DIGE-Based Protein Expression Analysis of B[a] - American Chemical

DIGE-Based Protein Expression Analysis of B[a]P-Exposed Hepatoma Cells Reveals a Complex Stress Response Including Alterations in Oxidative Stress, Cell Cycle Control, and Cytoskeleton Motility at Toxic and Subacute Concentrations Franziska Dautel,† Stefan Kalkhof,† Saskia Trump,‡ Jacob Michaelson,§ Andreas Beyer,§ Irina Lehmann,‡ and Martin von Bergen*,†,| Department of Proteomics, UFZ, Helmholtz-Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany, Department of Environmental Immunology, UFZ, Helmholtz-Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany, Biotechnology Center, Technische Universita¨t Dresden, Tatzberg 47/49, 01307 Dresden, Germany, and Department of Metabolomics, UFZ, Helmholtz-Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany Received July 13, 2010

Although the effects of high concentrations of the carcinogen benzo[a]pyrene (B[a]P) have been studied extensively, little is known about its effects at subacute toxic concentrations, which are typical for environmental pollutants. We exposed murine Hepa1c1c7 cells to a toxic concentration (5 µM) and a subacute concentration (50 nM) of B[a]P over a period of 2-24 h to differentiate between acute and pseudochronic effects and conducted a time-course analysis of B[a]P-influenced protein expression by DIGE. In total, a set of 120 spots were found to be significantly altered due to B[a]P exposure of which 112 were subsequently identified by mass spectrometry. Clustering and principal component analysis were conducted to identify sets of proteins responding in a concerted manner to the exposure. Our results indicate an immediate response to the contaminant at the protein level and demonstrate that B[a]P exposure alters the cellular response by disturbing proteins involved in oxidative stress, cell cycle regulation, apoptosis, and cytoskeleton organization. Furthermore, network analysis of protein-protein interactions revealed a complex network of interacting, B[a]P-regulated proteins mostly belonging to the cytoskeleton organization and several signal transduction pathways. Keywords: benzo[a]pyrene (B[a]P) • aryl hydrocarbon receptor (Ahr) • oxidative stress • human protein reference database (HPRD) • protein expression analysis • DIGE

1. Introduction Benzo[a]pyrene (B[a]P) belongs to the class of polycyclic aromatic hydrocarbons (PAH), a group of ubiquitous environmental contaminants. These compounds consist of two or more benzene rings and are formed by incomplete combustion of organic materials. They are characterized as carcinogens by the International Agency for Research on Cancer, as exposure to these chemicals may lead to the formation of lung cancer.1 In addition to its carcinogenic potential, B[a]P is also known to cause immune suppression, birth defects, and atherogenesis.2 The procarcinogen B[a]P is incorporated into all cytoplasmic membranes within several minutes3 and rapidly distributed to * To whom correspondence should be addressed. PD Dr. Martin von Bergen, UFZ, Helmholtz-Centre for Environmental Research, Department of Proteomics, Permoserstr. 15, 04318 Leipzig, Germany. Email: Martin. [email protected]. Fax: +49-341-2351786. † Department of Proteomics, UFZ, Helmholtz-Centre for Environmental Research. ‡ Department of Environmental Immunology, UFZ, Helmholtz-Centre for Environmental Research. § Technische Universita¨t Dresden. | Department of Metabolomics, UFZ, Helmholtz-Centre for Environmental Research. 10.1021/pr100723d

 2011 American Chemical Society

several tissues in vivo.4,5 After entering the cytoplasm, B[a]P binds the aryl hydrocarbon receptor (Ahr), a cytoplasmatic transcription factor, which is subsequently activated and translocated to the nucleus.6 As a result, a number of genes involved in phase 1 and 2 metabolism of B[a]P are reported to be transcribed, leading to the formation of several metabolites and the ultimate carcinogen 7,8-dihydrodiol-9,10-oxy-7,8,9,10tetrahydrobenzo[a]pyrene (BPDE).7 In this study, we analyzed murine hepatoma cells (Hepa1c1c7) cells, which actively metabolize B[a]P and thus are used as a model system to detect and characterize the complete B[a]P and B[a]P-metabolite induced cellular stress responses. The B[a]P (or active B[a]P-metabolites)-affected protein expression has been previously analyzed in different cellular models using one incubation time point and often several B[a]P or B[a]Pmetabolite concentrations.8-13 Our study provides the missing link with a B[a]P-concentration- and incubation-time dependent protein expression analysis. To precisely monitor proteomic changes over time, two early (2 and 4 h) and two late (12 and 24 h) incubation time points were chosen according to the results obtained by B[a]P-induced cytotoxicity measurements. Journal of Proteome Research 2011, 10, 379–393 379 Published on Web 11/20/2010

