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Keck Functional Proteomics Center, Department of Biochemistry and Biological Chemistry, Los Angeles, California 90095Department of Medicine, Division of Clinical Immunology and Allergy, David Geffen School of Medicine, UCLA, Los Angeles, California 90095
To whom correspondence should be addressed: Dept. of Medicine, Division of Clinical Immunology and Allergy, UCLA, Box 951680, 52175 CHS, Los Angeles, CA 90095-1680. Tel.: 310-825-6620; Fax: 310-206-8107;
* This work was supported by United States Public Health Service Grants RO-1 ES12053, RO-1 ES10553, and PO-1 AI50495 and by a grant (to J. A. L.) from the UCLA Molecular Biology Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Epidemiological studies demonstrate an association between short term exposure to ambient particulate matter (PM) and cardiorespiratory morbidity and mortality. Although the biological mechanisms of these adverse effects are unknown, emerging data suggest a key role for oxidative stress. Ambient PM and diesel exhaust particles (DEP) contain redox cycling organic chemicals that induce pro-oxidative and pro-inflammatory effects in the lung. These responses are suppressed by N-acetylcysteine (NAC), which directly complexes to electrophilic DEP chemicals and exert additional antioxidant effects at the cellular level. A proteomics approach was used to study DEP-induced responses in the macrophage cell line, RAW 264.7. We demonstrate that in the dose range 10–100 μg/ml, organic DEP extracts induce a progressive decline in the cellular GSH/GSSG ratio, in parallel with a linear increase in newly expressed proteins on the two-dimensional gel. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electrospray ionization-liquid chromatography/mass spectrometry/mass spectrometry analysis, 32 newly induced/NAC-suppressed proteins were identified. These include antioxidant enzymes (e.g. heme oxygenase-1 and catalase), pro-inflammatory components (e.g. p38MAPK and Rel A), and products of intermediary metabolism that are regulated by oxidative stress. Heme oxygenase-1 was induced at low extract dose and with minimal decline in the GSH/GSSG ratio, whereas MAP kinase activation required a higher chemical dose and incremental levels of oxidative stress. Moreover, at extract doses >50 μg/ml, there is a steep decline in cellular viability. These data suggest that DEP induce a hierarchical oxidative stress response in which some of these proteins may serve as markers for oxidative stress during PM exposures.
Epidemiological studies demonstrate an association between short term exposure to ambient particulate matter (PM)
). Even though the relative risks are small, there is considerable public health concern because of the large number of exposed people and the existence of high risk groups. People suffering from asthma constitute a susceptible group, as exemplified by acute symptomatic flares after a sudden surge in ambient PM levels (
). In addition to these short term effects, animal and human studies conducted with diesel exhaust particles (DEP) as a model air pollutant showed that these particles can enhance allergen-specific IgE production and airway allergic inflammation in parallel with increased Th2 cytokine production (
Committee on Research Priorities for Airborne Particulate Matter Research Priorities for Airborne Particulate Matter: I. Immediate Priorities and a Long-range Research Portfolio. National Academy Press, Washington,
). How does ambient PM induce oxidative stress? When exposed to intact DEP or organic extracts made from these particles, macrophages and epithelial cells respond by producing reactive oxygen species (ROS) (
). DEP contain a large number of organic chemical compounds among which the polycyclic aromatic hydrocarbons (PAH), nitro-derivatives of PAH, oxygenated PAH derivatives (ketones, quinones, diones), heterocyclic organic compounds, aldehydes, and aliphatic hydrocarbons are the most abundant (
Although much remains to be learned about the role of oxidative stress in PM-induced adverse health effects, we have demonstrated that organic DEP extracts induce a wide range of biological effects in epithelial cells and macrophages (
). We used this approach to test the premise of an incremental oxidative stress response in RAW 264.7 cells during exposure to organic DEP extracts. Our data show that methanol DEP extracts induce a linear increase in newly expressed proteins, >50% of which are suppressed by NAC. We have subjected 32 of these proteins to mass spectrometry, and used select candidates to show that there is a difference in the dose-response kinetics of antioxidant versus pro-inflammatory proteins. These results support the existence of a hierarchical oxidative stress model.
