Use of proteomics to demonstrate a hierarchical oxidative stress response to diesel exhaust particle chemicals in a macrophage cell line, GG Xiao, M Wang, N Li, JA Loo, AE Nel

Tags:
Content: JBC Papers in Press. Published on September 30, 2003 as Manuscript M306423200 Use of Proteomics to Demonstrate a Hierarchical Oxidative stress response to Diesel Exhaust Particle Chemicals in a Macrophage Cell Line Gary Guishan Xiao 1,2, Meiying Wang2, Ning Li2, Joseph A. Loo1, Andre E. Nel2,$ 1The Keck Functional Proteomics Center, Department of Biochemistry and Biological Chemistry, University of California Los Angeles, 1430 Molecular Science Building, 611 Charles E. Young Dr. E., Los Angeles, CA 90095 2Department of Medicine-Clinical Immunology and Allergy, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 Running title: Macrophage proteome analysis of DEP-induced Oxidative Stress Keywords: diesel exhaust particles, oxidative stress, proteomics, and macrophages $ To whom correspondence should be addressed: University of California Los Angeles, Division of Clinical Immunology & Allergy, Department of Medicine, Box 951680, 52-175 CHS, Los Angeles, CA 90095-1680. Tel: (0) 825-6620, Fax: (0) 206-8107 E-mail: [email protected] 1 Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
SUMMARY 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 DEPinduced 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 2-D 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/NACsuppressed 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, while 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. 2
INTRODUCTION Epidemiological studies demonstrate an association between short-term exposure to ambient particulate matter (PM) and cardiorespiratory morbidity and mortality (1-3). Even though the relative risks are small, there is considerable public health concern due to 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 (3). This is likely due to PM-induced airway inflammation and airway hyperreactivity (4-10). 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 (5,11-14). This raises the important question about the mechanism of these adverse health effects. Although the biological hypotheses for the mechanisms of PM action are just beginning to develop (15), most of the limited mechanistic data generated to date suggest that oxidative stress is a key biological event in causing the adverse health effects of ambient PM (5,16-20). 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) (16,17). In this regard, it is known that DEP and ambient PM contain transition metals (21,22) as well as redox cycling organic components which elicit ROS production in various cellular locations (19,23,24). For instance, organic DEP extracts induce superoxide production in lung microsomes through the action of NADPH-dependent P450 reductase (20), as well as through damage to the mitochondrial inner membrane (16,17,25). DEP contain a large number of organic chemical 3
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 (23,24,26). We have shown that there is a good correlation between the induction of oxidative stress and PAH content of ambient PM (19,25). Another chemical group that needs to be considered is quinones (27,28). Chemical derivatization of quinones diminished the effect of organic DEP extracts on superoxide production in lung microsomal preparations (20). Moreover, we have shown that polar chemical groups, fractionated from DEP and enriched in quinones, act as potent inducers of oxidative stress in macrophages and epithelial cells (26,29). In addition to being produced by the fuel combustion process, quinones are also generated during enzymatic conversion of PAH in the lung, including their conversion by cytochrome P450 1A1 (28,30). Although a lot 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 (16-18,21). This includes the induction of pro-inflammatory and cytotoxic effects, which can be suppressed by the thiol agent, N-acetylcysteine (NAC) (16,18). These pro-inflammatory effects include the production of cytokines and chemokines (13,14), while the cytotoxicity depends on the perturbation of mitochondrial function (16,17,29). This includes disruption of the mitochondrial inner membrane potential, cytochrome c release and caspase 9 activation (17). In addition to these harmful effects, organic DEP components have also been shown to induce cytoprotective responses, including the expression of an antioxidant enzyme, heme oxygenase 1 (HO-1) (26). Based on these diverse effects, we have postulated that DEP may induce a hierarchy of oxidative stress effects which range from cytoprotective to injurious (19,26). 4
Proteomics offers a unique means for analyzing the expressed genome, and has been successfully employed to look at the generation of oxidative stress at the cellular level (31-38). In addition to displaying oxidative modification of proteins (31,32,34-36,38), this approach can also be used to look at newly expressed proteins (33,37,38). 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 vs pro-inflammatory proteins. These results support the existence of a hierarchical oxidative stress model. 5
Reagents
MATERIALS AND METHODS
FBS was purchased from Irvine Scientific (Santa Ana, CA). DMEM, penicillin-streptomycin, and L-glutamine were purchased from Life Technologies (Gaithersburg, MD). DEP, collected from a low duty engine, was generously provided by Dr. Masaru Sagai (National Institute of Environmental Studies, Tsukuba, Japan) (20). NAC, EDTA, monoclonal anti-catalase, and propidum iodide (PI) were purchased from Sigma (St. Louis, MO). Anti-JNK1/2 and antip38MAPK Abs, anti-phospho-JNK and -p38MAPK Abs were purchased from Cell Signaling Technology (Cell Signaling Technology, Inc., Beverly, MA). Monoclonal mouse anti-GAPDH was purchased from Ambion Inc. (Austin, Texas). Monoclonal anti-Rel A (p65) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CAlifornia).