research articles Importantly, we measured the effects of one higher (5 µM) and one subacute concentration (50 nM), which realistically reflects environmental conditions. Although the proteome can be assessed by different means, we opted for the DIGE system because of its high reproducibility and accuracy in terms of quantification.14 Furthermore this approach allows concomitantly the global analysis of the proteome and the assessment of the post-translational modifications.15 Beyond the gene enrichment analysis, it is crucial to analyze the interactions among the regulated proteins and with other proteins involved in molecular pathways. To unravel functional and physical interactions between differentially expressed proteins and proteins not identified in this study, a network analysis was performed using HPRD (Human Protein Reference Database).16 By analyzing the proteomic changes, it is the aim of this study to unravel the cellular response mechanisms to B[a]P exposure over time. The comparison between the different concentrations reveals insights into the early cellular changes that are not necessarily identical with the observed effects at higher concentrations.

2. Material and Methods 2.1. Cell Culture and B[a]P Exposure. All operations were performed using personal protection measures defined by German law (safety glasses, gloves, and laboratory coat) as well as clean benches of the security level 2. All solutions containing DMSO or B[a]P were stored in appropriate safety cabinets. Wastes have been collected and disposal has been managed according to German law. Murine hepatoma cells (Hepa1c1c7, ATCC No. CRL-2026; LGC Promochem, Wesel, Germany) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 7% heat-inactivated FCS, 1% penicillin/streptomycin and 2 mmol L-alanyl-L-glutamine (Biochrom, Berlin, Germany) at 37 °C and 5% CO2. Cells at Passage 7, 8, and 16 were used for exposure experiments. About 1 × 104 cells/cm2 were seeded, medium changed 24 h later, and treatment started after 48 h. Before and after exposure, cell viability, and cell numbers were recorded by Trypan blue exclusion. Benzo[a]pyrene (B[a]P) (Sigma-Aldrich, Steinheim, Germany) was dissolved in dimethylsulfoxide (DMSO; Applichem, Darmstadt, Germany) to obtain a 10 mM stock solution. The cells were exposed to 50 nM B[a]P, 5 µM B[a]P or DMSO for 2, 4, 12, and 24 h. Three independent biological replicates of all treatments were prepared. 2.2. Cytotoxicity Measurements. To determine cytotoxicity, a Neutral Red-(NR) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), an Alamar Blue-(AB) (Invitrogen GmbH, Karlsruhe, Germany) and a lactate dehydrogenase (LDH) assay (Roche Applied Science, Mannheim, Germany) of five different B[a]P concentrations (50 nM, 250 nM, 500 nM, 1 µM, and 10 µM) and three exposure time points (4, 24, and 48 h) were performed. DMSO-treated cells served as a control. All incubations were conducted in triplicates. The experiments were conducted as described elsewhere.17,18 A one way analysis of variance (one-way ANOVA) was carried out and difference between groups determined by a Holm-Sidak posthoc test. For more information, please see supplementary methods (7.1.1, 7.1.2). 2.3. FACS-Analysis: Cell Cycle Alterations. For determination of the cell cycle status Hepa1c1c7 cells were stained using propidium iodine (PI) RNase staining buffer (BD Pharmigen, Beckton Dickinson GmbH, Heidelberg, Germany) according to 380

