Cytotoxicity Due To Pm-electrophoresis

Meiying Wang1 Gary Guishan Xiao1 Ning Li1 Yongming Xie2 Joseph A. Loo2 Andre E. Nel1 1

Department of Medicine, Division of Clinical Immunology and Allergy, and David Geffen School of Medicine 2 Department of Chemistry and Biochemistry, and Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA

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Use of a fluorescent phosphoprotein dye to characterize oxidative stress-induced signaling pathway components in macrophage and epithelial cultures exposed to diesel exhaust particle chemicals A large body of evidence has shown that exposure to ambient particulate matter (PM) leads to asthma exacerbation through an excitation of allergic inflammation. Utilizing diesel exhaust particles (DEPs) as a model air pollutant, we and others have demonstrated that PM contains redox-active chemicals that generate inflammation through an oxidative stress mechanism. Recently, the strengths of proteomics have enabled us to demonstrate that organic DEP extracts induce a hierarchical expression pattern of oxidative stress-induced proteins in macrophages and epithelial cells. As a further extension of this work, we now employ a new phosphosensor fluorescent dye, Pro-Q Diamond, to elucidate the induction of phosphoproteins and intracellular signaling cascades that may play a role in DEP-induced inflammation. We demonstrate that DEPs induced the phosphorylation of several phosphoproteins that belong to a number of signaling pathways as well as other oxidative stress pathways. In combination with cytokine array, phosphoproteome analysis using Pro-Q Diamond allowed us to characterize the aromatic and polar chemicals of DEPs that are involved in the activation of three different mitogen-activated protein (MAP) kinase signaling pathways. Keywords: Diesel exhaust particles / Inflammation / Mitogen-activated protein kinases / Oxidative stress / Phosphoproteome DOI 10.1002/elps.200410428

1 Introduction A number of studies have shown that exposure to ambient particulate matter (PM) leads to asthma exacerbation through an excitation of allergic airway inflammation [1– 6]. In order to elucidate the mechanisms by which PM induces inflammation, it is important to consider the contribution of the chemicals that are present on the particle surface [7]. Utilizing diesel exhaust particles (DEPs) as a model air pollutant, others and we have demonstrated that PM contains redox-active chemicals that generate

Correspondence: Dr. Andre E. Nel, Division of Clinical Immunology and Allergy, Department of Medicine, University of California, Los Angeles, CA 90095, USA E-mail: [email protected] Fax: 1310-206-8107 Abbreviations: DEP, diesel exhaust particle; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GSH, glutathione S-transferase; HRP, horseradish peroxidase; IL-8, interleukin-8; JNK, c-Jun amino-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MEKK-1, MAPK/ ERK kinase kinase 1; NAC, N-acetyl cysteine; PAH, polycyclic aromatic hydrocarbons; PM, particulate matter; ROS, reactive oxygen species; TNFa, tumor necrosis factor a

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inflammation through an oxidative stress mechanism [2, 8–12]. In this regard, DEPs and ambient PM contain transition metals [13, 14] and redox-cycling organic chemical components that are capable of producing reactive oxygen species (ROS) in target cells [15–17]. Using silica gel chromatography to fractionate organic DEP extracts into aliphatic, aromatic, and polar components, we have previously demonstrated that most of the redox-active chemicals reside in the polar fraction, which is enriched for quinones [15]. However, the aromatic fraction, which is enriched for polycyclic aromatic hydrocarbons (PAHs) was also active, while the aliphatic fraction was devoid of redox cycling chemicals [15, 18]. Our subsequent studies have shown a good correlation between the content of redox cycling organic chemicals in ambient PM and the induction of oxidative stress in macrophages and epithelial cells and the PAH content of ambient PM [11, 19]. Organic DEP extracts have been shown to induce superoxide production in lung microsomes through the action of NADPH-dependent P450 reductase [12]. In addition, organic DEP chemicals also exert effects on the mitochondrial inner membrane, where a disruption of electron flow leads to superoxide production [8, 19, 20]. Our major interest is how DEP chemicals exert their pro-inflamma-

General

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tory effects on airway target cells, such as macrophages and bronchial epithelial cells, and the signaling pathways involved in these biological responses. What are the major intracellular pathways by which oxidative stress induces pro-inflammatory effects? Utilizing the strengths of proteomics, we have recently shown that organic DEP extracts induce a hierarchical oxidative stress response in macrophages and epithelial cells [21]. According to this stratified response, the lowest level of oxidative stress (tier 1) leads to the induction of antioxidant and cytoprotective responses through the activation of the antioxidant response element (ARE) in the promoters of phase II enzymes [15, 23]. This genetic response element is transcriptionally activated by a basic leucine zipper transcription factor, Nrf2 [18, 24]. Recent evidence has shown that phase II enzymes, such as glutathione S-transferase (GST), protect against the adjuvant and pro-allergic effects of DEP in vivo [18, 21]. If the rate of ROS production exceeds the ability of tier 1 to protect, an escalation in the level of oxidative stress leads to the activation of pro-inflammatory cytokines and chemokines [21]. This event is partially dependent on activation of the Jun kinase (JNK) cascade, a well-known stress-activated protein kinase family [22]. Further escalation in the level of oxidative stress leads to mitochondrial perturbation and cytotoxicity (tier 3) [20]. In order to more completely follow the intracellular signaling pathways that are induced by oxidative stress, we were interested in a discovery tool which comprehensively displays the phosphoprotein content of tissue culture cells exposed to pro-oxidative DEP chemicals. Proteomics has proven to be powerful means of discerning macrophage and epithelial responses to oxidative stress [21, 25]. Utilizing two-dimensional (2-D) gels to screen for newly induced oxidative stress proteins, we were able to detect an incremental expression pattern. As a further extension of this work, we were interested in employing the new phosphosensor dye, Pro-Q Diamond, to screen for the induction of phosphoproteins under conditions of oxidative stress. Pro-Q Diamond is a unique fluorescence-based detection system for the specific and sensitive analysis of protein phosphorylation status on 1-D or 2-D polyacrylamide gels [26]. In this communication, we used Pro-Q Diamond staining of 2-D gels to elucidate phosphoproteins and intracellular signaling cascades that play a role in DEP-induced inflammation [2, 21, 22, 27]. This revealed the phosphorylation of several phosphoproteins which belong to a number of signaling cascades and functional groups of proteins. Moreover, we used the activation of three different mitogen-activated protein (MAP) kinase cascades to characterize the aromatic and polar chemical compounds that are involved in

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the activation of pro-inflammatory cascades. We supplemented this phosphoproteome analyses with cytokine arrays to elucidate pro-inflammatory products, which are induced by DEP chemicals in RAW 264.7 and BEAS-2B cells.

