1.30 NA) objectives equipped with differential interference contrast optics–with a SensiCam QE 12-bit, cooled CCD camera (Cooke, Romulus, MI), operated by SlideBook software (Intelligent Imaging Innovations, Denver, CO). Observations for Figures 4A–4C were made on a Zeiss Axiovert 405M inverted fluorescence microscope (40x objective, LD Achroplan, 0.60 NA) with a Microfire Megapixel Digital CCD camera, operated by PictureFrame software (Optronics, Goleta, CA). Z-plane images captured though the 40x objective were processed by SlideBook, with no neighbors deconvolution. Z-plane images captured though the 100x objective were processed by SlideBook with constrained iterative deconvolution to confirm co-localization of the BDS-associated fluorescence with pDsRed-Monomer ADFP. The 100x deconvolved images were combined to create a three-dimensional rotating video image of a single BDS-exposed BEAS-2B cell (supplemental movie). We used SlideBook to measure lipid droplet dimensions and Adobe Photoshop CS to process all images using only crop, screen, and levels commands.


Figure 1
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  		Figure 1. Time-dependent fluorescent responses of BEAS-2B cells exposed to sonicated petrochemical combustion-derived ultrafine particles (butadiene soot [BDS]). Diffuse fluorescence is visible at 30 minutes (A). Punctate fluorescence appears within the first 60 minutes of exposure to BDS (B). Punctate fluorescence is more prevalent by 4 hours of BDS exposure (C) and is further increased by 24 hours of exposure (D). All photomicrographs are presented at the same magnification (x40) without editing. Bars = 10 µm.

 

Figure 2
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  		Figure 2. Bronchoepithelial cells, alveolar macrophages, and adipocytes display characteristic BDS-associated fluorescence. Differential interference contrast (DIC) (A, B) and brightfield photomicrographs (C) paired with ultraviolet-fluorescence images of BEAS-2B human bronchoepithelial cells (A), MH-S murine alveolar macrophages (B), and 3T3-L1 murine adipocytes (C) after a 24-hour exposure to sonicated BDS. The fluorescent bodies appear as variably sized refractile bodies; peripheral membrane blebs are visible on some cells (A). In contrast to BEAS-2B cells, mouse alveolar macrophages displayed larger droplets, all of which were fluorescent (B). Large droplets in mouse adipocytes displayed BDS-associated fluorescence (C). Black BDS particles are visible in the brightfield panel of C, and the fluorescent panel demonstrates the lack of fluorescence associated with the particles. Bars = 10 µm (A), 7.5 µm (B), and 10 µm (C).

 

Figure 3
Figure 3
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  		Figure 3. Cellular proliferative ability is unchanged in BEAS-2B and MH-S cells after up to 24 hours of BDS exposure. (A) A formazan dye assay demonstrates BDS-exposed BEAS-2B cells present in numbers equal to unexposed BEAS-2B cells through 24 hours of exposure, indicating no reduction in viability as a result of BDS exposure. Blue line, diamonds: B2B control; red line, squares: B2B BDS. (B) The formazan dye assay shows parallel curves for BDS-exposed and non-exposed MH-S cells over 24 hours, indicating no viability change due to the BDS exposure. Blue line, diamonds, MH-S control; red line, squares, MH-S BDS. BDS-exposed MH-S cells were inadvertently plated at a lower dilution (evident at time zero).

 

Figure 4
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  		Figure 4. Colocalization assays of organelle-specific probes with BDS fluorescence in BEAS-2B cells. The left column shows responses to red fluorescent probes for lysosomes (LysoTracker Red, A), endosomes (dextran-tetramethylrhodamine [dextran-TMR] B), endoplasmic reticulum (ER Tracker Red, C), and lipid droplets (cholesterol-BODIPY, D; DsRed-ADFP, E). The central column shows the concurrent BDS-associated fluorescence after ultraviolet excitation. The right column shows the merged images. Bars = 10 µm (A–D) and 5 µm (E).