Journal of Proteome Research • Vol. 10, No. 2, 2011

Dautel et al. the manufacture’s protocol. Briefly, cells were treated with 50 nM, 5 µM B[a]P or DMSO for either 24 or 48 h. After the incubation period medium was collected and combined with the corresponding cell suspensions, which were derived using Accutase (PAA Laboratories GmbH, Pasching, Austria). After centrifugation at 750 rpm for 10 min, cell pellets were washed twice with PBS followed by fixation with ice-cold 70% ethanol. Cells (5 × 105) were aliquoted, washed twice with PBS containing 1% FCS, and subsequently stained with PI/RNase. Samples were kept on ice until the analysis on a FACSCalibur (Beckton Dickinson GmbH, Heidelberg, Germany). FACS measurements were analyzed using the Modfit LT software (Verity Software House, Topsham, USA). 2.4. DIGE and Data Analysis. 2.4.1. Difference Gel Electrophoresis. Cells were washed and lysed according to the procedure previously described.18 The acetone precipitated protein samples were reconstituted in 30 µL of labeling buffer (pH 9.0, 7 M urea, 2 M thio-urea, 4% CHAPS, 30 mM Tris/HCl). Solubility of the precipitate was facilitated by sonication for 30 s and the pH of the solution was adjusted to 8.5 with addition of labeling buffer. The precipitates were then labeled using the CyDye DIGE Fluor minimal dye (GE Healthcare, Uppsala, Sweden) following the manufacturer’s recommendation for minimal labeling. The samples of each group were labeled in such a way to avoid any color quenching and variation in dye-protein binding ratio. A Cy2-labeled common internal standard for all gels was prepared from a mixture of all samples. IPG strips (24 cm, pH range 3-10 NL; GE Healthcare, Freiburg, Germany) were rehydrated overnight and focused for 100 000 Vhrs using an Ettan IPGphor 3 isoelectric focusing unit (GE Healthcare, Freiburg, Germany) as described earlier.19 Strips were equilibrated for 15 min with 20 mg/mL DTT (GE Healthcare, Uppsala, Sweden) and proteins were subsequently alkylated for 15 min in 25 mg/mL iodoacetamide (GE Healthcare, Buckinghamshire, UK) (both dissolved in equilibration buffer, containing 6 M urea, 30% (v/v) glycerol, 4% (w/v) SDS, 0.05 M Tris/HCl (pH 8.8), bromophenol blue). The equilibrated strips were then carefully placed on top of 12% acrylamide gels and sealed with 1% (w/v) agarose. Second dimension separation was performed using an Ettan DALTtwelve electrophoresis system (GE Healthcare, Uppsala, Sweden). The LabAid Page Ruler Prestained Protein ladder 10-170 kDa was used as a molecular standard (Fermentas GmbH, St. Leon-Rot, Germany). Five gels according to the different incubation time points were run simultaneously to improve reproducibility. The proteins were separated initially at 12 °C, 1 W/gel for 12 h, followed by 12 W/gel until the dye front reached the bottom of the gel. The gels were scanned while they were still in the glass cassettes using the Ettan DIGE Imager Scanner (GE Healthcare, Uppsala, Sweden). Each fluorescent dye was consecutively excited and scanned at a resolution of at least 100 µm with their appropriate filter. Each gel image file was checked with the ImageQuant software (GE Healthcare, Uppsala, Sweden) by employment of the volume review, if the image did not exceed the linear dynamic range (Max. Val >100 000) to adjust the DIGE scanner accordingly. 2.4.2. DIGE Analysis. Gel images were analyzed in Delta 2D version 3.6 software (Decodon GmbH, Greifswald, Germany20). After warping the gels using the all-to-one strategy, a fusion gel was created including all gels of the experiment. Detected spots were manually edited and transferred to all gel pictures. Spot volumes (integrated staining intensity) were normalized to the total protein amount on each gel (excluding the largest