MATERIALS AND METHODS
Reagents—Fetal bovine serum was purchased from Irvine Scientific (Santa Ana, CA). Dulbecco's modified Eagle's medium, penicillin-streptomycin, and l-glutamine were purchased from Invitrogen. DEP, collected from a low duty engine, was generously provided by Dr. Masaru Sagai (National Institute of Environmental Studies, Tsukuba, Japan) (
). NAC, EDTA, monoclonal anti-catalase, and propidium iodide (PI) were purchased from Sigma. Anti-JNK1/2, anti-p38MAPK, and antiphospho-JNK Abs were purchased from Cell Signaling Technology (Beverly, MA). Monoclonal mouse anti-GAPDH was purchased from Ambion Inc. (Austin, TX). Monoclonal anti-Rel A (p65) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Western Blot Analysis—RAW 264.7 cells were harvested by scraping and lysed in radioimmune precipitation assay buffer (10 mm NaPO4,pH 7.2, 0.3 m NaCl, 0.1% SDS, 1%Triton X-100, 1% sodium deoxycholate, 2 mm EDTA) supplemented with phosphatase and protease inhibitor mixtures (sets II and III, Calbiochem-Novabiochem Corp., San Diego, CA). Eighty μg of total lysate protein was electrophoresed on SDS-polyacrylamide gels before transfer to polyvinylidene difluoride membranes. To determine HO-1 (
), GAPDH, Rel A (p65), and catalase expression, blots were probed with 0.3 μg/ml, 1:2000, 1:1000, and 1:1000 of the respective antibody, followed by 1:1000 dilution of a HRP-conjugated rabbit anti-mouse Ab. Phosphopeptide blotting for JNK and p38MAPK was performed with a 1:10000 dilution of the primary Ab, followed 1:1000 dilution of HRP-conjugated goat anti-rabbit Ab (1:1000). For β-actin immunoblotting, stripped membranes were overlaid with monoclonal anti-actin Ab (1: 200), followed by HRP-conjugated rabbit anti-mouse Ab (1:1000). All blots were developed by ECL.
Preparation of DEP Methanol Extracts—DEP were provided by Dr. Masura Sagai (Tsukuba, Japan). These particles were collected from the exhaust from a 4JB1-type LD, 2.74-liter, 4-cylinder Isuzu diesel engine under a load of 10 torque onto a cyclone impactor equipped with a dilution tunnel constant volume sampler (
). DEP was collected on high capacity glass fiber filters, from which the scraped particles were stored as a powder in a glass container under nitrogen gas. The particles consist of aggregates in which individual particles are <1 μm in diameter. The chemical composition of these particles, including PAH and quinone analysis, has been described previously (
). Briefly, 100 mg of DEP were suspended in 25 ml of methanol and sonicated for 2 min. The suspension was centrifuged at 425 × g for 10 min at 4 °C, and the supernatant transferred to a preweighed Eppendorf tube to determine the amount of extractable material. After drying under nitrogen gas, the dried material was completely dissolved in Me2SO and aliquots saved at -80 °C in the dark until use.