 
Cytotoxicity Assay
To determine whether BDS is overtly toxic to BEAS-2B or MH-S cells, we used the Cell Counting Kit-8 from the Dojindo Molecular Technologies (Gaithersburg, MD) to assess cell viability. The assay was performed according to the manufacturer's instructions on BEAS-2B and MH-S cells grown in 60-mm dishes as described previously for fluorescent assays. Cells were exposed to sonicated BDS for 1, 4, 8, or 24 hours. Unexposed sets of control cells were used for comparison at each time point.

RNA Isolation
BEAS-2B cells and MH-S cells at or near confluence were collected from three wells of a 6-well plate by scraping into 1 ml of TRIzol Reagent (Invitrogen) before passing them three times through a 23G needle attached to a 1-ml syringe. RNA was isolated using a TRIzol/chloroform extraction, followed by column purification using the Qiagen RNeasy Mini Kit. RNA concentrations were measured with a ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE). RNA quality and integrity was assessed using the Agilent RNA 6000 Nano Assay Kit and the 2100 BioAnalyzer (Agilent, Santa Clara, CA). Total RNA was converted to cDNA using TaqMan Reverse Transcriptase Reagents (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol.

pDsRed-Monomer-C-ADFP Plasmid Preparation and Transformation
Human ADFP PCR primers were as designed by Targett-Adams and colleagues (28) from the ADFP mRNA sequence in GenBank: 5'-GGGGCAGGTTTAATGAGTTTTATG-3' and 5'-CCAGGAAGAAAAAT GGCATCCGTT-3' (Integrated DNA Technologies, Coralville, IA). PCR was performed on 50 ng of BEAS-2B cDNA in Eppendorf MasterMix (Hamburg, Germany) for 30 cycles in a PTC-100 thermal cycler (MJ Research, Waltham, MA). To impart red fluorescence to ADFP expressed within cells, the ADFP PCR product was ligated into the pDsRed-Monomer-C In-Fusion Ready Vector with the In-Fusion Dry-Down PCR Cloning Kit and expanded in Fusion-Blue Competent Escherichia coli cells (Clontech, Mountain View, CA) according to the manufacturer's protocols. Plasmid DNA was isolated from the bacteria with the Wizard Plus SV Miniprep system (Promega, Madison, WI). Plasmid insert size was confirmed by double restriction enzyme digestion with EcoRI and SalI (New England Biolabs, Beverly, MA); the insert sequence was confirmed by GeneLab (Louisiana State University, School of Veterinary Medicine, Baton Rouge, LA) with the Applied BioSystems BigDye Terminator v3.1 Cycle Sequencing Kit. BEAS-2B cells grown on glass coverslips in 60-mm dishes were transformed with 500 ng of the plasmid, in the presence of Lipofectamine LTX and PLUS reagents (Invitrogen). After 48 hours, cells were observed for lipid droplet fluorescence, then exposed to BDS (20 µg/ml) for 24 hours before imaging for colocalization.

Gene Microarray Assay
Global gene expression in BEAS-2B cells was assessed from total RNA (see RNA ISOLATION above) on Affymetrix Human Genome U133 Plus 2.0 Arrays. 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 then biotinylated, fragmented, and added to a hybridization cocktail that included probe array controls, bovine serum albumin, and herring sperm DNA. This cocktail was then 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. Data 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).

Quantitative Real-Time PCR
Quantitative RT (qRT)-PCR was performed on cDNA samples from BEAS-2B and MH-S cells with inventoried TaqMan Gene Expression Assays primer-probe sets (Applied Biosystems) for the genes listed in Table 2. Reaction volumes were 25 µl and 40 reaction cycles were performed for each gene in an Applied Biosystems 7300 Real Time PCR System. Relative gene expression was determined by the comparative cycle threshold ({Delta}{Delta}CT) method, with each gene normalized to β-actin (ACTB) for human cells (29) or hypoxanthine guanine phosphoribosyl transferase (Hprt1) for mouse cells (30), and then compared with the 0-hour control. Results are reported as fold change over control. Standard error of the mean [(2–{Delta}{Delta}CT) ± SEM] was used to determine significant at {alpha} = 0.05.