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Analysis of B[a]P-Exposed Hepatoma Cells spots representing ∼5% of total intensity from the normalization). Thus, relative volumes of the spots were determined. Before proceeding with the statistical analysis, several technical artifacts were removed from the data. We first adjusted the (log2) ratio of the Cy3 and Cy5 intensities to the intensities of the internal standard on the Cy2 channel. For each spot, we then regressed out the effect of the dye type (Cy3 or Cy5) and took the residuals. Each gel then had a distribution of residual spot intensities, which were centered by subtracting the mean of each gel. Next, a random effects model was fitted to each spot to remove the spot-specific intragel correlation between the Cy3 and Cy5 signals. Finally, we took the residuals of this model and subtracted the mean of the DMSO samples at the respective time point, so that each final measurement reflects perturbation due only to B[a]P. With the unwanted technical variation accounted for, a twoway ANOVA model was fitted to each spot, with time and B[a]P concentration as the factors. P-values for the time main effect, the concentration main effect, and their interaction were corrected for multiple hypotheses testing using the false discovery rate (FDR). After the FDR adjustment, only the time main effect was significant for 120 spots at FDR < 0.05. Only these spots were considered in the subsequent analysis. For the HPRD (human protein reference database) analysis, we used data downloaded from http://www.hprd.org in September 2009. The files were dated August 21, 2007. 2.4.3. Preparation of 2D-Mastergels for Protein Identification. Since DIGE-gels only contain 300 µg protein/gel, only very large protein spots are visible on DIGE-gels after applying the blue silver staining method - a modified Neuhoff’s colloidal Coomassie Blue G-250 staining with sensitivity close to silver staining21 (data not shown). In order to detect all identified protein spots, mastergels with 2.0 and 2.5 mg protein (equal mix of all samples) were created for protein identification (procedure as described elsewhere18). To avoid mistakes in protein spot identification, the images of the mastergels were loaded in the Delta 2D-DIGE-project and warped to the DIGEimages. 2.5. Protein Identification by MALDI-MS or nano-HPLCESI-MS. Proteins were excised from dried master gels if they were found to be significantly up- or down-regulated as described previously. Tryptic digestion was carried out with porcine trypsin (100 ng/gel piece Sigma-Aldrich, Steinheim, Germany).22 The extracted peptides were reconstituted in 10 µL 0.1% formic acid and 1 µL of sample was mixed with 1 µL HCCA matrix (0.6 mg/mL) and spotted on a MALDI target. The measurement was performed with MALDI-TOF/TOF-MS (Ultraflex III, Bruker Daltonik, Bremen Daltonik, Bremen, Germany) according to Georgieva et al.23 Briefly, a MS spectrum as well as LIFT-MS/MS-spectra of the 20 most intensive ions (S/N > 20) were recorded. Alternatively, if no significant identification was obtained with MALDI-TOF-MS analysis (Mascot-Score cutoff 100), the samples were measured using nano-HPLC/nano-ESI-MS(/MS). Therefore, 5 µL of the reconstituted peptides were injected by the autosampler and concentrated on a trapping column (nano-HPLC column, C18, 180 µm × 2 cm, 5 µm, Eksigent, Dublin, CA) with water containing 0.1% formic acid at flow rates of 15 µL/min. After 4 min, the peptides were eluted onto the separation column (nano-HPLC column, C18, 75 µm × 10 cm, 5.0 µm, Eksigent, Dublin, CA). Chromatography was performed using 0.1% formic acid in solvents A (97% water, 3% ACN) and B (97% ACN, 3% water) (Sigma-Aldrich, Steinheim, Germany) with peptides eluted over

Figure 1. Cell toxicity assays. Cell toxicity of B[a]P treatments with different concentrations (50 nM-10 µM) and varying incubation times (4 h (green), 24 h (yellow), and 48 h (red)) were measured in triplicates using Neutral Red (NR), Alamar Blue (AB), and lactate dehydrogenase (LDH) release assay. A treatment with 50 nM B[a]P did not result in reduced cell viability whereas cell toxic effects were observed with all assays for concentrations higher than 1 µM.

30 min with a 8-40% solvent B gradient using a nano-HPLC system (2D-nano-HPLC, Eksigent, Dublin, CA, USA) coupled to an LTQ-Orbitrap XL ETD hybrid mass spectrometer (Thermo Fisher Scientific, U.S.A.). Continuous scanning of eluted peptide ions was carried out between m/z 350-2000, automatically switching to CID-MS/MS mode on ions exceeding an intensity of 2000. MALDI-MS or ESI-MS raw spectra were analyzed with the Biotools software (Bruker Daltonik, Bremen, Germany) or ProteomDiscoverer 1.0 software (Thermo Fisher Scientific, U.S.A.), respectively. Mascot searches (http://www.matrixscience. com, version 2.2.06) were conducted on the Swiss-Prot database (http://expasy.org/sprot) against Mus musculus protein sequences, tolerating up to one proteolytic miscleavage, a mass tolerance of 100 ppm (MALDI-TOF MS) or 10 ppm (nano-ESIMS) for precursor ions, 0.5 Da for MS/MS product ions allowing for methionine oxidation (variable modification), and cysteine carbamidomethylation (static modification). Proteins were specified as unambiguously identified if the MOWSE score was higher than 100 and at least 2 different peptides (p < 0.05) were used for identification. Additional protein hits were considered if the MASCOT score of the second hit was more than 33% of the first hit but still higher than the MASCOT significance threshold. Molecular weight and pI of the identified protein were crosschecked with the gel position of the excised spot.