Western blot analysis RAW 264.7 cells were harvested by scraping and lysed in RIPA 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 cocktails (sets II & III, Calbiochem-Novabiochem Corp., San Diego, CA). Eighty µg of total lysate protein was electrophoresed on SDS polyacrylamide gels before transfer to PVDF membranes. To determine HO-1 (26), 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 -
6
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 l, 4-cylinder Isuzu diesel engine under a load of 10 torque onto a cyclone impactor equipped with a dilution tunnel constant volume sampler (21). 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, was previously described (26). Methanol extraction of DEP was performed as previously described (17). Briefly, 100 mg DEP were suspended in 25 ml methanol and sonicated for 2 min. The suspension was centrifuged at 425 Ч 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 DMSO 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 DMEM supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). For cellular stimulation, 2 x 106 cells in 3 ml of culture medium were treated with the indicated amounts of the DEP extract in six-well culture plates for 6 hrs at 37 °C in a humidified CO2 incubator. Control cultures received 0.1% of the DMSO carrier. Some cultures received 20 mM NAC from a 1M stock made 7
in HEPES buffer immediately before use. NAC was added independently prior to, concomitant with or following the addition of the DEP extract as indicated. In order to determine if NAC interacts directly with electrophilic chemicals in the extract, we premixed 10 mg NAC with 1 mg of the DEP extract in a small volume (50 µl). This mixture was incubated at room temperature for an hour 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 hrs prior to the addition of the DEP extract at the indicated concentrations. The cells were harvested 6 hrs later and used for HO-1 immunoblotting as previously described (26). Determination of cellular GSH/GSSG ratios Total glutathione (GSH plus GSSG) and GSSG were measured in a recycling assay that uses 5,5'-dithio-bis(2-nitrobenzoic acid) and glutathione reductase (39). Briefly, cells were lysed and deproteinized in 3 % 5-sulfosalicylic acid. Whole cell lysates were cleared at 4 °C by centrifugation at 20,800 Ч 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 supernatant was incubated with 2 µl 2vinylpyridine and 6 µl 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 8
Cells (3 x 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 µl of 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 Ч 107 RAW 264.7 cells were washed twice with ice-cold PBS containing protease inhibitors and sonicated in ice-cold RIPA 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 cocktail 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 cocktail set II (200 mM imidazole/100 mM sodium fluoride/115 mM sodium molybdate/100mM sodium orthovanadate/400 mM sodium tartrate dihydrate) (CalBiochem, La Jolla, CA) for 10 sec. Lysates were centrifuged at 1000 x g for 5 minutes. To remove the salt from the lysates, the supernatant proteins were precipitated with TCA (10% w/v)/20mM DTT for 30 min on ice. The precipitate was collected at 20,800 Ч g for 10 min at 4°C and washed 3x with 10% TCA/20 mM DTT. TCA in the precipitate was removed through the extraction with diethyl ether or acetone /10mM DTT. After drying, the pellet was resuspended by sonication in a buffer containing 7 M urea, 2M thiourea, 4% w/v CHAPS, 100 mM DTT, 0.2% w/v Bio-Lyte pH 3/10:4/6:5/8 (1:0.5:0.5), 5% glycerol, and protease/phosphatase inhibitors (cocktail sets II and III). After standing for 1 h at room temperature, the sample was centrifuged at 23,800 Ч g for 10 min at 15°C, and the supernatants 9
stored at ­80°C until use for 2D-PAGE. Protein concentration in these samples was estimated by using a commercial Bradford kit (DC reagent kit, Bio-Rad), and BSA as standard. Two-dimensional gel electrophoresis (2D-PAGE) Two-dimensional gel electrophoresis was performed with the Bio-Rad (Hercules, CA) system as described by Jungblut (40). 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% Bromophenol 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 hr, held at 500 V for 1.5 hr. Formal IEF was then performed with a linear increase up to 10,000 V over 2 hrs and then held at 10,000 V for 7 hrs 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 2D 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 two-fold increase in staining intensity as a criterion for a significant increase in protein expression. For the purpose 10