2 Materials and methods 2.1 Reagents Dulbecco’s modified Eagle medium (DMEM), penicillinstreptomycin, and L-glutamine were obtained from GIBCO Life Technologies (Baltimore, MD, USA). Bronchial epithelial growth medium (BEGM) was purchased from Clonetics (San Diego, CA, USA). Type I rat tail collagen was from Collaborative Research (Bedford, MA, USA). Fetal bovine serum (FBS) was purchased from Irvine Scientific (Santa Ana, CA, USA). Anti-phospho-SAPK/JNK, anti-JNK, anti-phospho-p38 and anti-p38 MAPK, antiphospho-p44/42 MAPK, and p44/42 MAPK Abs were from Cell Signaling (Beverly, MA, USA). Biotinylated swine anti-rabbit and rabbit anti-mice Abs were obtained from Dako (Carpinteria, CA, USA). Horseradish peroxidase (HRP)-conjugated sheep anti-mouse immunoglobulin G (IgG) and donkey anti-rabbit Ig were purchased from Amersham (Piscataway, NJ, USA). N-Acetylcysteine (NAC) was obtained from Sigma (St. Louis, MO, USA). MAPK inhibitors: SB203580 (p38 MAPK inhibitor), PD98059 (p44/42 MAPK kinase inhibitor), and SP600125 (JNK inhibitor), were purchased from Calbiochem (San Diego, CA, USA). ECL reagents were from Pierce (Rockford, IL, USA). Pro-Q Diamond phophoprotein stain was purchased from Molecular Probes (Eugene, OR, USA). DryStrip cover fluid was from Pharmacia Biotech (Uppsala, Sweden). Rehydration buffer, 11 cm or 17 cm IPG strips (3/10NL), SDS precasted gels (12.5%), 106TGS (Tris-glycine SDS buffer), DC reagent kit, and bovine serum albumin (BSA) were obtained from Bio-Rad Laboratories (Hercules, CA, USA). SYPRO Ruby was purchased from Molecular Probes (Eugene, OR, USA). Poly(dI-dC) was from Pharmacia Biotech (Piscataway, NJ, USA). Protease inhibitor cocktail set III was obtained from CalBiochem (La Jolla, CA, USA).

2.2 Cell culture and stimulation with DEP chemicals The human bronchial epithelial (BEAS-2B) and the murine macrophage (RAW 264.7) cell lines were obtained from ATCC. All cell cultures were carried out in 5% CO2 at 377C in a humidified incubator. BEAS-2B cells were cultured in BEGM in type I rat tail collagen-coated flasks or plates as

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previously described [22]. RAW 264.7 cells were cultured in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS. Where indicated, cells were pre-incubated with NAC (20 mM) for 2 h before stimulation with the DEP extract or the chemical fractions at the indicated concentrations. MAP kinase inhibitors (PD98059, SB203580, and SP600125) were added 1 h prior to the addition of the DEP extract at the indicated concentrations. In all experiments, an equal volume of the carrier (DMSO) was used as control.

2.3 Western blotting analysis One hundred to 150 mg of total protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane. After blocking the membranes were incubated with primary antibodies against phospho-ERK1 & 2, ERK1 & 2, phospho-JNK or JNK. The membranes were overlayed with either biotinylated rabbit anti-mouse or swine antirabbit secondary Ab before the addition of the HRP-conjugated avidin-biotin complex. p38 MAPK was detected using monoclonal anti-phospho-p38 MAPK and polyclonal anti-p38 MAPK Abs, followed by HRP-conjugated secondary Ab. The proteins were detected using ECL reagent according to the manufacturer’s instructions. Protein abundance was quantified by densitometric scanning using a laser Personal Densitometer SI and Image Quant software (Amersham Biosciences).

2.4 Measurement of IL-8 and TNFÆ production BEAS-2B and RAW 264.7 cells were pretreated with NAC and the MAPK inhibitors as indicated, followed by stimulation with the crude DEP extract at the indicated concentrations for 6 h. The culture media were collected, centrifuged to remove all debris, and sent frozen to the Cytokine Core Laboratories at the University of Maryland (Baltimore, MD, USA) for measuring interleukin-8 (IL-8) and tumor necrosis factor a (TNFa) levels by ELISA.

2.5 Antibody-based protein array system Human Cytokine Array I and Mouse Cytokine Array I kits were purchased from RayBiotech (NorCross, GA, USA). The cytokine array membranes were used according to the manufacturer’s instructions. Briefly, membranes were blocked for 30 min with 2 mL blocking buffer at room temperature. One mL culture medium was added and incubated for 16 h at 47C. After repeated washes the membranes were incubated with biotin-conjugated antibodies (1:250) for 2 h followed by incubation with HRPconjugated streptavidin at room temperature for 1 h.

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2.6 Preparation of DEP methanol extracts DEPs were generously provided by Dr. Masura Sagai (National Institute of Environment Studies, Tsukuba, Japan). These particles were collected as previously described [22]. Methanol extraction of DEPs was performed as described by Hiura et al. [9]. Briefly, 100 mg DEPs was suspended in 25 mL methanol and sonicated for 2 min on ice. The suspension was centrifuged at 4256g for 10 min at 47C, and the supernatant transferred to a preweighed polypropylene tube to determine the amount of extractable material. After drying under nitrogen gas, the dried material was completely dissolved in DMSO and aliquots stored at 2807C in the dark until use. The chemical composition of these particles, including PAHs and quinone analysis have been previously reported [15]. The signature PAHs which have been detected in the aromatic fraction include phenanthrene, 2-methylfluorene, 3,6-dimethylphenanthrene, 1-methylphenanthrene, chrysene, pyrene, benz[a]anthracene, benzo[k]fluoranthene, benzo[b]fluoranthene, and benzo[a]pyrene, while the polar fraction includes oxygenated compounds, such as 9-H-fluorene-9-one, anthracenone/phenanthrenol, and 9,10-anthracenedione. The aliphatic DEP fraction contains mainly normal alkanes [15].