3. Results and Discussion 3.1. Toxicity Studies. We analyzed the cell toxicity of B[a]P in Hepa1c1c7 cells using three different cytotoxicity assays to determine relevant B[a]P treatment concentrations and incubation time points (Figure 1). Neutral Red (NR) uptake, Alamar Blue (AB) reduction and lactate dehydrogenase (LDH) release Journal of Proteome Research • Vol. 10, No. 2, 2011 381

research articles were measured to investigate the mechanism of cytotoxicity. Cells were treated for 4, 24, and 48 h with 50 nM, 250 nM, 500 nM, 1 µM, and 10 µM of B[a]P. Each value obtained was normalized to the corresponding DMSO measurements and expressed as percent of vehicle control. Although NR and AB are both viability assays, they are based on different mechanisms. AB serves as an indicator of the energy metabolism of a cell24 whereas NR incorporation into the cell is dependent on intact membranes in particular those of lysosomes.25 The LDH assay assesses the activity of this enzyme released into the cell culture medium through damaged cell membranes.26 All assays showed very similar results. However, NR and AB proved to be more sensitive to B[a]P treatment in comparison to the LDH assay. While all three assays did not show any cytotoxicity after 4 h of treatment, both, NR and AB determined an increased cytotoxic effect at 24 h for B[a]P concentrations higher than 500 nM (Figure 1). After 48 h of treatment in the NR and the AB assay all B[a]P concentrations induced a significant cytotoxic effect. However, the LDH leakage test only showed a significant difference from control for 1 µM and 10 µM B[a]P treatments for 24 and 48 h, respectively (Figure 1). These results indicate a threshold concentration of 1 µM B[a]P causing cytotoxicity. The only concentration not inducing any toxic effects after 24 h of B[a]P exposure in all three assays was 50 nM B[a]P. Thus, all B[a]P levels below 50 nM were assumed to be subacute for Hepa1c1c7-cells. An increased number of apoptotic murine Hepa1c1c7 cells was observed after 24 h of 10 µM B[a]P exposure.27 Furthermore, 5 µM of B[a]P decreased cell viability by 23% compared to control Hepa1c1c7 cells when measured by the MTT assay.28 For Jurkat T-cells, a concentration of more than 2,5 µM B[a]P for 48 h caused an increase in the cytotoxic effects and a reduced cell viability.9 Comparing to our results, Hepa1c1c7 cells seem to be more susceptible to B[a]P-induced cytotoxicity, possibly due to their origin as liver cells and their consequently active B[a]P-metabolism resulting in more reactive B[a]Pmetabolites, even though the opposite, an increased detoxification of the B[a]P-metabolites, is also possible.29 Based on these results, we chose one toxic (5 µM) and one subacute (50 nM) B[a]P-concentration and 4 different incubation periods (2, 4, 12, and 24 h) to analyze B[a]P-induced changes of the proteome using DIGE technology. 3.2. Treatment with B[a]P Leads to Significant Changes in Protein Expression. We analyzed the time- and concentration dependent B[a]P-induced alterations in protein expression by DIGE. We detected and quantified the expression of 1,227 protein spots of murine Hepa1c1c7 cells with a pI between 3 and 10 using the Delta 2D software (Figure 2A). The normalized data including the concentration and the time factor as well as their interaction was analyzed using a 2-way ANOVA model. The concentration main effect has more or less uniformly distributed P-values, suggesting that there are no spots with significant differences between the concentrations. The time effect shows high enrichment for small p-values. Thus, B[a]P (at any concentration) has a significant time effect on many spots. After adjusting with the FDR < 0.05, 120 spots showed significant abundance changes for a B[a]P-induced time effect. Of those, 112 were identified by tryptic digestion and mass spectrometric analysis of the proteolytic peptides (Table 1). The identified proteins were related to the protein expression values and grouped according their protein function (Table 2, Supplementary Table 1, Supporting Information). Twenty identifica382