Cellular Stimulation with DEP Extracts—RAW 264.7 cells were cultured in complete medium, which consisted of Dulbecco's modified Eagle's medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum. For cellular stimulation, 2 × 106 cells in 3 ml of culture medium were treated with the indicated amounts of the DEP extract in 6-well culture plates for 6 h at 37 °C in a humidified CO2 incubator. Control cultures received 0.1% of the Me2SO carrier. Some cultures received 20 mm NAC from a 1 m stock made in HEPES buffer immediately before use. NAC was added independently prior to, concomitant with or following the addition of the DEP extract as indicated. To determine whether NAC interacts directly with electrophilic chemicals in the extract, we premixed 10 mg of NAC with 1 mg of the DEP extract in a small volume (50 μl). This mixture was incubated at room temperature for 1 h before addition to the cell culture at a final extract concentration of 10–50 μg/ml, while limiting the NAC concentration in the medium to 61.5 μm. The controls consisted of cells receiving DEP chemicals only, or 20 mm NAC added to the culture medium for 2 h prior to the addition of the DEP extract at the indicated concentrations. The cells were harvested 6 h later and used for HO-1 immunoblotting as described previously (
). Briefly, cells were lysed and deproteinized in 3% 5-sulfosalicylic acid. Whole cell lysates were cleared at 4 °C by centrifugation at 20,800 × g in an Eppendorf centrifuge. The supernatant was used to measure total and oxidized glutathione. Total glutathione was read from a GSH standard curve, prepared in 5-sulfosalicylic acid. For the GSSG assay, 100 μl of supernatant was incubated with 2 μl of 2-vinylpyridine and 6 μl of triethanolamine for 60 min on ice. GSSG standards were treated in the same way, and the GSSG content of the samples was calculated from a GSSG standard curve. Reduced GSH was calculated by subtracting GSSG from total glutathione.
Determining Cell Viability by Propidium Iodide (PI) Staining—Cells (3 × 106) were plated into 3.5-cm plates in 3 ml of medium and rested for 4 h. Some cultures were preincubated with 20 mm NAC for 2 h. Varying concentrations of DEP were added to these cultures for 18 h. Cells were collected, washed twice in PBS, and resuspended in 500 μlof PBS containing 0.5 μg/ml PI for 5 min. Cells were analyzed in a FACScan (Becton Dickinson, Mountain View, CA) equipped with a single 488-nm argon laser. Dead cell fragments were gated out by forward and side scatter, and PI analysis was performed at excitation and emission settings of 488 and 575 nm, respectively.
Protein Extraction and Sample Preparation—Aliquots of 2 × 107 RAW 264.7 cells were washed twice with ice-cold PBS containing protease inhibitors and sonicated in ice-cold radioimmune precipitation assay buffer containing 10 mm NaPO4, pH 7.2, 0.3 m NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 2 mm EDTA, protease inhibitor mixture set III (100 mm AEBSF, 80 μm aprotinin, 5 mm bestatin, 1.5 mm E-64, 2 mm leupeptin, 1 mm pepstatin), and phosphatase inhibitor mixture set II (200 mm imidazole, 100 mm sodium fluoride, 115 mm sodium molybdate, 100 mm sodium orthovanadate, 400 mm sodium tartrate dihydrate) (Calbiochem, La Jolla, CA) for 10 s. Lysates were centrifuged at 1000 × g for 5 min. To remove the salt from the lysates, the supernatant proteins were precipitated with trichloroacetic acid (10% w/v), 20 mm DTT for 30 min on ice. The precipitate was collected at 20,800 × g for 10 min at 4 °C and washed three times with 10% trichloroacetic acid, 20 mm DTT. Trichloroacetic acid in the precipitate was removed through the extraction with diethyl ether or acetone plus 10 mm DTT. After drying, the pellet was resuspended by sonication in a buffer containing 7 m urea, 2 m thiourea, 4% w/v CHAPS, 100 mm DTT, 0.2% v/v Bio-Lyte pH 3/10:4/6:5/8 (1:0.5:0.5), 5% glycerol, and protease/phosphatase inhibitors (mixture sets II and III). After standing for 1 h at room temperature, the sample was centrifuged at 23,800 × g for 10 min at 15 °C, and the supernatants stored at -80 °C until use for two-dimensional PAGE. Protein concentration in these samples was estimated by using a commercial Bradford kit (DC reagent kit, Bio-Rad), and bovine serum albumin as standard.