2.7 Preparation of DEP fractions DEPs (0.5 g) were extracted by sonication with 100 mL of methylene chloride. The extract was filtered using a Millipore filtration system with a 0.45 mm nylon filter. Preparation of DEP fractions was carried out as previously described with some modifications [15]. The methylene chloride extract was concentrated by rotary evaporation and asphaltenes (insoluble, polar chemicals with S and O heteroatoms) were precipitated by hexane. The contents were left overnight in the freezer, centrifuged, and the hexane supernatant was collected. The precipitate was washed twice with hexane and the washings were combined with the first hexane extract, concentrated, and dried over anhydrous sodium sulfate. The extract thus prepared was subjected to gravity-fed silica gel chromatography. Aliphatic, aromatic, and polar fractions were collected by successive elution with hexane, hexane:methylene chloride (3:2 v/v), and methylene chloride:methanol (1:1 v/v), respectively [15]. The elution of the aromatic fraction was monitored by a UV lamp at long wavelength (365 nm). The eluates were concentrated by roto-evaporation. The aliphatic fraction was in hexane, the others in methylene chloride and they were stored at 2807C until further use. The weight of the fractions was determined in a microbalance after evaporating off the solvents from a known sample volume. Alkanes in the

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aliphatic fraction were characterized by a GC (Varian 3400 with a SPI injector) equipped with flame ionization detector and a DB-5 column (30 m, 0.25 mm ID, 0.25 mm film). The fractions were dried with N2 gas and redissolved in DMSO for biological studies. A signature group of 16 PAHs was measured by HPLC fluorescence as previously described [18, 19]. Some of those compounds are listed above. Quinone content was analyzed in their most stable diacetyl derivatives by GC-MS as previously reported [18]. Four signature quinones, 1,2-naphthoquinone, 1,4naphthoquinone, phenanthrenequinone, and anthraquinone, were measured by the electron impact (EI) GC-MS technique, using an HP MSD equipped with an automatic sampler [18]. PAH and quinone analyses of the various silica gel fractions also comfirmed that PAHs were confined to the aromatic fraction, while all four signature quinones were confined to the polar fraction.

2.8 DTT assay The DTT assay was used to quantitate the redox activity of PM as previously reported [12, 28]. This assay measures DTT oxidation by quinones in the following net reaction: DTT 1 2O2 ? DTT-disulfide 1 2 O22. Briefly, the crude DEP extract or its fractions were incubated with 100 mM DTT in a phosphate buffer at pH 7.4 for 15–60 min. Aliquots of the incubation mixture were mixed with Tris buffer, pH 8.9 and the 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) solution and the optical density was read at 412 nm.

2.9 Protein extraction and sample preparation for 2-D electrophoresis Aliquots of 26107 RAW 264.7 or 161076 BEAS-2B 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 mM 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/100 mM sodium orthovanadate/ 400 mM sodium tartrate dihydrate) for 10 s. Lysates were centrifuged at 10006g for 5 min. To remove the salt from the lysates, the supernatant proteins were precipitated with TCA (10% w/v)/20 mM DTT for 30 min on ice. The precipitate was collected at 20 8006g for 10 min at 47C and washed 36with 10% TCA/20 mM DTT. TCA in the precipitate was removed through the extraction with diethyl ether or acetone/10 mM DTT. After drying, the pellet was resuspended by sonication in a buffer containing 7 M

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urea, 2 M 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 8006g for 10 min at 157C, and the supernatants stored at 2807C until use for 2-D PAGE. Protein concentration in these samples was estimated by using a commercial Bradford kit (DC reagent kit; BioRad), and BSA as standard.

2.10 Two-dimensional gel electrophoresis (2-D PAGE) 2-D PAGE was performed with the Bio-Rad system as described previously [21]. Whole-cell lysate protein, 350 mg for RAW 264.7 and 250 mg for BEAS-2B cells, was added to each immobilized pH gradient (IPG) strip, which was rehydrated in 8 M urea, 2% CHAPS, 50 mM Bio-Lyte 3/10 ampholyte, and 0.001% bromophenol blue. The preisoelectric focusing and isoelectric focusing (IEF) were performed using pre-made 17 cm IPG strips (pH 3–10 NL) or 11 cm length IPG strips (pH 3–6) on the Protean IEF cell. For 17 cm IPG strips (pH 3–10 NL), the pre-IEF was performed linearly up to 500 V for 1 h, held at 500 V for 1.5 h. Formal IEF was 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 kVh. For 11 cm IPG strips (pH 3–6), the pre-IEF was performed linearly up to 250 V for 1 h, held at 250 V for 1.5 h. Formal IEF was performed with a linear increase up to 8000 V over 2 h, and then held at 8000 V for 7 h a total of 57 kVh. 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, followed by 8–16% SDS-PAGE (20 cm gels) on a Protean Plus Dodeca Cell (Bio-Rad). Gels were stained with SYPRO Ruby and visualized under ultraviolet light with a Molecular Imager FX Pro Plus (Bio-Rad). To check the reproducibility of the data, three independent 2-D analyses were performed on each cellular lysate.

2.11 Phosphoprotein identification Fluorescent staining of 2-D SDS-polyacrylamide gels using Pro-Q Diamond phosphoprotein gel stain was performed by fixing the gels overnight in 50% methanol/10% acetic acid, followed by washing three times with deionized water. Gels were incubated in Pro-Q Diamond stain for 120 min, and destained by washing 3 times in 20% acetonitrile plus 50 mM sodium acetate, pH 4.0, for 30– 60 min. Before imaging, the gels were rehydrated in deionized water for 30 min. Following image acquisition, gels were stained for total protein with SYPRO Ruby protein gel stain for serial dichromatic detection. This permits

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comparison of phosphoprotein and total protein profiles. Images were acquired on a Molecular Imager FX Pro Plus (Bio-Rad). Pro-Q Diamond and SYPRO Ruby staining were detected with 532 nm excitation and 580 nm long pass emission filters or 473 nm excitation and 580 nm long pass emission filters, respectively. Computer-generated differential display maps of protein phosphorylation and protein expression patterns were generated using PDQuest, Version 7.2.0 software (Bio-Rad). This software uses raw-image-based computation of registration, region-based matching, and a complementary pseudocolor visualization technique. With this system, spots of the reference gel appear green (SYPRO Ruby) and those of the comparative gel appear red (Pro-Q Diamond). When images are aligned, similarly intense spots in the overlay image appear yellow, while those that differ in intensity levels appear green or red. The yellow spots shown on the pseudocolor images 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, USA), and the resulting tryptic peptides were extracted using standard protocols [29]. 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, USA). MALDI peptide fingerprint mass spectra were acquired with a MALDI time-of-flight (TOF) instrument (M@LDI-R, Micromass-Waters, Beverly, MA, USA), using a-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, USA), interfaced to an ion trap mass spectrometer (LC-MSD Trap SL; Agilent Technologies). A reverse-phase column (75 mm6150 mm, C18 Zorbax StableBond) was used as the analytical column. A Zorbax 300SB enrichment precolumn (0.365 mm) was used to concentrate and desalt the peptide mixtures. Additional measurements were made with an LC-quadrupole TOF system (Dionex/LC Packing nano-LC and Applied Biosystems/Sciex QSTAR Pulsar XL mass spectrometer). 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 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.