Dautel et al. tions with multiple hits were excluded from further analysis to avoid the inclusion of nonregulated proteins. Eleven proteins were identified in different spots. For the interpretation, the spot with the maximum regulation or single identifications were chosen. In total, 58 (5 µM) and 57 (50 nM) out of the 120 protein spots with a significant time-effect were up-regulated at the time point of maximum regulation, while 62 (5 µM) and 63 (50 nM) were down-regulated (Figure 2B). For 79 spots and thus 65.8% of all time-dependent regulated spots, the maximum level of regulation was observed using the higher B[a]Pconcentration of 5 µM. The maximum level of expression modulation was reached for 33, 35, 22, and 30 spots at 2, 4, 12, and 24 h of B[a]P-incubation, respectively. These results show an even distribution of maximum induction and repression among the experimental time scale. However, out of the 92 unambigiously identified proteins, 74 respond before 2 h, 17 between 2 and 4 h, and only one protein between 12 and 24 h of B[a]P-exposure, thus indicating an immediate response to the contaminant on the protein level. 3.2.1. B[a]P Causes Oxidative Stress. Cells express a number of antioxidant enzymes including superoxide dismutases (SOD), glutathione (GSH) peroxidases, catalases, and peroxiredoxins, as well as nonenzymatic antioxidants such as GSH to protect against the harmful consequences of oxidative stress.30 B[a]P is known to induce oxidative stress in vitro and in vivo, which has been linked to oxidative DNA damage.31-33 Intriguingly, our data shows the expression regulation of 4 redox-sensitive proteins namely superoxide dismutase 2 (SOD2) (Figure 3A), peroxiredoxin-6 (Prdx6), glutathione S-transferase pi (GSTpi) (Figure 3B) and glyoxalase domain containing protein 4 known to protect the cell from the aftermath of oxidative stress. SOD2 plays a crucial role in the detoxification of superoxide free radicals, which protects cells from reactive oxygen speciesinduced oxidative damage.34 SOD2 protein expression steadily increased over time to a maximum fold change of 1.73 at 5 µM and 1.62 at 50 nM B[a]P-exposure (24 h) (Figure 3A, Table 2). Cu-Zn SOD and thioredoxin were up-regulated in rat LEC cells as a result of 100 nM B[a]P-exposure.13 Although perioxiredoxin-6 (Prdx6) expression was slightly decreased (0.72) in the 50 nM group after a 2 h exposure, levels of this protein also increased for both B[a]P-concentrations at later timepoints. Prdx6 is a 1-cys enzyme that utilizes GSH as the physiological reductant to reduce H2O2 and organic hydroperoxides.35 Expression of Prdx6 increased by a factor of 3 after a 12 h treatment with 0.05 µM BPDE, the ultimate carcinogen of the B[a]P, in amniotic FL-cells.10,11 It has been described earlier that many detoxification genes are transcriptionally induced as a consequence of Ahr activation, among them are several phase II enzymes like the GSTs.36 Glutathione Stransferase pi (GSTpi) showed the same time-dependent expression curve as Prdx6 resulting in a maximum induction of 1.32 (5 µM) and 1.48 (50 nM) fold change at 12 h of incubation (Figure 3B, Table 2). S-Glutathionylation of proteins is critical as a response to cellular stress such as B[a]Ptreatment. GSTpi potentiates S-glutathionylation reactions following oxidative and nitrosative stress in vitro and in vivo.37 Furthermore, glyoxalase domain containing protein 4 was slightly induced by 50 nM B[a]P treatment after 2 h by a factor of 1.30 but its concentration decreased with further B[a]Pincubation to 0.75 (5 µM, 24 h) and 0.91 (50 nM, 12 h) fold change. This protein belongs to the GSH dependent glyoxalase I family (Glo1) involved in the detoxification of glyoxal and

Analysis of B[a]P-Exposed Hepatoma Cells

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Figure 2. 2D-DIGE-analysis. (A) Representative 2D-DIGE-gel of murine Hepa1c1c7 cells (here treated with 5 µM B[a]P 24 h, DMSO was used as a control) The 120 protein spots which were found to be significantly regulated over time course are labeled (identification data are shown in Table 1). The protein spots of two representative proteins per discussed pathway were highlighted. The time curves of those are shown in Figure 3. The LabAid Page Ruler Prestained Protein ladder 10-170 kDa was used. The pI values were added relatively according to the pIs of the identified proteins.)B) Protein spots (1227) were detected and quantified using DIGE. Maximum protein expression ratios in Log2 of the 120 regulated proteins species are shown for both B[a]P concentrations. Fifty-seven and 58 protein spots were found to be down-regulated (negative protein numbers); 63 and 62 protein spots were up-regulated (positive numbers) by a treatment with a concentration of 50 nM and 5 µM B[a]P, respectively.