Two-dimensional Polyacrylamide Gel Electrophoresis (PAGE)—Two-dimensional gel electrophoresis was performed with the Bio-Rad system as described by Jungblut and Thiede (
). 350 μg of whole cell lysate was added to each immobilized pH gradient (IPG) strip, which was rehydrated in 8 m urea, 2% CHAPS, 50 mm, 0.2% Bio-Lyte 3/10 ampholyte, 0.001% bromphenol blue. The pre-isoelectric focusing and isoelectric focusing (IEF) were performed using pre-made 17-cm length IPG strips (pH 3–10 NL) on the Protean IEF cell. The pre-isoelectric focusing was performed linearly up to 500 V for 1 h, held at 500 V for 1.5 h. Formal IEF was then performed with a linear increase up to 10,000 V over 2 h and then held at 10,000 V for 7 h a total of 90 KV-h. For the second dimension, the IPG strips were equilibrated in a buffer containing 37.5 mm Tris-HCl, pH 8.8, 20% glycerol, 2% SDS, and 6 m urea with 2% dithiothreitol (Sigma), followed by 8–16% SDS-PAGE on a Protean Plus Dodeca Cell (Bio-Rad). Gels were stained with Sypro-Ruby (Molecular Probes, Eugene, OR) and visualized under ultraviolet light with a Molecular Imager FX Pro Plus (Bio-Rad). To check the reproducibility of the data, three independent two-dimensional analyses were performed on each cellular lysate.
Protein Identification—Protein spots were selected based on staining intensity of the Sypro Ruby as determined by the PDQuest software (Bio-Rad). This sensitivity of the software is set to detect a 2-fold increase in staining intensity as a criterion for a significant increase in protein expression. For the purpose of this study, we increased the stringency to 8-fold. Spots were excised by a spot-excision robot (Proteome Works, Bio-Rad) and deposited into 96-well plates. Gel spots were washed and digested with sequencing-grade trypsin (Promega, Madison, WI), and the resulting tryptic peptides were extracted using standard protocols (
). Trypsin digestion and extraction, and peptide spotting onto a matrix-assisted laser desorption ionization (MALDI) targets, were accomplished by a robotic liquid handling work station (MassPrep, Micromass-Waters, Beverly, MA). MALDI peptide fingerprint mass spectra were acquired with a MALDI time-of-flight instrument ([email protected], Micromass-Waters), using α-cyano-4-hydroxycinnamic acid (Sigma) as the matrix. Peptide sequencing was accomplished with nanoflow high performance liquid chromatography with electronic flow control (1100 Series nanoflow liquid chromatography system, Agilent Technologies, Palo Alto, CA) interfaced to an ion trap mass spectrometer (LC-MSD Trap SL, Agilent Technologies). A reverse phase column (75 μm × 150 mm, C18 Zorbax StableBond) was used as the analytical column. A Zorbax 300SB enrichment pre-column (0.3 × 5 mm) was used to concentrate and desalt the peptide mixtures. The MS data from both tandem mass spectra from the LC-MS/MS experiments and the MALDI-MS peptide fingerprint mass spectra were searched against a subset of rodent proteins in the SWISS-PROT protein sequence data base, using the Mascot search program (Matrix Science, London, United Kingdom) (www.matrixscience.com). Positive protein identification was based on standard Mascot criteria for statistical analysis of the MALDI peptide fingerprint mass spectra and the LC-MS/MS data. A -10log(P) score, where P is the probability that the observed match is a random event, of >72 was regarded as significant.
Data Analysis—GSH/GSSG ratio, cell viability, and newly induced protein data are expressed as the mean ± S.E. One-way analysis of variance was used to determine differences between groups with post hoc comparisons by the method of Fisher. Significance was assumed at p < 0.05.
Organic DEP Extracts Induce Oxidative Stress and a Range of Biological Responses—Reduced glutathione (GSH) plays an important role in ROS scavenging and maintenance of cellular redox equilibrium (
). When exposed to incremental amounts of a methanol DEP extract, RAW 264.7 cells show a progressive and statistically significant decline in the GSH/GSSG ratio at doses >10 μg/ml (Fig. 1). This effect was diminished by pre-treating the cells with NAC (Fig. 1).