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2.12 Statistical analyses Data were analyzed with either Excel, using Student’s t-test, or SPSS software, using analysis of variance (ANOVA). p , 0.05 is considered statistically significant.

3 Results 3.1 DEP-induced phosphoprotein profile as determined by 2-D gel electrophoresis, Pro-Q staining, and protein MS There is a great need to comprehensively identify cellular signaling cascades which regulate cellular function in response to a wide range of stimuli, including oxidative stress. It is now possible through the use of MS and protein chemistry techniques to characterize most phosphoproteins from a whole-cell lysate in one experiment [26]. The Pro-Q Diamond stain has been developed to identify phosphoproteins for phosphoproteome and signal transduction studies [26, 30]. This fluorescent stain allows for direct, in-gel detection of phosphate groups attached to tyrosine, serine or threonine residues. Utilizing this approach in 2-D gels on which lysates from the macrophage cell line, RAW 264.7, were resolved at pI 3.0–10, a number of phosphoprotein spots were obtained in cells treated with the crude DEP organic extract (Figs. 1A, B). All of the Pro-Q-positive spots (left panels) corresponded to polypeptides which could be counterstained with a sensitive protein dye, SYPRO Ruby (right panels), allowing protein identification by protein MS. Exposure to 50 mg/mL of the crude extract induced at least 14 spots, which were excised from the gel and subjected to MALDI-MS and nanoflow liquid chromatography-tandem MS (Fig. 1B, Table 1). The induction of Pro-Q staining spots was dose-dependent, with a limited number of spots appearing at 10 mg/mL of the extract, followed by a precipitous increase in spot number and staining intensity at doses  50 mg/mL (Fig. 1B, bottom panel). The magnitude of change, as measured by the number of Pro-Q spots, with increasing dose is shown in Fig. 1C. This shows a small increase in the number of phosphoproteins at an extract dose of 10 mg/mL (previously characterized as tier 1), followed by a bigger increase at an extract dose of 50 g/mL (previously characterized as tier 2) [21]. Because most phosphoproteins migrated to the acidic side of the gel, where spots were compressed, the procedure was repeated using narrow pI-range IEF gels that were resolved at pI 3.0–6.0 (Fig. 2). The improved 2-D resolution helped confirm the identity of the phosphoprotein spots in Fig. 1, and led to the identification of two additional phosphoproteins (Fig. 2B, spots #15 and 16).

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Figure 1. Phosphoprotein analysis using Pro-Q staining of 2-D gels in DEP-treated RAW 264.7 cells. RAW 264.7 cells were treated with the indicated extract concentrations for 1 h. See Section 2 for experimental details regarding performance of 2-D electrophoresis and gel staining. 350 mg lysate proteins were separated by IEF (pI 3–10, 17 cm IPG strips) in the first dimension followed by SDS-PAGE in the second dimension. (A) Analysis of unstimulated cell lysates on a 2-D gel stained with Pro-Q Diamond (left panel), followed by SYPRO Ruby staining (right panel). (B) Similar analysis in cells treated with the indicated amounts of the crude DEP extract (in mg/mL). The two panels at the top show the Pro-Q Diamond (left-hand side) and the SYPRO Ruby profiles (right hand side) of macrophages treated with 50 mg/mL of the extract. In the four panels at the bottom, the Pro-Q Diamond staining profiles of different extract doses are compared. (C) Bar graph representing the number of phosphopeptides detected on the Pro-Q Diamond stained gels at incremental doses of the organic DEP extracts. The dashed lines on the gels represent the acidic side, which was further expanded by the narrow pH (11 cm IPG) shown in Fig. 2. The numbered spots in (B) are the phophoproteins identified by MS and outlined in Table 1. The 2-D gels were run in triplicate for each sample; average protein abundance as indicated by the stained gels was estimated to vary by less than 10% for the three gels.

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Table 1. Phosphoproteome analysis of RAW 264.7 cells Spot ID

Access. number

MW (kDa)

pI

Sequence coverage (%)

Kinases: ERK-1 ERK-2 MAPKAP-2 ErbB-2 p38 MAPK a p38 MAPK b JNK1 JNK2

1 2 13 10 9 4 8 15

P21708 P27703 P49136 P70424 Q9DGE2 Q9WUI1 Q9U6D2 P49186

42.9 41.3 38.0 33.5 38.0 38.5 41.2 52.6

5.22 5.11 4.55 5.18 5.13 4.76 5.25 5.41

36 32 35 10 22 72 34 13

Other signaling components: MAGUK p55 p65/RelA Multistep phosphorelay regulator 1 Protein phosphatase 2A (B 56) regulatory g

5 16 11 7

Q7ZVT6 Q04207 O94321 Q99N67

58.7 65.0 33.6 54.0

4.67 5.61 5.06 5.32

54 16 15 23

Oxidative stress proteins: HSP27 TNFa convertase TNF ligand (member 6) Proteosome a (20S) Protein disulfide isomerase precursor

14 3 12 7 6

P14602 Q9Z0F8 P29965 Q8BKU2 P36897

23.1 93.1 34.0 49.0 56.5

4.60 5.11 5.10 5.33 4.95

24 73 16 24 56

Scrutiny of the list of Pro-Q staining polypeptides (Table 1) shows that these phosphoproteins belong to distinct groups. The largest category is protein kinases, including members of the MAP kinase cascades (ERK1, ERK2, p38 MAPKa and -b, 45 and 54 kDa JNK isoforms), as well as MAPKAP-2 and Erb-2. The analysis also identified a regulatory phosphatase subunit (B56g) that plays a role in determining the subcellular localization of PP2a activity, as well as proteins that play a role in the NF-kB pathway, proteosomal degradation and cellular stress (Table 1). Similar phosphoprotein analysis of the human bronchial epithelial cell line, BEAS-2B, confirmed the induction of Pro-Q positive peptides by the crude DEP extract (Figs. 3A, B). Protein MS identified ERK1, ERK2, TNFa, p38 MAPKa, 46 kDa JNK, p65/RelA, TNFa, and the regulatory PP2a subunit, B56g (Table 2). Other Pro-Q stained spots above pI 6 appeared on the gel. However, we were unable to unambiguously determine their identities at this time because of their low relative abundance. Future work will focus on developing more sensitive methods for identifying neutral and basic phosphoproteins stimulated by DEP. Taken together, 2-D gel electrophoresis and Pro-Q staining identified a number of phosphoproteins that could play a role in the pro-inflammatory response to organic

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DEP chemicals. This includes the identification of several MAP kinases. The precipitous increase in the number of phosphoproteins in the 50 mg/mL dose-range is compatible with the appearance of pro-inflammatory polypeptides on 2-D gels in that same dose range [21]. We have previously demonstrated that these pro-inflammatory effects are congruent with tier 2 of the hierarchical oxidative stress response [21].