of this study, we increased the stringency to 8-fold. Spots were excised by a spot-excision robot (Proteome Works, BioRad) and deposited into 96-well plates. Gel spots were washed, digested with sequencing-grade trypsin (Promega, Madison, WI), and the resulting tryptic peptides were extracted using standard protocols (41). The trypsin digestion and extraction, and peptide spotting onto a matrix-assisted laser desorption ionization (MALDI) targets was accomplished by a robotic liquid handling workstation (MassPrep, Micromass-Waters, Beverly, MA). MALDI peptide fingerprint mass spectra were acquired with a MALDI time-of-flight (TOF) instrument ([email protected], Micromass-Waters, Beverly, MA), using -cyano-4-hydroxycinnamic acid (Sigma) as the matrix. Peptide sequencing was accomplished with a nanoflow HPLC with electronic flow control (1100 Series nanoflow LC 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 Ч 150 mm, C18 Zorbax StableBond) was used as the analytical column. A Zorbax 300SB enrichment pre-column (0.3 Ч 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 database, using the Mascot search program (Matrix Science, London, UK) (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 11
GSH/GSSG ratio, cell viability, and newly induced protein data are expressed as the mean ± SEM. One-way ANOVA was used to determine differences between groups with post hoc comparisons by the method of Fisher. Significance was assumed at p < 0.05. 12
RESULTS 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 (42). A decline in the ratio of reduced to oxidized glutathione (GSSG) is a sensitive parameter for cellular oxidative stress (42). 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 pretreating the cells with NAC (Fig. 1). Cells respond to oxidative stress in a variety of ways, including the activation of intracellular signaling pathways which exert pro-inflammatory effects in the lung (5). 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-kD JNK isoforms on allosteric sites that lead to their activation (Fig. 2B). While 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 due to 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. 2A and B). 13
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 (16,17). 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 since not all oxidative effects are injurious in nature (19,26). This necessitated the use of a discovery tool that is more appropriate for revealing an extensive dose-response relationship. 2D 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 2DPAGE 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 14
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 1 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 of the extract, while 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 approximately 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 of the DEP extract (Fig. 5A). Compared to untreated cells, GAPDH expression increased > 8-fold, while 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 1). 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 1, the tyrosine kinase, ErbB-2, as well as the detoxification enzyme, alcohol dehydrogenase (Table 1). 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 2D gels which utilized a narrower focusing range (pH 5.5-6.7) 15
(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. 6A-C). To examine the fidelity of these newly induced proteins and to confirm the 2D-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 2D-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 (43). 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. In order to discern between these different modes of action, we demonstrated that NAC addition 2 hours 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 can could also be demonstrated that 16
NAC addition prior to the delivery of the stimulus can prevent HO-1 induction (Fig. 7A, lanes 57); the effect was more prominent in unwashed cell cultures (lane 7) compared to cells where the thiol was added for two hours 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 (lane 7-9). This is compatible with the data in Fig. 4. Although the extent to which a direct electrophilic interactions vs 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. 4-7 demonstrate that organic DEP extracts induce the expression of a range of proteins, approximately 50% of which are suppressed by NAC. While 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 vs injurious cellular effects at different extract doses and at different levels of oxidative stress. 17
DISCUSSION 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, while 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 (31-38). Typically, proteome analysis of oxidative stress markers requires the identification of protein S-nitrosation, tyrosine nitration, glutathionylation or methionine oxidation (31,32,34,35,38). While these posttranslational 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 18
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 (Fig. 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 (42,44,45). The inhibitory effect of NAC is particularly relevant to the induction of oxidative stress by organic DEP chemicals (Figs. 1-7). 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 (18). 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 (46-48) are induced at the lower (10 µg/ml) extract dose (Figs. 6 and 7, Table 1), while prominent MAP kinase activation (Fig. 2) and induction of cellular toxicity require extract doses > 10 µg/ml (Fig. 3). HO-1 and catalase are antioxidant enzymes (49), 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 (29,50), 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 (ARE) in the promoter of that gene (Fig. 8) (26,50). This genetic response element is transcriptionally activated by a basic leucine zipper 19