Cells respond to oxidative stress in a variety of ways, including the activation of intracellular signaling pathways that exert pro-inflammatory effects in the lung (
). One example is the activation of the JNK and p38MAPK cascades by the organic DEP extract in RAW 264.7 cells (Fig. 2). This effect is demonstrated by the increased phosphorylation of p38MAPK (Fig. 2A) and the 46- and 54-kDa JNK isoforms on allosteric sites that lead to their activation (Fig. 2B). Although an extract dose of 10 μg/ml failed to induce JNK activation, doses of ≥50 μg/ml did induce kinase activation as determined by anti-phosphopeptide immunoblotting (Fig. 2B). Prominent p38MAPK activation also required an extract dose of ≥50 μg/ml, while registering a smaller effect at 10 μg/ml (Fig. 2A). The increase in site-specific phosphorylation was not the result of changes in kinase abundance, as demonstrated by parallel immunoblotting for kinase protein (Fig. 2, bottom panels). Prior treatment with NAC interfered in these phosphorylation events, confirming that these MAP kinase cascades are activated under conditions of oxidative stress (Fig. 2, A and B).
In addition to activating these signaling cascades, extract doses ≥10 μg/ml induced cellular toxicity as shown by increased PI uptake (Fig. 3). This increase in cell death was more noticeable at doses ≥50 μg/ml (Fig. 3). We have previously shown that this cytotoxic effect is a programmed cell death event that involves mitochondrial perturbation and release of cytochrome c (
). The involvement of oxidative stress is confirmed by the ability of NAC to interfere in cytotoxicity (Fig. 3). Taken together with the data in Fig. 2, these findings demonstrate that at doses ≥10 μg/ml, organic DEP extracts induce a progressive increase in injurious cellular responses. However, elucidation of cellular responses at 10 μg/ml is important because not all oxidative effects are injurious in nature (
). This necessitated the use of a discovery tool that is more appropriate for revealing an extensive dose-response relationship.
Two-dimensional Gel Electrophoresis and Mass Spectrometry Reveal a Hierarchical Response to Organic DEP Extracts—Changes in the proteome of RAW 264.7 macrophage cells were examined in cell populations exposed to incremental amounts of the DEP extract. Protein expression was displayed by two-dimensional PAGE and yielded >1200 individual polypeptides in unstimulated cells. The addition of DEP extracts induced new protein expression, which was defined as >8-fold increase (p < 0.01) in the staining intensity of each individual Sypro Ruby-stained polypeptide (Fig. 5). The number of newly expressed proteins increased linearly as the extract dose increased, and yielded 10, 65, and 100 new proteins at DEP extract concentrations of 10, 50, and 100 μg/ml, respectively (Fig. 4A). Linear regression analysis showed an excellent correlation (r2 = 0.982) between extract dose and the number of newly induced proteins (Fig. 4B). There was some overlap as well as unique expression profiles for each extract dose (Fig. 4C). Thus, six new proteins were expressed at all extract doses and are listed in Fig. 4D. These include proteins that play a role in antioxidant defense (HO-1, catalase, and metallothionein), a signaling pathway component (the α1 subunit of p38MAPK), a transcription factor (Rel A), and a component of the Emden Meyerhoff pathway (GAPDH). An additional 45 newly expressed proteins were shared in cell populations treated with 50 and 100 μg/ml extract, whereas the respective cell populations treated with doses of 10, 50, and 100 μg/ml showed 4, 14, and 51 uniquely expressed proteins (Fig. 4D).