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  		TABLE 2. TIME-DEPENDENT MICROARRAY CHANGES IN ARYL HYDROCARBON RECEPTOR– AND NRF2-RELATED GENE EXPRESSION IN BUTADIENE SOOT–EXPOSED BEAS-2B CELLS*

 
Statistical Analysis
We used the general linear model procedure of the SAS statistical package (version 9.1.3; SAS Institute, Inc., Cary, NC) to compare RT-PCR data and used the Dunnett's t test to determine statistical differences. Gene chip data were filtered for removal of data from invariant (nonfunctional) probe sets. These filtered data were then analyzed by pairwise comparisons of gene expression from each BDS-treated time point to gene expression of untreated control cells (Expression Analysis, Durham, NC). Gene expression was expressed as log ratio for these comparisons. Gene expression data were analyzed with the network- and pathway-building software, Ingenuity Pathways Analysis 4.0; gene networks and canonical pathways were examined using the Ingenuity Analysis Knowledge Database (Ingenuity Systems, Redwood City, CA).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
BDS-Associated Fluorescence in Bronchial Epithelial Cells Is Time Dependent
We followed the time-dependent development of BDS-associated fluorescence in human bronchial epithelial cells by fluorescence microscopy. Figure 1 shows the fluorescent responses of BEAS-2B cells exposed to BDS (20 µg/ml) for periods ranging from 30 minutes to 24 hours. Responses progressed from diffuse fluorescence visible 30 minutes after exposure (Figure 1A) to the appearance of bright punctuate perinuclear blue fluorescence visible by 60 minutes (Figure 1B). The extent and intensity of the punctuate fluorescence increased through 24 hours of exposure (Figures 1C and 1D). Cells not exposed to BDS did not fluoresce. The differential interference contrast image in Figure 2A demonstrates that in BEAS-2B cells the punctuate fluorescent bodies are submicron (0.78 ± 0.05 µm) refractile entities.

Alveolar Macrophages and Adipocytes Display BDS-Associated Fluorescence
Murine alveolar macrophages exposed to BDS generally displayed the same overall characteristic distribution of punctate blue fluorescence as did the BEAS-2B cells. However, there were cytoplasmic differences between macrophages and BEAS-2B cells, with the former displaying large (1.8 ± 0.2 µm) fluorescent perinuclear bodies (Figure 2B).

Cultured adipocytes exposed to BDS are shown in Figure 2C. BDS-associated fluorescence is localized within the large (2.6 ± 0.3 µm) spherical cytoplasmic droplets within these cells. The development of fluorescence in these cells also was time dependent with the fluorescence intensity increasing with time of exposure to BDS.

Viability of BEAS-2B and MH-S Cells Is Unchanged through 24 Hours of BDS Exposure
In some BDS-exposed BEAS-2B cells, peripheral membrane blebs were noted after 24 hours of BDS exposure. Therefore, the affect of BDS exposure on cell viability was assessed. Results of the Dojindo Cell Counting Assay indicate that viability is unaffected in BEAS-2B and MH-S cells after 24 hours of BDS exposure (Figures 3A and 3B). BEAS-2B cell do not show any decrease in viability relative to BDS exposure up to 24 hours. BDS-exposed MH-S cells demonstrate no decrease in viability throughout the time course, as indicated by the parallel cell count curves. BDS-exposed MH-S cell were inadvertently started at a lower density than non-exposed MH-S cells (evidenced at time zero). No membrane blebs or other potential indications of cellular injury were detected in MH-S cells by light microscopic observation through 24 hours.