3.2 Activation of MAP kinase cascades by specific organic chemical groups present in DEPs In order to identify the major chemical group(s) present in the organic DEP extract that is/are responsible for MAP kinase activation, we used anti-phosphopeptide immunoblotting to assess the activation of the terminal ERK, JNK, and p38 MAPK components. These antibodies discern allosteric effector sites that are phosphorylated by upstream MAPKKs. Similar to what was previously demonstrated for the crude extract in RAW 264.7 cells [22], 10 mg/mL of this extract failed to induce JNK activation in BEAS-2B cells, while doses of 50 and 100 mg/mL were able to activate both the 46- and 54 kDa JNK isoforms (Fig. 4A). The increased phosphorylation was not due to changes in kinase abundance, as demonstrated

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Figure 2. Phosphoprotein analysis using narrow pH-range focusing gels (11 cm) to improve the resolution of phosphoproteins. See Section 2 for experimental details regarding performance of narrowrange gels. 350 mg RAW 264.7 lysate proteins was separated on pH 3–6 IEF gels in the first dimension, followed by SDS-PAGE in the second dimension. (A) 2-D gel stained with Pro-Q Diamond (left panel), followed by staining with SYPRO Ruby protein (right panel) in unstimulated cells. (B) Similar analysis in cells treated with 50 mg/mL of the crude DEP extract. The numbered spots on the gel are the phosphoproteins identified by MS and outlined in Table 1. These data were reproduced 3 times, during which the variability in protein expression was , 10%. The 2-D gels were run in triplicate for each sample.

by parallel protein immunoblotting (Fig. 4A, lower panel). Similar to JNK, p38 MAPK activation required extract doses of 50 and 100 mg/mL in RAW 264.7 and BEAS-2B cells, with a more prominent response in the latter cell type (Fig. 5A). p38 MAPK abundance did not change with DEP treatment (Fig. 5A, lower panel). In contrast to acti-

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vation of these stress-activated protein kinases, the ERK cascade was not activated by the crude extract in RAW 264.7 cells, with only a weak response at the highest extract dose in BEAS-2B cells (Fig. 6). Bacterial lipopolysaccharide (LPS) did induce ERK activation in RAW 264.7 cells (Fig. 6).

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Figure 3. Phosphoprotein analysis using Pro-Q staining of 2-D gels in DEP-treated BEAS-2B cells. See Section 2 for experimental details regarding performance of 2-D electrophoresis and gel staining. 250 mg BEAS-2B lysate proteins was separated by narrow pH-range (pH 3–6) IEF gels in the first dimension, followed by SDS-PAGE in the second dimension. (A) 2-D gel stained with Pro-Q Diamond (left panel), followed by staining with SYPRO Ruby protein (right panel) in unstimulated cells. (B) Similar analysis in cells treated with 50 mg/mL of the crude DEP extract. The numbered spots are the phosphoproteins identified by MS and shown in Table 2. These data were reproduced 3 times, during which the variability in protein expression was , 10%.

DEPs contain a host of organic chemicals, among which the aliphatic hydrocarbons, heterocyclic compounds, aromatic hydrocarbons (e.g., PAHs) and polar compounds (e.g., quinones) are major groups [16, 17, 31]. In order to determine which chemical group(s) is/are responsible for MAP kinase activation, silica gel chromatography was used to fractionate the crude DEP extract into aliphatic, aromatic, and polar fractions (Table 3) [15]. Chemical analysis confirmed that the aromatic fraction contained an abundance of PAHs, while the polar fraction

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was devoid of PAHs but was enriched for quinones [15]. Utilizing these fractions, the polar fraction was effective in activating the stress-activated protein kinases with roughly the same efficiency as the crude DEP extract in RAW 264.7 and BEAS-2B cells (Figs. 4B and 5B). In contrast to the polar fraction, the aromatic material induced weak or no phosphorylation of JNK or p38 MAPK in BEAS-2B cells (Figs. 4B and 5B). The aromatic material induced a comparable p38-MAPK response than the polar material in RAW 264.7 cells (Fig. 5B). The aromatic

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Table 2. Phosphoproteome analysis of BEAS-2B cells Spot ID

Access. Number

MW (KDa)

pI

Sequence coverage (%)

Kinases: ERK-1 ERK-2 MAPK 14a NJK-1

1 2 5 8

P21708 P27703 Q90336 Q9U6D2

42.9 41.3 38.0 41.2

5.22 5.11 5.23 5.25

36 32 22 28

Other signaling components: p65/Rel A Protein phosphatase 2A (B56) regulatory g

6 7

Q04207 Q99N67

65.0 54.0

5.61 5.32

18 24

Oxidative stress proteins: TNFa TNF receptor 11A

3 4

P0804 Q9Y6Q6

23.5 74.0

5.01 5.21

23 19

Figure 4. Phosphopeptide immunoblotting showing JNK activation by DEP chemicals in RAW 264.7 and BEAS-2B cells. (A) Dose-dependent JNK activation by the indicated concentrations of the crude extract. RAW 264.7 and BEAS-2B cells were treated with the extract for 1 h. LPS (10 mg/mL) and TNFa (25 ng/mL) were used as controls. The upper panel depicts the phosphopeptide and the lower panel the kinase immunoblot. (B) Differential effects of DEP fractions on JNK phosphorylation. Cells were stimulated with 50 mg/mL of crude DEP extract or the DEP silica gel fractions for 1 h. The upper panel depicts the phosphopeptide and the lower panel the kinase immunoblot. Immunoblotting for phospho-JNK and total kinase protein were performed as described in Section 2. These experiments were reproduced once with similar results. These data were confirmed in a separate experiment. (C) JNK immunoblotting to demonstrate that NAC can inhibit response induction by the aromatic and polar chemical fractions. “NAC medium” refers to adding 20 mM NAC to the cell culture medium 1 h prior to the addition of the chemical fractions. “NAC wash” refers to adding 20 mM of NAc to the cell culture medium for 1 h, prior to washing, and then the addition of the DEP chemical fractions for 1 h. Density ratio: density of 54 and 46 kDa bands, respectively, compared to the control.