transcription factor, Nrf2 (Fig. 8) (51). It is interesting that oxidative DNA damage and the accumulation of 8-hydroxydeoxyguanosine in the lungs of Nrf 2 knockout mice is exaggerated during exposure to diesel exhaust fumes (52). Although increased expression of the p38MAPK 1-isoform can be seen to occur at 10 µg/ml of the extract (Fig. 4D), prominent activation of the p38MAPK and Jun kinase cascades required > 10 µg/ml 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 (53), 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 which are induced or activated in this zone, play a role in the pro-inflammatory and adjuvant effects of DEP in the lung (5,10-13). This notion is strengthened by increased expression or oxidative modification of proteins that play a role in the regulation of inflammation, e.g. Rel A (54), GM-CSF precursor (55), TNF receptor 2 (56), glucocorticoid receptor (54), EGR-4 (57), and acetyl-CoA carboxylase 2 (58) (Table 1). While 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 (59). This could be related to to the fact that this receptor is a Zn-finger transcription factor and can be oxidatively inactivated by crosslinking of critical cysteine groups (54). Among the pharmacologic agents tested to curb the pro-inflammatory effects of DEP, only NAC was fully effective in suppressing the adjuvant effects of DEP in a murine allergen challenge model (18). 20
The final proposed tier or superimposed level of oxidative stress is cytotoxicity, including the initiation of programmed cell death (Fig. 8) (19,29). We have previously demonstrated that this effect is dependent on mitochondrial perturbation, including effects on the mitochondrial membrane potential and cytochrome c release (16,17,25). This notion is strengthened by the induced expression of proteins that regulate mitochondrial function and apoptosis, including mitochondrial fumarate hydratase (60), voltage-dependent anion-selective channel protein 1 (VDAC-1) (61,62), MEKK-1 (63), and diacylglycerol kinase (64) (Table 1). It is interesting that the DEP extracts also induced 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 (65), phospho-enolase (66), and glyceraldehyde 3-phophate dehydrogenase (66-68) (Table 1). We are in the process of analyzing these pathways in more detail. The pro-oxidative effects of organic DEP extracts likely reflect the presence of redox cycling chemicals (10,24,26). 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 (26,29). Chemical analysis has shown that the aromatic fraction is enriched for PAH while the polar fractions are enriched for quinines (26). 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 21
DTT (25). Both parameters were linearly correlated with the HO-1 expression in RAW 264.7 cells (25). 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 (43). In addition to its radical scavenging effects, GSH directly conjugates to some of the quinone species presenting DEP, including benzo- and naphthoquinones (69). In addition, NAC itself can participate in electrophilic interactions, thereby establishing multiple mechanisms by which is this thiol agent can interfere in the oxidative stress effects of DEP chemicals. Whatever the exact contribution of direct electrophilic interactions vs 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 (16,17). In fact, the specificity of the NAC antioxidant effects (18) 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 to in vivo PM exposures in humans? While 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 what aerodynamic diameter 2.5 µm) deposition at airway bifurcation points is comparable to the in vitro tissue culture concentrations recalculated as extract dose/cm2 (70). Thus, we have shown that 1-100 µg/ml of the DEP extract is equivalent to 0.14-14 µg/cm2 in a 22
tissue culture dish, while an asthmatic person with airway stasis, breathing polluted air in Rubidoux, California, can deposit 2.3 µg/cm2 PM2.5 at tracheobronchial bifurcation sites (70). 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 (70). 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. 23
REFERENCES 1. Samet, J. M., Dominici, F., Curriero FC, Coursac I, Zeger SL. (2000) New Engl J Med 343, 1742-1749 2. Dockery, D. W., Pope, C.A., Xu, X.P., Spengler, J.D., Ware, J.H., Fay, M.E., Ferris, B.G. and Speizer, F.E. (1993) New Engl. J. Med. 329, 1753­1759 3. Pope, C. A., and Dockery, D.W. (1998) in air pollution and Health. (Holgate, S., Koren, Maynard, ed) Vol. 31, pp. 673-705, Academic Press 4. Bonvallot, V., Baeza-Squiban, A., Baulig, A., Brulant, S., Boland, S., Muzeau, F., Barouki, R., and Marano, F. (2001) Am J Respir Cell Mol Biol 25, 515-521 5. Nel, A. E., Diaz-Sanchez, D., Ng, D., Hiura, T., and Saxon, A. (1998) J Allergy Clin Immunol 102, 539-554 6. Miyabara, Y., Takano, H., Ichinose, T., Lim, H. B., and Sagai, M. (1998) Am J Respir Crit Care Med 157, 1138-1144 7. Miyabara, Y., Ichinose, T., Takano, H., Lim, H. B., and Sagai, M. (1998) J Allergy Clin Immunol 102, 805-812 8. Miyabara, Y., Ichinose, T., Takano, H., and Sagai, M.,. (1998) Int Arch Allergy Immunol 116, 124-131 9. Takano, H., Yoshikawa, T., Ichinose, T., Miyabara, Y., Imaoka, K., and Sagai, M. (1997) Am J Respir Crit Care Med 156, 36-42 10. Saldiva, P. H., Clarke, R.W., Coull, B.A., Stearns, R.C. Lawrence, J., Murthy, G.G., et al. (2002) Am J Respir Crit Care Med 165, 1610-1617 24