BDS-Associated Fluorescence Localizes to Lipid Droplets in Bronchial Epithelial Cells
Based on light and fluorescence microscope observations of size, shape, and perinuclear distribution of the fluorescent cytoplasmic bodies, candidate organelles were selected and tested by fluorescence co-localization to identify the subcellular compartment(s) in which the BDS-associated punctuate blue fluorescence became concentrated. Due to the wide excitation and emission spectra of the BDS-associated fluorescence, special consideration was necessary in the selection of fluorophores to avoid overlaps in excitation and emission. We determined that fluorophore excitation wavelengths greater than 500 nm were required to avoid eliciting fluorescence in BDS-exposed cells. Long UV excitation wavelengths (350–400 nm) were ideal for providing maximal, consistent fluorescent blue BDS-associated emissions (~450–500 nm; data not presented). Thus, we were limited to red fluorophores for co-localization.

LysoTracker Red is a membrane permeant fluorescent probe that selectively labels intracellular compartments with low pH (e.g., lysosomes [31]). Lysosomes also sequester lipid-containing molecules (e.g., low-density lipoproteins) acquired through endocytic mechanisms (32). Rare colocalization of LysoTracker Red and BDS-associated fluorescence is visible in the merge of Figure 4A. D-TMR is a 10-kD dextran moiety fused to the tetramethylrhodamine fluorophore. D-TMR preferentially enters cells via endocytosis (33), thus serving to label primary endosomes and later, secondary lysosomes. Figure 4B shows the images of D-TMR and BDS fluorescence. No colocalization of the fluorophore with BDS-associated fluorescence was detected. In BEAS-2B cells transfected with the fluorescent peroxisome marker, DsRed, expression was visualized as evenly distributed fluorescent red cytoplasmic inclusions less than or equal to 0.5 µm in diameter. Peroxisome fluorescence did not colocalize with BDS-associated fluorescence (data not shown).

Cholesteryl-BODIPY C11 concentrates in hydrophobic compartments within cells via the "selective" transport pathway, in which cholesteryl esters from high-density lipoproteins are routed directly to lipid droplets without prior processing in lysosomes or the Golgi apparatus (34). Figure 4D shows the colocalization of the red cholesteryl-BODIPY C11 and the blue BDS fluorescence as evidenced by the predominance of purple vesicles in the merged image.

To confirm the localization of BDS-associated fluorescence specifically to lipid droplets, we transfected BEAS-2B cells with a plasmid whose protein product is a fusion of DsRed-Monomer-C with the lipid droplet-specific protein ADFP. The transfected cells displayed fluorescent red perinuclear inclusions ~1 µm in size. The merged image of Figure 4E shows that the blue fluorescence associated with BDS colocalizes completely with the red fluorescence of the lipid droplets in BEAS-2B cells. The supplementary video is a three-dimensional rotating representation of the same cell shown in the Figure 3E merge. This video demonstrates the heterogeneity in size of the fluorescent lipid droplets (0.3–1.4 µm), their perinuclear distribution, and their uniform dispersion throughout the cytoplasm of a BEAS-2B cell. The role of lipid droplets in sequestration and/or trafficking of PAHs within the cell is the focus of continuing experiments. Colocalization of BDS-associated fluorescence with the ER-specific marker ER-Tracker Red was explored. Colocalization was extensive by 24 hours (Figure 4C).

BDS Stimulates Expression of Aryl Hydrocarbon Receptor–Induced and Nuclear Erythroid-2 Related Factor 2–Mediated Oxidative Stress Response Genes in BEAS-2B Cells
Microarray data revealed time-dependent changes in gene expression in BEAS-2B cells exposed to BDS for 1 to 24 hours. At 24 hours, there were 448 genes that were differentially regulated relative to control cells (70 down-regulated and 378 up-regulated). Up-regulated genes included those for aryl hydrocarbon receptor (AhR) signaling, xenobiotic metabolism, acute-phase enzyme responses, and nuclear erythroid-2 related factor 2 (NRF2)-mediated oxidative stress responses (Table 2). The microarray data showing time-dependent up-regulation of AhR- and NRF2-associated genes are presented in Table 2.