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Figure 5. Phosphopeptide immunoblotting showing p38 MAPK activation by DEP chemicals in RAW 264.7 and BEAS2B cells. (A) Dose-dependent p38 MAPK activation by the crude DEP extract. RAW 264.7 and BEAS-2B cells were treated with the crude DEP extract at the indicated concentrations for 30 min. LPS (10 mg/mL) and TNFa (25 ng/mL) were used as controls. The upper panel depict the phosphopeptide and the lower panel the kinase immunoblot. (B) Differential effects of DEP fractions on p38 MAPK phosphorylation. Cells were stimulated with 50 mg/mL crude DEP extract or the DEP silica gel fractions for 30 min. The upper panel depicts the phosphopeptide and the lower panel the kinase immunoblot. These data were confirmed in a separate experiment.

Figure 6. Phosphopeptide immunoblotting showing dosedependent ERK activation by the crude DEP extract in RAW 264.7 and BEAS-2B cells. RAW 264.7 and BEAS-2B cells were treated with the crude DEP extract at the indicated concentrations for 30 min. LPS (10 mg/mL) and TNFa (25 ng/mL) were used as controls. The upper panel depicts the phosphopeptide and the lower panel the kinase immunoblot. These data were confirmed in a separate experiment. Density ratio: density of 44 and 42 kDa bands, respectively, compared to the control.

Table 3. Recovery of major organic fractions from 0.5 g DEPs Fraction

Elution solvent

Amount Recovery (mg) (%)a)

Aliphatic

Hexane

117.5

24

Aromatic

Hexane/methylene chloride (3:2)b)

52.5

11

Polar

Methylene chloride/ methanol (1:1)b)

50

10

220

45

Total

a) The amount of asphaltene from 0.5 g DEPs = 145 mg, which represents 29% of particle mass b) Vol:Vol

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and polar material induced weak ERK activation in BEAS2B cells (data not shown). The aliphatic fraction was inactive under all test conditions (Figs. 4B, 5B, and 6). Differences in the potency of the various organic fractions could relate to differences in their content of redox cycling chemicals. In order to demonstrate this, we used an in vitro assay which measures the capacity of intact PM and DEP organic compounds to participate in redox cycling interactions with DTT (see Section 2). Quinones, and possibly other redox cycling chemicals, lead to DTT oxidation and O22 generation in this assay [15, 28]. When tested in the DTT assay, the polar was considerably more potent than the aromatic fraction, while aliphatic material was inactive (Table 4), which is in agreement with our

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Table 4. DTT assay to compare the in vitro redox activity of DEP fractions DEP fraction

Redox activity (nmol DTT/min/mg protein)

Crude extract Aliphatic Aromatic Polar

102 6 5 0 361 53 6 11

DTT assays were performed as described in Section 2. Values represent mean 6 SD of three readings. Please note that the aromatic plus polar fractions do not add up to the redox activity in the crude extract. The difference resides in redox cycling chemicals in the asphaltenes, which are precipitated by hexane before loading of the silica gel columns, as well as possible polar components, which remain on the column. previous demonstrations that polar chemicals are the most potent inducers of oxidative stress in intact cells [15, 18]. In order to confirm the role of oxidative stress at the cellular level, we used our previous demonstration that a thiol antioxidant, NAC, couples electrophilically to redox cycling DEP chemical groups to prevent ROS production [21]. Preloading of BEAS-2B with NAC or adding this thiol antioxidant to the culture medium, suppressed JNK activation by the polar and aromatic fractions (Fig. 4C). Similar effects were seen on p38 MAPK activation (not shown). Taken together, these results indicate that specific organic chemical groups from DEP induce MAP kinase activation in a cell-specific manner. The quinone-enriched polar fraction reproduced to large extent the effects of the crude extract. While the aromatic fraction exerted some effects, it was less potent than the polar material. This is in keeping with the ability of the quinones to participate in redox cycling reactions after cellular uptake, while PAHs need to be enzymatically converted to oxy-derivatives before being rendered functionally active [32, 33]. Previously published studies demonstrating that environmental polycyclic aromatic hydrocarbons, including DEP extracts, are capable of inducing cytochrome P450 1A1 and 1B1 expression in human macrophages and bronchial epithelial cells supports this notion [1, 34, 35]. While evidence has been provided that this is also true in BEAS-2B cells, the same needs to be demonstrated for RAW 264.7 cells.

3.3 Protein array and ELISA analyses show MAPK-dependent cytokine production in RAW 264.7 and BEAS-2B cells Organic DEP chemicals induce a hierarchical oxidative stress response that includes antioxidant (tier 1), proinflammatory (tier 2), and cytotoxic (tier 3) effects [21]. In

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order to determine whether the MAP kinases are involved in the pro-inflammatory responses seen in tier 2, we used commercially available mouse and human cytokine arrays to screen for cytokine production in the supernatants of DEP extract-treated cells. Using the RayBio mouse cytokine array (array I, which measures 22 cytokines, www.raybiotech.com) to probe for cytokine production in RAW 264.7 cells, we demonstrated TNFa production in response to the crude DEP extract (Fig. 7A). Increased cytokine production was confirmed by ELISA, which also demonstrated that this occurs in the tier 2 dose-range ( 50 mg/mL) (Fig. 7A, lower panel). Similar results were obtained with VEGF production (Fig. 7A, upper panel). NAC interfered with TNFa production (Fig. 7B). In order to demonstrate MAP kinase involvement in this cytokine response, we used the previous demonstration that all three MAPK cascades are involved in the transcriptional activation of the TNFa promoter [36– 41]. Use of inhibitors which target the ERK kinase (PD98059), p38 MAPK (SB203580), and JNK (SP600125) cascades, interfered with TNFa production in RAW 264.7 cells (Fig. 7B). These inhibitors also interfered with the activation of the kinases, as determined by phosphopeptide immunoblotting (not shown). These results are compatible with previous demonstration that these inhibitors as well as mutagenic alteration of key AP-1 response elements in the TNFa promoter, interfere in TNFa production [36–41]. Similar use of the RayBio human cytokine array (array I) in BEAS-2B cells demonstrated increased IL-8 production, which could be confirmed by ELISA (Fig. 8). NAC suppressed this response (Fig. 8). Since it has previously been demonstrated that PD98059, SB203580, and SP600125 and dominant-negative MEKK1 can interfere in IL-8 production [42–47], we ask whether these inhibitors could interfere in IL-8 production in BEAS-2B cells (Fig. 8). The cytokine array analysis also showed IL-6 expression (Fig. 8), which was sensitive to SB203580 in an ELISA (not shown).