11. Muranaka, M., Suzuki, S., Koizumi, K., Takafuji, S., Miyamoto, T., Ikemori, R., and Tokiwa, H. (1986) J Allergy Clin Immunol 77, 616-623 12. Takenaka, H., Zhang, K., Diaz-Sanchez, D., Tsien, A., and Saxon, A. (1995) J Allergy Clin Immunol 95, 103-115 13. Diaz-Sanchez, D., Tsien, A., Fleming, J., and Saxon, A. (1997) J Immunol 158, 24062413 14. Diaz-Sanchez, D., Jyrala, M., Ng, D., Nel, A., and Saxon, A. (2000) Clin Immunol 97, 140-145 15. NRC. (1998) (National Acad Sci National Research Council), National Academy Press, Washington DC. 16. Hiura, T. S., Kaszubowski, M. P., Li, N., and Nel, A. E. (1999) J Immunol 163, 55825591 17. Hiura, T. S., Li, N., Kaplan, R., Horwitz, M., Seagrave, J. C., and Nel, A. E. (2000) J Immunol 165, 2703-2711 18. Whitekus, M. J., Li, N., Zhang, M., Wang, M., Horwitz, M. A., Nelson, S. K., Horwitz, L. D., Brechun, N., Diaz-Sanchez, D., and Nel, A. E. (2002) J Immunol 168, 2560-2567 19. Li, N., Kim, S., Wang, M., Froines, J., Sioutas, C., and Nel, A. (2002) Inhal Toxicol 14, 459-486 20. Kumagai, Y., Arimoto, T., Shinyashiki, M., Shimojo, N., Nakai, Y., Yoshikawa, T., and Sagai, M. (1997) Free Radic Biol Med 22, 479-487 21. Jacqueline D. Carter, A. J. G., James M. Samet, and Robert B. Devlin. (1997) Toxicology and applied pharmacology 146, 180-188 25
22. Andrew J. Ghio, J. S., Lisa A. Dailey, Jacqueline D. Carter. (1999) Inhalation Toxicology 11, 37-49 23. Schuetzle, D., Lee, F. S., and Prater, T. J. (1981) Int J Environ Anal Chem 9, 93-144 24. Cohen, A. J., Nikula, K. (1998) The health effects of diesel exhaust: Laboratory and epidemiologic studies. In: Air Pollution and Health (Holgate, S. T., H. S. Koren, J. M. Samet and R. L. Maynard, Ed.), 32, Academic Press 25. Li, N., Sioutas, C., Cho, A., Schmitz, D., Misra, C., Sempf, J., Wang, M., Oberley, T., Froines, J., and Nel, A. (2003) Environ Health Perspect 111, 455-460 26. Li, N., Venkatesan, M. I., Miguel, A., Kaplan, R., Gujuluva, C., Alam, J., and Nel, A. (2000) J Immunol 165, 3393-3401 27. Monks, T. J., Hanzlik, R.P., Cohen, G.M., Ross, D., Graham, D.G. (1992) Toxicol Appl Pharmacol. 112, 2-16 28. Penning, T. M., Burczynski, M. E., Hung, C. F., McCoull, K. D., Palackal, N. T., Tsuruda, L. S. (1999) Chem Res Toxicol 12, 1-18 29. Li, N., Wang, M., Oberley, T. D., Sempf, J. M., and Nel, A. E. (2002) J Immunol 169, 4531-4541 30. Takano, H., Yanagisawa, R., Ichinose, T., Sadakane, K., Inoue, K., Yoshida, S., Takeda, K., Yoshino, S., Yoshikawa, T., Morita, M. (2002) Arch Toxicol. 76, 146-151 31. Fratelli, M., Demol, H., Puype, M., Casagrande, S., Eberini, I., Salmona, M., Bonetto, V., Mengozzi, M., Duffieux, F., Miclet, E., Bachi, A., Vandekerckhove, J., Gianazza, E., Ghezzi, P. (2002) Proc Natl Acad Sci U S A 99, 3505-3510 32. Aulak, K. S., Miyagi, M., Yan, L., West, K. A., Massillon, D., Crabb, J. W., and Stuehr, D. J. (2001) Proc Natl Acad Sci U S A 98, 12056-12061 26
33. Hoang, V. M., Foulk, R., Clauser, K., Burlingame, A., Gibson, B.W., Fisher, S.J. (2001) Biochemistry 40, 4077-4086 34. Whitelegge, J. P., Penn, B., To, T., Johnson, J., Waring, A., Sherman, M., Stevens, R. L., Fluharty, C. B., Faull, K. F., and Fluharty, A. L. (2000) Protein Sci 9, 1618-1630 35. Gow, A. J., Chen, Q., Hess, D. T., Day, B. J., Ischiropoulos, H., and Stamler, J. S. (2002) J Biol Chem 277, 9637-9640 36. Rabilloud, T., Heller, M., Gasnier, F., Luche, S., Rey, C., Aebersold, R., Benahmed, M., Louisot, P., and Lunardi, J. (2002) J Biol Chem 277, 19396-19401 37. Loo, J. A., DeJohn, D. E., Du, P., Stevenson, T. I., and Ogorzalek Loo, R. R. (1999) Med Res Rev 19, 307-319 38. Conrad, C. C., Choi, J., Malakowsky, C. A., Talent, J. M., Dai, R., Marshall, P., and Gracy, R. W. (2001) Proteomics 1, 829-834 39. Tietze, F. (1969) Anal Biochem 27, 502-522 40. Jungblut, P., and Thiede, B. (1997) Mass Spectrom Rev 16, 145-162 41. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Anal. Chem. 68, 850-858 42. Halliwell, B., Gutteridge, J.M. (1984) Biochem. J. 219, 1 43. Gillissen, A. and Nowak, D. (1998) Respiratory Medicine 92, 609-623 44. Susin, S. A., Zamzami, N., Kroemer, G. (1998) Biochim Biophys Acta 1366, 151-165 45. Zoratti, M., Szabo, I. (1995) Biochim Biophys Acta 1241, 139-176 46. Andrews, G. K. (2000) Biochem. Pharmcol. 59, 95-104 47. Bernstein, C., Payne, C.M., Berstain, H., Garewal, H. (2002) Pharmacology 65, 2-9 48. Takano, H., Satoh, M., Shimada, A., Sagai, M., Yoshikawa, T., Tohyama, C. (2000) Lab Invest. 80, 371-377 27