BDS Induces AhR-Responsive Gene Transcription in MH-S Cells
We used RT-PCR to confirm the expression of AhR-responsive genes, including the aryl hydrocarbon receptor repressor (Ahrr), aldehyde dehydrogenase 3A1 (Aldh3a1), cytochrome P450 IA1 (Cyp1a1) and IB1 (Cyp1b1), and TCDD-inducible poly(ADP-ribose) polymerase (Tiparp), in MH-S cells exposed to S-BDS for 24 hours (Table 3). Although there was no change in Ahr expression in the macrophages (MH-S), there was greater than a 3-fold increase in Ahrr expression across all time points examined. This is consistent with a report that expression of Ahrr and the AhR nuclear transporter increase after exposure of cells to the PAH carcinogen 3-methylcholanthrene, even though there is no increase in AhR transcript expression (35). We also measured significant increases for Cyp1a1, Cyp1b1, and Tiparp. Expression of Aldh3a1 was increased, but it was not significantly different from expression in the control cells (Table 3).


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  		TABLE 3. TIME-DEPENDENT QUANTITATIVE REAL-TIME–PCR EXPRESSION CHANGES IN ARYL HYDROCARBON RECEPTOR–RELATED GENES OF BEAS-2B AND MH-S CELLS EXPOSED TO BUTADIENE SOOT FOR 24 h*

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The objectives of this study were to identify the cytoplasmic compartment where PAHs reside after delivery by inhalable combustion-derived ultrafine particles and to determine whether this compartmentalization is associated with altered expression of Phase I biotransformation enzyme and/or cytokine genes. We used organelle-specific probes and fluorescence imaging techniques to identify lipid droplets as the subcellular sites of localization of PAH-associated fluorescence in respiratory epithelial cells. This investigation represents the first time that this organelle has been identified as a site of accumulation and potential repository for organic toxicants associated with combustion-derived ultrafine particles.

We used fluorescence microscopy to follow the time course of intracellular compartmentalization of PAHs from ultrafine combustion particles in respiratory system cells. Fluorescent responses were detected within the first 30 minutes of exposure and gene expression changes within the first hour. The fluorescence and gene expression changes we report precede the first visible evidence of cell injury by many hours. Even after 24 hours of continuous exposure to BDS, there was no significant decrease in cell viability of BEAS-2B cells (Figures 3A and 3B), although there was membrane blebbing. The viability assay is based on maintenance of mitochondrial integrity and thus may be a less sensitive and later measure of cell responses to PAHs than are membrane based events. The alveolar macrophage MH-S cells exhibited similar responses and displayed no membrane changes even after 24 hours of exposure to BDS.

We used qRT-PCR to determine that Phase I biotransformation genes were up-regulated in a time-dependent manner in a murine alveolar macrophage cell line (MH-S) and in a human bronchoepithelial cell line (BEAS-2B) after BDS exposure. We further investigated gene expression changes using gene microarrays in BEAS-2B cells after BDS exposure. The results from both cell lines demonstrated BDS induction of the AhR signaling pathway and up-regulation of Phase I biotransformation genes. Microarray data in BEAS-2B cells indicated up-regulation of NRF2-mediated oxidative stress response genes in addition to the AhR-induced/Phase I biotransformation genes.

A role for carbonaceous particles as a PAH delivery system to cells has been previously considered. Bevan and colleagues (36) and Lakowicz and colleagues (37) demonstrated that cellular uptake of PAHs is enhanced when the PAHs are adsorbed to high-surface-area solids, such as carbon black particles or asbestos fibers. In an earlier study, we reported that aggregates of nonsonicated BDS particles occasionally were found apposed to cells but that endocytosis of the particles was not detected. We also demonstrated that organic solvent-extracted BDS particles did not produce the characteristic fluorescent responses and that the soot-derived PAHs that produce those responses can be extracted from the cells (7). Based on these observations, we propose that BDS particles physically deliver the hydrophobic PAHs to the cells and that the PAHs are distributed by diffusion to lipid bilayers in the plasma membrane.