4 Discussion We used a proteomics approach to demonstrate the dose-dependent induction of protein phosphorylation in macrophage and epithelial cell lines by organic DEP chemicals. This phosphoprotein profile includes the activation of a number of MAP kinases at an intermediary dose level. This is in agreement with the induction of a hierarchical oxidative stress response by pro-oxidative DEP chemicals in this dose range [21]. Regarding the chemical components which play a role in this response, we demonstrate that the quinone-containing polar frac-

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Figure 7. Induction of cytokine production in RAW 264.7 cells. (A) Mouse cytokine array analysis (upper panel) and ELISA (lower panels) in RAW 264.7 cells. For the cytokine array analysis, cells were either left unstimulated or treated with 50 mg/mL of the DEP extract for 6 h, before harvesting of the culture medium and conducting Mouse Cytokine Array I as described by the manufacturer. For the ELISA, cells were treated with the indicated concentrations of the DEP extract for 6 h, before the culture media were collected, frozen and sent to a commercial laboratory for measurement of TNFa levels. (B) ELISA showing the effects of NAC and the MAP kinase inhibitors. Cells were pre-incubated with MAP kinase inhibitors or 20 mM NAC for 1 and 2 h, respectively, before the addition of 50 mg/mL of the DEP extract. Values represent means 6 SD; n = 3. (a) p , 0.001 compared with control; (b) p , 0.001 compared with DEP extract only. * PD = PD98059, used at 25 mM; SB = SB203580, used at 3 mM; SP = SP600125, used at 10 mM.

tion, and to a lesser extent the PAH-containing aromatic fraction, mimic the effects of the crude organic extract in the activation of the p38 MAPK, JNK, and ERK cascades. Activation of these cascades was associated with the induction of pro-inflammatory responses, as determined by cytokine ELISA and array assays. In particular, we demonstrated increased production of TNFa in RAW 264.7 cells; these responses were suppressed by MAP kinase inhibitors. Similarly, we found that the MAP kinase inhibitors suppressed IL-8 production in BEAS-2B cells in response to the crude organic extract. The relationship of these cytokine responses to oxidative stress was confirmed by suppressing their production with NAC. Inclusion of a regulatory PP2a subunit in the phosphoprotein profile, suggests that this MAPK phosphatase could be involved in the regulation of MAP kinase activation by prooxidative DEP chemicals. Taken together, these results demonstrate that DEP induce pro-inflammatory effects through the activation of the MAP kinase cascades.

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Recently developed proteomic approaches have advanced beyond identification of co- and post-translational modification of proteins [48]. However, defining the state of post-translational modifications, such as phosphorylation, still remains a challenging aspect of proteomics. Several strategies have been employed to elucidate the “phosphoproteome.” Enrichment of phosphoproteins using antibodies specific to phosphoamino acids (pSer, pThr, pTyr) have demonstrated success [49], particularly for detection of phosphotyrosine proteins. However, antibodies that specifically recognize pSer and pThr residues are typically sensitive to specific amino acid microdomains and do not provide universal detection of proteins phosphorylated at these sites. Another method to enrich for phosphorylated proteins is immobilized metal-affinity chromatography (IMAC), in which negatively charged phosphate groups bind to positively charged metal ions (e.g., Fe31, Ga31) immobilized to a chromatographic support. Previous limitations of IMAC

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use of radiolabels [26]. Patton and co-workers [30] identified several phosphorylated proteins from the mitochondria using Pro-Q staining. The dye indiscriminately binds to all phosphoamino acids, and is compatible with the MS analysis. We show the advantages of coupling narrow-range pI separations prior to the second-dimensional SDS separation with Pro-Q staining to identify a number of phosphorylation events as a result of DEP stimulation.

Figure 8. Induction of cytokine production in BEAS-2B cells. (A) Human cytokine array analysis (upper panel) and ELISA for IL-8 (lower panels) in BEAS-2B cells. For the cytokine array analysis, cells were either left unstimulated or treated with 25 mg/mL DEP extract for 6 h, before harvesting of the culture medium and conducting the array analysis as described in Fig. 7. For the ELISA, cells were pre-incubated with MAP kinase inhibitors or 20 mM NAC for 1 and 2 h, respectively, before addition of 25 mg/mL DEP extract. The culture media were collected 6 h later, frozen, and sent to a commercial laboratory for measurement of IL-8 levels. Values represent means 6 SD; n = 3. (a) p , 0.001 compared with control; (b) p , 0 001 compared with DEP extract only. * PD = PD98059, used at 25 mM; SB = SB203580, used at 3 mM; SP = SP600125, used at 10 mM.

included nonspecific binding of acidic Glu and Asp amino acids. However, methyl esterification of acidic residues prior to IMAC enrichment has demonstrated impressive results; coupled with LC-MS/MS identification, more than 383 sites of phosphorylation were determined for the analysis of a whole-cell lysate from Saccharomyces cerevisiae [50]. Two-dimensional gel electrophoresis remains a popular means to display global protein expression and can be used for displaying the phosphoproteome when coupled with 32P-radiolabeling. The recent development of a small molecule fluorophore dye, Pro-Q Diamond that binds selectively to phosphoproteins, complements the

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Because protein phosphorylation is such an important biochemical event, researchers have developed numerous technologies to detect phosphorylation, such as selective staining, antibody-based methods, and affinity selection that involve multiple platforms, such as gel electrophoresis, microarrays, and MS. Currently, there is not one technology that can address all questions regarding protein phosphorylation, because each method has their unique advantages and disadvantages. Performance issues, such as sensitivity, throughput, selectivity, and dynamic range, are addressed with varying success by each of the methods. For this study, we chose to apply the relatively new Pro-Q/2D-gel electrophoresis method because of its demonstrated potential for global phosphoprotein detection. The cytokine arrays provide the supplemental information needed to address specific protein targets. This combination strategy allows us to confirm and validate our interpretation of the importance phosphorylation events upon DEP stimulation. Although activation of the MAP kinase cascades is best delineated in response to extracellular stimuli, there is growing evidence that these cascades are also activated by oxidative stress [51]. A number of studies have shown that bolus addition of exogenous H2O2 or intracellular ROS generation by drugs and radiation exposure lead to MAP kinase activation [52, 53]. Depletion of intracellular GSH leads to JNK and the p38 MAPK activation, as demonstrated by treatment with alkylating agents as well as organic DEP extracts [21, 54, 55]. These organic extracts lead to a decline in reduced to oxidized glutathione ratio (GSH/GSSG) in epithelial cells and macrophages, leading to MAP kinase activation at an intermediary level of oxidative stress [21, 22]. In spite of the accruing evidence for oxidative stress in MAPK activation, the mechanism, by which ROS activate these cascades remain unclear [51]. Possible explanations include the activation of Src kinases and the apoptosis stimulating kinase 1 (ASK1), as well as the modulation of phosphatase activity [51, 56]. While we found no evidence for Src activation in our phosphoprotein profile, our 2-D gels elucidated Pro-Q staining of the receptor tyrosine protein kinase, ErbB2 (Table 1). Oxidative stress leads to tyrosine phosphorylation of the EGFR, while ErbB2, 3, and 4 have