NAC addition diminished protein expression by ∼50%, confirming the possible relationship to oxidative stress (Fig. 4B). NAC suppression or subtraction was defined as a 50% decrease in staining intensity of an inducible protein. This suppression by NAC was used as a criterion to select response markers for further analysis by protein mass spectrometry (Fig. 5). GAPDH is an example of an oxidative stress protein that was induced in RAW 264.7 cells during exposure to 50 μg/ml DEP extract (Fig. 5A). Compared with untreated cells, GAPDH expression increased >8-fold, whereas the inclusion of NAC decreased that response by 70% (Fig. 5B). Use of this approach led to the identification of an additional 31 proteins by mass spectrometry (Table I). These proteins were distributed into two zones according to pI values (Fig. 5A). The first zone falls in the pI range 4.5–5.5, and includes a signaling component, p38MAPK α1, the tyrosine kinase, ErbB-2, as well as the detoxification enzyme, alcohol dehydrogenase (Table I). The second zone, spanning pI 5.8–9.0, contains several proteins involved in intermediary metabolism, ATP production, and oxidative stress (e.g. GAPDH), a transcription factor (e.g. Rel A), and antioxidant defense proteins (e.g. HO-1, catalase, and metallothionein) (Fig. 5). To increase the protein resolution in this zone of the gel, cellular extracts were further analyzed on two-dimensional gels, which utilized a narrower focusing range (pH 5.5–6.7) (Fig. 6). These zoom gels helped to confirm the induction of HO-1 and catalase expression by the DEP extract, as well as the ability of NAC to suppress their expression (Fig. 6, A–C).
Table IProtein assignments from whole RAW 264.7 cells exposed to both 50 and 100 μg/ml DEP extracts*, Pro-inflammatory, the potential to contribute to biological events culminating in or regularly inflammation; oxi, oxidative.
To examine the fidelity of these newly induced proteins and to confirm the two-dimensional PAGE analysis, Western blotting was performed. GAPDH immunoblotting confirmed its expression at all DEP extract doses tested (Fig. 5C). Interestingly, GAPDH expression was fully induced at the lowest DEP extract dose, and showed increased sensitivity to NAC suppression at higher extract doses (Fig. 5C). Similar subtractive protein expression, making use of two-dimensional PAGE and Western blotting, was demonstrated for catalase and HO-1 (Fig. 6D), as well as Rel A (p65) and metallothionein (Fig. 6E).
The Suppressive Effect of NAC Is Dependent on Cellular Antioxidant Effects as Well as Direct Electrophilic Interactions with DEP Chemicals—NAC is the N-acetyl derivative of the naturally occurring amino acid, l-cysteine, and functions as a radical scavenger as well as a precursor for glutathione synthesis (
). In addition to these cellular antioxidant effects, NAC also utilizes its SH group to directly complex to electrophilic DEP chemicals. This interaction could take place in the tissue culture medium as well as intracellularly. To discern between these different modes of action, we demonstrated that NAC addition 2 h after the introduction of the DEP extract could suppress HO-1 expression, provided that the stimulus was removed before the addition of NAC (Fig. 7A, lane 2). However, if not removed from the culture medium, the stimulating effects of the DEP chemicals were unopposed (lane 3). These data suggest that NAC interfere in the pro-oxidative effects of DEP chemicals at a cellular level. In the same experiment, it could also be demonstrated that NAC addition prior to the delivery of the stimulus can prevent HO-1 induction (Fig. 7A, lanes 5–7); the effect was more prominent in unwashed cell cultures (lane 7) compared with cells where the thiol was added for 2 h and then washed away (lane 6). This raises the possibility that NAC may also interfere in the effects of the inducing chemicals by direct chemical interactions, some of which may occur in the culture medium. This possibility was further explored by premixing a weight excess of NAC with the DEP extract in a small volume before adding the mix to the cell culture medium (Fig. 7B). In this experiment, in which the final NAC concentration in the culture medium was <100 μm, the interference in HO-1 expression (lanes 4–6) was equivalent to the effect of 20 mm NAC introduced with the stimulus (lanes 7–9). This is compatible with the data in Fig. 4. Although the extent to which a direct electrophilic interactions versus cellular antioxidant effects contribute to the NAC effect is difficult to quantify, the net effect is to prevent a decline in the cellular GSH/GSSG ratio as well as new protein expression (Fig. 1).