Investigators have used B(a)P-associated fluorescence as a marker for intracellular PAH accumulation and have described the uptake and partitioning kinetics of B(a)P delivered to cells. Lakowicz and colleagues (37) showed that the rate-limiting step of B(a)P transfer from a particulate is desorption of the PAH from the particle into the aqueous environment and that its subsequent incorporation into a lipid phase is very rapid. Subsequently, Plant and colleagues (38) reported that the rate-limiting step in B(a)P movement intracellularly is desorption from the inner leaflet of the plasma membrane into the aqueous cytosol. B(a)P would then partition to lipophilic compartments including the membranes of the Golgi apparatus and endoplasmic reticulum and lipid droplets. B(a)P was used in these earlier descriptions of PAH movement within cells because of its inherent fluorescence and well known carcinogenicity. The results presented here with BDS, a complex mixture of PAHs, are consistent with the earlier B(a)P results and suggest that consideration of hydrophobic compound movement and compartmentalization within cells should be applied to a broader range of compounds, including lipophilic toxins, other xenobiotics and pharmacologic agents. Lipid droplets might act as repositories for such compounds preventing them from exerting their toxic or therapeutic effects, in effect "protecting" the cell. Alternatively, the sequestration of compounds in lipid droplets may perpetuate their availability to the cell. The degree of availability may depend upon the characteristics of the lipid droplet membrane and its associated proteins charged with regulation of access to the droplets' cargo.

The results presented here indicate that inhalable combustion-derived ultrafine particles can deliver the PAHs adsorbed to their surface to intracellular depots (i.e., lipid droplets within target cells). The lipid droplets then serve as reservoirs for accumulation and possible delayed release of these compounds. By gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry, we have identified BDS-associated PAHs spanning the size range from 100 amu to more than 400 amu (7, 8). Among those identified specifically were anthracene, chrysene, benzopyrenes [including B(a)P], and perylene (8). PAHs en route to lipid droplets likely are available to biotransformation enzymes, especially those enzymes induced by PAH interaction with the AhR. In support of this, we report here that AhR-induced genes, including CYP1A1 and CYP1B1, are up-regulated in BEAS-2B cells in a time-dependent manner after exposure to BDS. We have observed similar AhR-induced and NRF2-mediated expression changes in lungs of mice exposed to BDS by inhalation (52).

BDS-associated fluorescence localization to the ER, which is evident at 24 hours, suggests availability of at least some of the PAHs for biotransformation. AhR-induced gene expression increases before there is evidence of colocalization, consistent with up-regulation of Phase I biotransformation enzymes within the first hour of exposure. Elevated expression of these genes at 24 hours correlates with increased colocalization of ER marker and BDS-associated fluorescence, which is indicative of PAH biotransformation. Up-regulation of NRF2-mediated oxidative stress response genes is evident after 24 hours of BDS exposure in BEAS-2B cells (Table 2). The role of NRF2 in activation of antioxidant response elements has been described for macrophages and epithelial cell lines exposed to diesel exhaust PAHs (39). NRF2, in conjunction with small v-maf musculoaponeurotic fibrosarcoma oncogene homolog proteins, activate antioxidant response elements in promoters of a variety of genes having antioxidant and cytoprotective functions; these include Phase II biotransformation enzymes (NQO1, ALDH1A3) and protective proteins such as DNAJB6 (40). PAHs have also been shown to stimulate mitogen-activated protein kinases (MAPKs), resulting in expression of immediate early genes (JUN, FOS), and subsequent NRF2-mediated activation of Phase II responses (41). The identification of NRF2-mediated oxidative stress response genes in microarray data from BDS-exposed BEAS-2B cells further supports the availability of BDS-associated PAHs for biotransformation, and indicates increased cellular oxidative stress as a result of BDS exposure.