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been shown to suppress oxidative burst activity in microglial cells in a dose-dependent fashion [57, 58]. ASK1 is a MAPKKK that can activate both JNK and p38 MAP kinase pathways [59]. This kinase is negatively regulated by thioredoxin, which binds directly to ASK1 [60]. ROS leads to the release of thioredoxin, followed by ASK1 activation [60]. Using in vitro kinase assay for ASK1, we could not obtain evidence for its activation by DEP chemicals in either cell type (data not shown). MAP kinase activation needs to be terminated with some precision to regulate the biological consequences of JNK, p38 MAPK, and ERK activation [61]. MAP kinase activity is dependent on the dynamic equilibrium between activating upstream kinases and inhibitory phosphatases. A major site of regulation is at the level of the terminal cascade components by constitutively active serine/threonine as well as inducible dual-specificity phosphatases. An example of the former is PP2a, which has been shown to be a key negative regulator of MAP kinase activity in the brain [62]. It is interesting, therefore, that the phosphoprotein analysis in Fig. 1 reveals Pro-Q staining of one of the four B subunits (B56g) that regulate PP2a activity (Table 1). Several PP2a regulatory subunits participate in the formation of a series of holoenzymes, which differ with respect to substrate specificity and subcellular localization [63]. Phosphorylation of the B61 subfamily is involved in the regulation of PP2a activity in vivo and substrate affinity in vitro [64]. We propose that phosphorylation of the B56g subunit (Table 1) plays a role in regulating PP2a activity in macrophages and epithelial cells under conditions of DEP-induced oxidative stress. This hypothesis is currently under study. The MAP kinases play an important role in the regulation of gene expression, including activation of cytokine, chemokine, and adhesion molecule promoters, and could be a key ingredient in the pro-inflammatory effects of DEPs and PM in the respiratory tract [65]. We demonstrate that organic DEP compounds induce the production of TNFa and IL-8 in RAW 264.7 and BEAS-2B cells, respectively (Table 1, Figs. 7 and 8). Above cytokines are relevant to asthma and airway inflammation, with direct evidence that DEP induce IL-8 and TNFa production in vitro [66, 67]. TNFa is a potent multifunctional cytokine that plays a central role in the pathogenesis of asthma and allergic rhinitis [68]. Elevated TNFa levels can be detected in the airways and bronchoalveolar lavage (BAL) fluid lavage from asthmatics. TNFa is produced by a variety of cell types and is involved in airway hyperresponsiveness, a pathophysiological hallmark of asthma [68]. TNFa also upregulates adhesion molecules and is directly responsible for transendothelial migration of neutrophils and monocytes [68]. Although only one time point was shown

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Electrophoresis 2005, 26, 2092–2108 in Figs. 4–8 for protein phosphorylation (30 min for p38 and ERK, and 1 h for JNK) and cytokine production (6 h for TNFa and IL-8), our previous kinetic studies have demonstrated that in RAW 264.7 the phosphorylation of p38 and JNK begins 30 min after cellular exposure to DEP extracts and lasting up to 4 h, while ERK is constitutively phosphorylated. Phosphorylation of all three MAP kinases in BEAS-2B began 30 min after DEP stimulation. While phospho-p38 started to decrease after 2 h, the phosphorylation of JNK and ERK lasted for 4 h. The increase in TNFa from RAW 264.7 began at 2 h and peaked at 6 h, whereas the increase in IL-8 production in BEAS-2B started at 2 h and peaked 4 h after the addition of DEP extract (data not shown). A key finding in this study, which extends out previous results with crude DEP extracts, is that the aromatic and polar compounds, fractionated from DEP by silica gel chromatography, mimic the effect of the crude DEP extract on JNK activation (Figs. 4–6). These fractions are toxicologically relevant, because the aromatic fraction is enriched in PAHs, while the polar fraction contains several oxy-PAH compounds, including quinones [15, 69]. Quinones are able to redox cycle, leading to ROS production, while PAHs can be converted to quinones [32, 38]. A number of studies have shown that specific PAHs, such as benzo[a]pyrene and b-napthoflavone, can induce MAP kinase activation in macrophages, epidermal cells, and epithelial cells [70–72]. Similarly, quinones such as menadione, 1,4-naphthoquinone, and benzo(a)pyrene quinones, induce MAP kinase activation secondary to the generation of oxidative stress [73, 74]. The higher potency of the polar compared to the aromatic fractions in MAP kinase activation (Figs. 4, 5), can be explained by the presence of redox cycling quinones in the former but not the latter fraction. This is in agreement with the increased activity of the polar fraction in the DTT assay [18, 20]. It is important to clarify that not all PM adverse health effects are mediated by PAHs or quinones, because transition metals also play a role in PM-induced ROS generation [14]. Taken together, we show that a fluorescent dye that is specific for phosphoproteins, can be used to identify oxidative stress signaling pathways that are induced by organic DEP chemicals. This includes activation of the MAP kinase cascades which are involved in cytokine production. These data are of considerable importance in advancing our understanding of the mechanism of toxicity of air pollutants, and identifies specific chemical groups that may need to be monitored for regulatory purposes. We thank Charisa Cottonham and Dr. James Kerwin for their help with the gel image analysis, protein digestion, and protein identifications, and Dr. Rachel Ogorzalek Loo

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for advice on sample preparation and gel electrophoresis. We thank Indira Venkatesan, Arantza Eiguren-Fernandez, Debra Schmitz, Emma Di Stefano, Antonio H. Miguel, and Arthur Cho for their help in preparing and analyzing the DEP chemical fractions. This work was funded by the US Public Health Sciences grants, RO-1 ES12053, RO-1 ES10553, and RO-1 ES 013432. 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.

[19]

[20]

[21] [22] [23] [24]

Received December 22, 2004

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