Taken together, the data depicted in Figs. 4, 5, 6, 7 demonstrate that organic DEP extracts induce the expression of a range of proteins, ∼50% of which are suppressed by NAC. Although prominent MAPK activation and the induction of cellular toxicity require DEP extract doses >10 μg/ml, the antioxidant enzymes (HO-1, catalase, and metallothionein) and GAPDH were induced at lower extract doses. This suggests a segregation of protective versus injurious cellular effects at different extract doses and at different levels of oxidative stress.
We demonstrate that organic DEP extracts induce a dose-dependent decrease in the GSH/GSSG ratio in RAW 264.7 cells, in parallel with a linear increase in the number of newly expressed proteins. More than half of these proteins were suppressed in the presence of NAC. Using mass spectrometry analysis, 32 newly induced/NAC-suppressed proteins were identified. These include antioxidant enzymes, e.g. HO-1 and catalase, as well as proteins that play a role in pulmonary inflammation, namely p38MAPK and Rel A. HO-1 was induced at a low extract dose and with minimal decline in the GSH/GSSG ratio, whereas prominent Jun and p38MAPK activation required higher extract amounts and incremental levels of oxidative stress. Moreover, at extract doses >50 μg/ml, there is an increase in the rate of cytotoxicity. These data suggest that organic DEP chemicals induce a hierarchical oxidative stress response, which is reflected by the types of proteins being expressed.
Mass spectrometry and proteome analysis have been useful in identifying oxidative stress markers under a variety of disease conditions, including cellular hypoxemia, T-cell dysfunction in setting of AIDS, Alzheimer's disease, and tissue inflammation (
). Although these post-translational modifications are helpful as a qualitative display of oxidative stress, this approach is not helpful in quantifying the cellular response to oxidative stress. We therefore used an alternative proteomics approach, which looks at the total number of newly expressed proteins as well as their NAC subtraction, to quantify the oxidative stress response. This showed that increasing amounts of the organic DEP extract induce a progressive decline in the cellular GSH/GSSG ratio, in parallel with a linear increase in the number of newly expressed proteins (Figs. 1 and 4). The decline in the GSH/GSSG ratio is a representative cellular marker for oxidative stress (Fig. 1) and is directly involved in eliciting cellular responses, including antioxidant defense and protection of the mitochondrial PT pore (
). The inhibitory effect of NAC is particularly relevant to the induction of oxidative stress by organic DEP chemicals (Figs. 1, 2, 3, 4, 5, 6, 7). Among a wide range of antioxidants tested, thiol antioxidants were the most specific in interfering in the pro-oxidative effects of organic DEP chemicals in vitro and in vivo (
What conclusions can be drawn from the proteome analysis of DEP-treated RAW 264.7 cells? The linear increase in new protein expression with increasing extract doses suggests an escalating cellular response to oxidative stress (Fig. 4). This notion is supported by the fact that HO-1, catalase, and metallothionein (
), suggesting that cytoprotective pathways are induced at the lowest levels of oxidative stress (Fig. 8). This may constitute the first tier of a hierarchical oxidative stress response (Fig. 8). HO-1 expression is also a very sensitive marker for oxidative stress in bronchial epithelial cells (
), another key cellular target for PM. The induction of HO-1 expression by redox cycling chemicals, including cadmium and organic DEP compounds, is dependent on the anti-oxidant response element in the promoter of that gene (Fig. 8) (
Although increased expression of the p38MAPK α1 isoform can be seen to occur at 10 μg/ml extract (Fig. 4D), prominent activation of the p38MAPK and Jun kinase cascades required >10 μg/ml amounts of the same material (Fig. 2). These stress-activated protein kinases play a role in the expression and transcriptional activation of several AP-1 proteins (