49. Maines, M. D. (1997) Annu Rev Pharmacol Toxicol 37, 517-554 50. Choi, A. M., Alam, J. (1996) Am J Respir Cell Mol Biol 15, 9-19 51. Enomoto, A., Itoh, K., Nagayoshi, E., Haruta, J., Kimura, T., O'Connor, T., Harada, T., and Yamamoto, M. (2001) Toxicol Sci 59, 169-177 52. Aoki, Y., Sato, S., Nishimura, N., Takahashi, S., Itoh, K. Yamamoto, M. (2001) Toxcol. Appl. Pharmacol. 173, 154-160 53. Johnson, G. L., and Lapadat, R. (2002) Science 298, 1911-1912 54. Webster, K. A., Prentice, H., Bishoperic, N.H. (2001) Antioxidants & Redox Signaling 3, 535-548 55. Laan, M., Prause, O., Miyamoto, M., Sjostrand, M., Hytonen, A.M., Kaneko, T., Lotvall, J., Linden, A. (2003) Eur. Respir. J. 21, 387-393 56. Naismith, J. H., Spring, S.R. (1995-96) J. Inflamm. 47, 1-7 57. Decker, E. L., Nehmann, N., kampen, E., Eibel, H., Zipfel, P.F., Skerka, C. (2003) Nucleic Acids Res. 31, 911-921 58. Grunfeld, C., Soued, M., Adi, S., Moser, A.H., Fiers, W., Dinarello, C.A., Feingold, K.R. (1991) Cancer Res. 51, 2803-2807 59. Diaz-Sanchez, D., Tsien, A., Fleming, J., and Saxon, A. (1999) Clin Immunol 90, 313- 322 60. Eng, C., Kiuru, M.,Fernandez, M.J., Aaltonen, L.A. (2003) Natrue 3, 193-202 61. Madesh, M., Hajnoczky, G. (2001) J.Cell.Biol. 155, 1003-1015 62. Crompton, M. (1999) Biochem. J. 341, 233-249 63. Cassarino, D. S., Halvorsen, E.M., Swerdlow, R.H., Abramova, N.N., Parker, W.D. Jr., Sturgill, T.W., Bennett, J.P. Jr. (2000) J. Neurochem. 74, 1384-1392 28
64. Lavrentiadou, S. N., Chan, C., Kawcak, T., Ravid, T., Tsaba, A., van der Vliet, A., Rasooly, R., Goldkorn,T. (2001) Am J Respir Cell Mol Biol. 25, 676-684 65. Fernandez, V., Videla, L.A. (1996) Biol. Res. 29, 177-182 66. Shenton, D., Grant, C.M. (2003) Biochem. J., Epub ahead of print 67. Ito, Y., Pagano, P.J., Tornheim, K., Brecher, P., Cohen, R.A. (1996) Am. J. Physiol. 270, H81-87 68. Eaton, P., Wright, N., Hearse D.J., Shattock, M.J. (2002) J. Mol. Cell Cardiol. 34, 1549- 1560 69. O'Brien, P.J. (1991) Chem. -Biol. Interactions 80, 1-41 70. Li, N., Hao, M., Phalen, R.F., Hinds, W.C., Nel, A.E. (2003) Clin Immunol., In Press 71. Luo, J. D., Chen, A.F. (2003) Curr. Med. Chem. 10, 1593-1601 72. Ohsawa, I., Takamura, C., Kohsaka, S. (2001) J. Neurochem. 76, 1411-1420 73. Dimayuga, F. O., Ding, Q., Keller, J.N., Marchionni, M.A., Seroogy, K.B., Bruce-Keller, A.J. (2003) J. Neuroimmunol. 136, 67-74 74. Hook, V. Y., Affolter, H.U. (1988) FEBS lett. 238, 338-342 75. Hook, V. Y. (1988) Cell. Mol. Neurobiol. 8, 49-55 76. Demaurex, N., Distelhorst, C. (2003) science 300, 65-67 77. McQueen, K. L., Freeman, J.D., Takei, F., Mager, D.L. (1998) Immunogenetics 48, 174- 183 29
ACKNOWLEDGEMENTS We thank Sheng Yin and Dr. James Kerwin for their help with the gel image analysis, protein digestion, and protein identifications, and Dr. Rachel Ogorzalek Loo for advice on sample preparation and gel electrophoresis. This work was funded by the US Public Health Sciences grants, RO-1 ES12053, RO-1 ES10553 and PO-1 AI50495. The support from Agilent Technologies in the operation of the ion trap mass spectrometer is acknowledged. J. A. L. also acknowledges support from the UCLA Molecular Biology Institute. The UCLA Functional Proteomics Center was established and equipped with a grant from the W. M. Keck Foundation. 30
ABBREVIATIONS ARE, antioxidant response element; DAGK, diacylglycerol kinase; DEP, diesel exhaust particles; DTT, dithiothreitol; HPLC, high liquid chromatography; IPG, immobilized pH gradient; IEF, isoelectric focusing; LC-tandem MS/MS, liquid chromatograph tandem mass spectrometers; MEKK-1, MAPK/ERK kinase kinase 1; MALDI, matrix-assisted laser desorption/ionization; NAC, N-acetyl cysteine; PAH, polycyclic aromatic hydrocarbons; PM, particulate matter; ROS, reactive oxygen species; TOF, time-of-flight; 2D-PAGE, two-dimensional polyacrylamide electrophoresis; VDAC-1, voltage-dependent anion-selective channel protein 1; FIGURE LEGENDS 31
Fig. 1. Glutathione assay showing a dose-dependent decline in cellular GSH/GSSG ratios in RAW 264.7 cells treated with organic DEP extracts. RAW 264.7 cells were exposed to the indicated concentrations of DEP extract (solid line) for 6 hr in the absence or the presence of 20 mM NAC (broken line). Determination of total and oxidized glutathione and GSH/GSSG ratios was performed as described in Materials and Methods. Values represent the mean ± SEM. p < 0.05 at extract doses 50 µg/ml. These data were confirmed in an independent experiment. Fig. 2. Phosphopeptide immunoblotting to show dose-dependent activation of the JNK and p38MAPK cascades by DEP extracts and interference by NAC. (A) p38MAPK phosphopeptide (top panel) and protein (bottom panel) immunoblotting; (B) JNK phosphopeptide (top panel) and protein (bottom panel) immunoblotting. RAW 264.7 cells were treated with the indicated amounts of the DEP extract for 6 hrs, in the absence or the presence of 20 mM NAC. Phosphopeptide and protein blotting was performed as described in the Materials and Methods. These data were confirmed in an independent experiment. Fig. 3. PI staining and flow cytometry showing a dose-dependent increase in cytotoxicity during treatment with the organic DEP extract. Cellular treatment and assessment of PI staining by flow cytometry is described in the Materials and Methods. Fig. 4. Dose-dependent increase in new protein expression in RAW 264.7 cells as determined by 2D gels. (A) Dose-dependent increase in new protein expression in response to organic DEP extracts; protein expression was suppressed by NAC; (B) Regression analysis showing the linear correlation between extract dose and the number of newly expressed proteins; 32