Microsomal cytochrome P450 enzyme activity is responsible for the Phase I metabolism of many PAHs, whereby oxidation of a toxic substrate prepares the substrate for Phase II metabolism and eventual excretion. For some compounds, including B(a)P, Phase I metabolism can transform substrates into highly reactive compounds (bioactivation). The most bioactive metabolite of B(a)P is benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE), which forms guanine adducts in DNA (e.g., at specific mutational loci of the p53 gene). These loci often are altered in human lung cancer (42). Buening and colleagues (43) reported that intraperitoneal injection of BPDE significantly increases pulmonary tumor occurrence in rats. In studies with normal mammary epithelial cells exposed to B(a)P in vitro, Keshava and colleagues (44) demonstrated that cytochrome P450 IA1 and IB1 expression is highly variable between individuals, but that expression of these genes is generally induced by exposure to B(a)P, regardless of baseline expression. Although those data seemed to strongly support bioactivation of B(a)P, there was only a weak correlation between CYP1A1 and CYP1B1 induction and B(a)P-DNA adduction. We suggest that sequestration of PAHs in lipid droplets may perpetuate their interaction with the AhR, and thus prolong activation of the xenobiotic metabolism pathways.

The involvement of the smooth ("agranular") endoplasmic reticulum in metabolism of lipid-soluble drugs, cholesterol, and steroid hormones was recognized over 40 years ago (45). Members of the cytochrome P450 family are vital for detoxification of xenobiotics, including PAHs. Inducible microsomal cytochrome P450 enzymes accumulate on the endoplasmic reticulum and induce proliferation of smooth endoplasmic reticulum enzymes (46). Our results indicate that, over time, lipid droplets can deliver their PAH cargo to the endoplasmic reticulum. It is unlikely that any PAH metabolism occurs within lipid droplets. The Drosophila lipid droplet proteome has been investigated extensively and is considered an excellent model for the mammalian lipid droplet proteome (47). Of the 248 proteins currently identified in the Drosophila lipid droplet proteome, only one is a member of the cytochrome P450 system (48).

The potential health relevance of PAH localization to lipid droplets in respiratory system cells extends beyond ultrafine particles that arise during industrial petrochemical combustion. Two other common environmental sources of readily respirable particles—cigarette smoke and diesel exhaust—are likely sources of PAHs that would be expected to localize within lipid droplets. At least six of the most prevalent PAHs in BDS (anthracene, benzopyrenes, benzperylene, chrysene, fluoranthene, and pyrene (7) are major components of the particulate phase of mainstream and sidestream cigarette smoke (49).

Experiments in dogs have shown that organic solvent-denuded diesel exhaust particles coated with B(a)P are stripped of one third of their B(a)P load within 30 minutes of initial exposure. The parent B(a)P is detected quickly in the systemic circulation followed by rapid appearance of systemic metabolites. This "quick" release avoids initial metabolism of the B(a)P by respiratory epithelium. Two thirds of the original B(a)P load can be recovered adhered to particles in the lungs more than 5 months after exposure. This B(a)P has apparently been unavailable or inaccessible for biotransformation (50). Thus, long-term retention of PAH-laden particles in the lung could allow for long-term concentration of PAHs into the lipid droplets of alveolar macrophages and pulmonary epithelial cells. The availability of these PAHs to the AhR once they are sequestered in lipid droplets remains unknown. Persistent irritation from particle retention in the lung interstitium plus long-term activation of the AhR pathway, constant stimulation of xenobiotic metabolism enzymes, and perpetual inflammation could provide a procancerous environment in the lung parenchyma (51).

In conclusion, our results demonstrate that fluorescent PAH compounds adsorbed to inhalable combustion-derived ultrafine particles traffic to lipid droplets of adipocytes, as well as respiratory cells, including bronchial epithelial cells and alveolar macrophages. Furthermore, in vitro exposure of epithelial cells and alveolar macrophages to BDS stimulates AhR-responsive xenobiotic metabolism pathways known to potentiate the toxicity of certain PAHs, including several found in BDS and in cigarette smoke.


   Acknowledgments
 
The authors thank Dr. David Burk of the Socolofsky Microscopy Facility at Louisiana State University for his assistance with SlideBook software and Leica microscope operation and Mr. Michael Kearney for the statistical analyses.


   Footnotes
 
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.2007-0204OC on December 13, 2007

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 June 6, 2007

Accepted in final form November 21, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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