), and are often linked to pro-inflammatory and injurious cellular responses (Fig. 8). This includes the transcriptional activation of cytokine and chemokine genes (Fig. 8). We propose that these pro-inflammatory effects constitute a second tier or a superimposed level of oxidative stress, and that proteins that are induced or activated in this zone play a role in the pro-inflammatory and adjuvant effects of DEP in the lung (
) (Table I). Although increased expression of the glucocorticoid receptor may be important for the treatment of allergic disease, it is interesting that steroid administration does not reverse the pro-inflammatory effects of DEP in the nasal mucosa (
) (Table I). It is interesting that the DEP extracts also induced Fas-associating death domain-containing protein (FADD) expression, which may play a role in receptor-induced apoptosis, as well as the expression of proteins that play a role in intermediary metabolism and are linked to regulation of oxidative stress, e.g. phosphoenolpyruvate carboxykinase (
). In this regard, we have recently demonstrated that the use of increasing polar elutants to fractionate organic DEP extracts by silica gel chromatography yielded aromatic and polar chemical groups, which mimic the effect of the crude extract in cellular toxicity assays (
). We are currently investigating the hypothesis that these chemical groups are responsible for the pro-oxidative and pro-inflammatory effects of PM. It may be relevant that analysis of ambient PM with aerodynamic diameter <0.15 μm (ultrafine particles), collected by particle concentrators in the Los Angeles basin, demonstrated an excellent correlation between PAH content and their capacity to generate ROS in the presence of DTT (
The inhibitory effects of NAC on protein expression is interesting from a number of different perspectives. The ROS scavenging effects of NAC is explained by its SH-group, which has the potential to directly interact with oxidants such as H2O2, leading to the formation of H2O and O2. Deacetylation of NAC also leads to the formation of cysteine, which is a precursor for glutathione synthesis (
). In addition, NAC itself can participate in electrophilic interactions, thereby establishing multiple mechanisms by which this thiol agent can interfere in the oxidative stress effects of DEP chemicals. Whatever the exact contribution of direct electrophilic interactions versus effects on GSH synthesis and radical scavenging may be, the net effect of NAC is to prevent a drop in the cellular GSH/GSSG ratio (Fig. 1) as well as to interfere in ROS generation (
) may prove useful in identifying the major chemical groups in DEP that is responsible for ROS generation.
How does exposure to 1–100 μg/ml of the DEP extract compare with in vivo PM exposures in humans? Although it is difficult to directly extrapolate from the in vitro to the in vivo exposure amounts, it is possible to demonstrate using human dosimetry models that the dose of PM2.5 (particulate matter with aerodynamic diameter ≤ 2.5 μm) deposition at airway bifurcation points is comparable with the in vitro tissue culture concentrations recalculated as extract dose/cm2 (
). Thus, we have shown that 1–100 μg/ml DEP extract is equivalent to 0.14–14 μg/cm2 in a tissue culture dish, whereas an asthmatic person with airway stasis, breathing polluted air in Rubidoux, California, can deposit 2.3 μg/cm2 PM2.5 at tracheobronchial bifurcation sites (
). It is possible, therefore, that at these so-called hot spots of deposition (airway bifurcations), the bronchial mucosa may be exposed to DEP chemical doses that are toxicologically relevant from an oxidative stress perspective (
In summary, we have shown that proteomics analysis can be used to study the linear increase in new protein expression in parallel with increased levels of DEP-induced oxidative stress.
We thank Sheng Yin and Dr. James Kerwin for help with the gel image analysis, protein digestion, and protein identifications, and Dr. Rachel Ogorzalek Loo for advice on sample preparation and gel electrophoresis. We acknowledge the support from Agilent Technologies in the operation of the ion trap mass spectrometer. The UCLA Functional Proteomics Center was established and equipped with a grant from the W. M. Keck Foundation.
Committee on Research Priorities for Airborne Particulate Matter Research Priorities for Airborne Particulate Matter: I. Immediate Priorities and a Long-range Research Portfolio. National Academy Press, Washington,