(C) Venn-diagram to show the overlapping and unique expression profiles at different doses of the DEP extract; (D) List of 6 new proteins induced at all extract concentrations. RAW 264.7 cells were exposed to DEP extracts at indicated concentrations, in the absence or the presence of 20 mM NAC, for 6 hrs before cellular extraction and analysis of the soluble proteins by 2D electrophoresis. These data were reproduced 3 times, during which the variability in protein expression was < 10%. Fig. 5. Two-dimensional gel electrophoresis profile in the presence of 50 µg/ml of the organic DEP extract. (A) Proteins that were induced > 8-fold and subtracted in the presence of NAC were selected as oxidative stress markers that were identified by MS. Those proteins are numbered and their identities disclosed in Table 1; (B) Exert of of the 2D profile to show how above criteria led to the identification of GAPDH as an oxidative stress marker: the top panel shows background expression in untreated cells, the middle panel shows increased expression by the extract, and the bottom panel shows the subtracted response in the presence of NAC; (C) GAPDH immunoblotting shows the subtractive expression of this protein in crude cell lysates. See Materials and Methods section for experimental details. These data were reproduced 3 times, during which the variability in protein expression was < 10%. Fig. 6. Narrow pH-range focusing gels improve differentiation of oxidative stress-related proteins. Heme oxygenase-1 (HO-1) and catalase are shown in the 2D gel with the pH range of 5.5-6.7 (shown as dashed lines in Fig. 5A). (A) Control sample (RAW 264.7 cells exposed to the DMSO carrier); (B) Cells exposed to 50 µg/ml dose of DEP, showing induction of HO-1 and catalase. (C) Cells exposed to the same dose of the extract in the presence of NAC to demonstrate the suppression of HO-1 and catalase expression; (D) HO-1 and catalase 33
immunoblotting to confirm the expression of these proteins in crude cell lysates. (E) Rel A p65 and metallothioneins immunoblotting to confirm the expression of these proteins in crude cell lysates. * MTT1 isoform identified by proteomics; Tesmin = metallothionein-like protein also identified in our immunoblot. Fig. 7. NAC suppression of protein expression depends on antioxidant effects as well as direct electrophilic interactions with DEP chemicals. (A) RAW 264.7 cells were treated with 50 µg/ml of the DEP extract for 2 hours before the cells were washed and then returned to the culture dish for an additional 4 hours in the presence of 20 mM NAC (lanes 2). The controls in this experiment were untreated cells (lane 1), DEP-treated cells which received the NAC addition without washing away the DEP chemicals (lane 3), and DEP-treated cells which were washed and then treated with the carrier (Hepes) only (lane 4). In the same experiment, we also tested the effect of prior NAC addition before adding 50 µg/ml the DEP extract for 6 hrs (see legend for lanes 5-7). HO-1 immunoblotting was conducted as described in Fig. 6. (B) HO-1 immunoblotting was used to demonstrate that premixing of NAC and the DEP extract is effective in suppressing the pro-oxidative effects of electrophilic DEP chemicals. 10 mg NAC was premixed with 1 mg of the DEP extract in a small volume (50 µl). This mixture was added to the cell culture for 6 hrs to give a final DEP extract concentration of 10-50 µg/ml, while limiting the NAC concentration to 61.5 µM (lanes 4-6). The controls consisted of cells receiving DEP chemicals and either no NAC (lanes 1-3) or 20 mM NAC added to the culture medium 2 hrs before the addition of the DEP extract (lanes 9-12). The cellular extracts were used for HO-1 immunoblotting as described in Materials and Methods section for experimental details. 34
Fig. 8. Schematic to explain the hierarchical oxidative stress model in response to redox cycling DEP components. Activation of antioxidant enzymes HO-1 and catalase reflects the first tier oxidative stress response, activation of the p38MAPK and Jun kinase cascades constitutes the second tier of oxidative stress responses, while the final tier of oxidative stress response, mediated by mitochondrial perturbation, leads to cytotoxic effects. Please note that the suggested tiers are not rigidly demarcated, but represent an escalating trend, in which cytoprotective yield to pro-inflammatory and cytotoxic responses. The data in Table 1 indicate some overlap and intermingling of protective vs. injurious effects at the interface of these zones. Table 1. Protein assignments from whole RAW 264.7 cells exposed to both 50 µg/ml and 100 µg/ml of DEP extracts. DEP dose: 10 only = 10 µg/ml; 10 = 10, 50 & 100 µg/ml; 50 only = 50 µg/ml; 50 = 50 & 100 µg/ml; 100 only = 100 µg/ml. **NAC suppressibility: "+" = 25% decrease in intensity; "++" = 50%; "+++" = 75%; "++++" = 100%. "Pro-inflammatory" = the potential to contribute to biological events culminating in or regularly inflammation "Oxi " = oxidative 35
Table 1. Protein assignments from whole RAW 264.7 cells exposed to both 50 µg/ml and
100 µg/ml of DEP extracts.
Protein Assigned Heme oxygenase-1 Catalase Metallothionein
Spots *DEP No. dose (µg/ml)
Protein Database code
23
10
P14901
12
10
P00432
18
10 BAB24517
Glyceraldehyde 3phophate dehydrogenase Nuclear factor kappa B (Rel A) p38 MAPK 1 Granulocytemacrophage colonystimulating factor precursor Tumor necrosis factor receptor 2 Putative Rho/Rac guanine nucleotide Early growth response protein 4 (EGR-4) Acetyl-CoA carboxylase 2 Glucocorticoid receptor (GR)
17
10
P16858
2
10
P98150
24
10 Q99MG4
9
50
P01587
29 50 only P25119
27
50
P52734
4
50
Q00911
20
50
O00763
13
50
P49115
Alcohol dehydrogenase Fibulin precursor
10 50 only Q09007
8
50
Q08879
Receptor proteintyrosine kinase ERBB-2
22
50
Q60553
Carboxypeptidase H 11
50
P37892
precursor
Phosphoenolpyruvate 3
50
P07379
carboxykinase
Observed Mr (kDa) 42.0 57.5 23.2 35.0 23.4 41.4 16.1 50.3 106.0 49.6 27.9 84.8 31.0 78.0 138.2 51.4 69.4
Observed pI value 6.05 6.40 9.20 8.40 6.05 5.60 5.80 6.62 6.2 6.70 7.80 7.12 4.99 5.02 5.98 5.00 6.09
**NAC suppressibility ++++ +++ +++ +++ ++++ +++ ++ ++++ ++ ++ +++ ++ +++ ++ +++ ++ +++
Sequence Coverage (MS) % 38 66 73 26 18 13 13 13 14 12 69 38 25 29 22 17 32
Possible oxidative stress role Cytoprotective/ARE driven Cytoprotective/ARE driven Cytoprotective/oxi stress inducible (46- 48) abundance but function with oxi stress (56,57,66) Anti-apoptic/proinflammatory (54) Pro-inflammatory Pro-inflammatory (55) Pro-inflammatory (56) Pro-inflammatory (71) Transcriptional cytokine inducer (57) Regulated by proinflam cytokines, IL- 1,4 &6 (58) Anti-inflam transcriptional factor sensitive to oxi stress (54) Detoxification/ abundance but function with oxi stress (66) Regulation of amyloid precursor protein (72) Induced by oxi stress/ROS release from activated microglial cells (73) Proteolytic processing/Znmetallopeptidase (74,75) Intermediary metabolism regulates oxi stress (65)
36
Table 1. (cont.)
-phospho-enolase
15
50
P17182
47.0
7.05
+++
2-phosphopyruvate-
16
50 CAA59331
47
7.01
++
hydratase -enolase
Neurturin receptor A 7
50
O08842
51.6
8.11
++
precursor
Janus-atracotoxin-
28
50
P82226
36.4
4.77
+++
HV1B (J-ACTX-
HV1B) Disks large-
25
50
Q9P1A6
113.7
6.52
+++
associated protein 2
(DAP-2) Plasminogen
5
50
P12545
90.1
6.25
++
precursor
Proliferating cell
26
50
Q9DDF1
28.9
4.6
++
nuclear antigen
Transitional ER
21
50
P46462
89.2
5.14
+++
ATPase
Natural killer cell
19
50 13021834
30.7
8.79
++
receptor LY49M
FADD protein (FAS- 1
50
Q61160
23.0
5.77
+++
associating death
domain-containing
protein) Glutamate
6
50
P00366
55.5
8.50
+++
dehydrogenase
Mitochondrial
14
50
P14408
55.0
8.01
++
fumarate hydratase
Voltage-dependent
31
100
Q60932
32.4
8.55
+++
anion-selective
only
channel protein 1
(VDAC-1)
MAPK/ERK kinase
32
100
Q62925
160.0
8
+++
kinase 1 (MEKK-1)
only
Diacylglycerol
30
100
Q64398
126.0
6.2
+++
Kinase (DAGK)
only
20
Intermediary
metabolism regulates
oxi stress (65)/
function with oxi
stress (66)
14
Intermediary
metabolism regulates
oxi stress (65)/
function with oxi
stress(66)
15
?
22
?
27
?
26
?
18
?
13
Regulation of
apoptosis (76)
15
? Role in NK cell
killing (77)
22
Pro-apoptotic
34
Marker for cellular
injury/oxi damage of
mitochondria (62,63)
33
Mitochondrial
complex II activity
(60)
22
PT pore and
apoptosis regulator
(61,62)
24
Mitochondrial
apoptosis regulator
(63)
32
Ceramide/DAGK-
induced apoptosis is
sensitive to GSH (64)
DEP dose: 10 only = 10 µg/ml; 10 = 10, 50 & 100 µg/ml; 50 only = 50 µg/ml; 50 = 50 & 100 µg/ml; 100 only = 100 µg/ml. **NAC suppressibility: "+" = 25% decrease in intensity; "++" = 50%; "+++" = 75%; "++++" = 100%.
37

GG Xiao, M Wang, N Li, JA Loo, AE Nel

File: use-of-proteomics-to-demonstrate-a-hierarchical-oxidative-stress.pdf
Author: GG Xiao, M Wang, N Li, JA Loo, AE Nel
Published: Fri Sep 26 22:04:23 2003
Pages: 47
File size: 2 Mb


, pages, 0 Mb

Polymer handbook, 6 pages, 0.29 Mb

Crossing the chasm, 61 pages, 1.68 Mb

Al-Tusi, connaissez-vous, 12 pages, 0.42 Mb
Copyright © 2018 doc.uments.com