methylglyoxal in physiological systems. Glycation is indirectly linked to oxidative stress since the depletion of GSH and NADPH in oxidative stress also decreases the enzymatic activities of Glo1 family members thereby increasing the concentrations of glyoxal and methylglyoxal and associated glycation reactions. Glycation of proteins by Glo1 substrates may directly increase reactive oxygen species (ROS) by the glycation of mitochondrial proteins via direct electron leakage38 and thereby be a cause or contributory factor to oxidative stress.39 The most prominently described effect of activating the Ahr pathway is the induction of enzymes that metabolize xenobiotics. Accordingly, an active metabolism of B[a]P was found in murine Hepa1c1c7-cells. B[a]P-quinones, produced by peroxidases and by CYP enzymes in combination with dihydrodiol dehydrogenases, can undergo one electron redox cycling with their semiquinone radicals, resulting in ROS formation.40 Previously it has been shown that B[a]P-exposure was found to increase ROS levels in rat liver progenitor cells WB-F344.41 In addition, it was suggested that ligand-activated Ahr mediates oxidative stress through the induction of cytochrome P450 enzymes.6 Thus, B[a]P can induce oxidative stress by various

mechanisms. As a response of the stressed cells, several proteins known to be involved in oxidative stress protection were found to be up-regulated. 3.2.2. B[a]P Leads to Blockage of Transition to G2Phase. Several studies have pointed out the role of Ahr in cell cycle control, even though the exact mechanisms still remain unclear.37,43,44 There is evidence that in the absence of an exogenous ligand, cell cycle progression is promoted through the Ahr,36 while the presence of an exogenous ligand caused the activated Ahr to inhibit cell proliferation and to induce cell cycle arrest. In addition, Ahr-mediated oxidative stress as stated above might play a critical role upstream in the apoptosis cascade.6 Our study sheds more light on this by identifying several proteins involved in cell cycle- and apoptosis-control responding to B[a]P-treatment. Cyclin-dependent kinase 4 (Cdk4) (Figure 3C), calmodulin (CaM) (Figure 3D), nucleophosmin 1 (NPM), annexin 1 (ANXA1) and the eukaryotic translation initiation factor 5A (elF5A) were all up-regulated after 2 or 4 h of B[a]P treatment respectively. Cdk4 expression was up-regulated by a factor of 1.44 (5 µM) and 1.46 (50 nM) at 4 h of B[a]P-incubation (Figure 3C, Table Journal of Proteome Research • Vol. 10, No. 2, 2011 383

384

Early endosome antigen 1 Nonsense mRNA reducing factor 1 NORF1 Eukaryotic translation elongation factor 2 Heat shock protein 105 Ubiquitin-activating enzyme E1 Alanyl-tRNA synthetase Alanyl-tRNA synthetase, cytoplasmic Alpha glucosidase 2 alpha neutral subunit Lon protease homologue, mitochondrial Iron response element binding protein Eukaryotic translation elongation factor 2 Eukaryotic translation elongation factor 2 Lon protease homologue, mitochondrial Heat shock 70 kDa protein 4 L Annexin A1 Far upstream element-binding protein 2 Ezrin Radixin Moesin Heat shock protein HSP 90-alpha Polyadenylate-binding protein 1 Far upstream element-binding protein 1 Chaperonin containing Tcp1, subunit 3 (gamma) Transketolase Plastin-3 Bifunctional purine biosynthesis protein PURH NADP-dependent malic enzyme MKIAA0002 protein p21-activated kinase 2 Chaperonin subunit 8 (theta) Protein disulfide-isomerase A3 60 kDa heat shock protein, mitochondrial T-complex protein 1 subunit alpha A Vimentin Vimentin Leucine aminopeptidase 3 UDP-glucose dehydrogenase Inosine 5′-phosphate dehydrogenase 2 Aldehyde dehydrogenase 7 family, member A1 isoform a Aspartyl-tRNA synthetase Peptidyl-prolyl cis-trans isomerase Retinal dehydrogenase 1 ATP synthase subunit alpha, mitochondrial MNCb-1930 protein; Cytosolic nonspecific dipeptidase Aldehyde dehydrogenase, mitochondrial hypothetical protein LOC433182, EG433182 protein, Alpha enolase

2 3 4 6


41 42 43

28 29 30 31 32 33 34 35 36 37 38 39 40

27

23 24 25 26

12 13 14 15 16 17 18 19 20 21

7 8 9 10 11

identification (Swiss-Prot database)

ID

607 1171 701 1995 1083 1118 1534 483 1704 991 857 2582 877 486 365 2043 494 306 594 472 551 241 2226 375 732 484 709 1091 370 3480 220 895 1355 442 1403 1092 1085 95 1254 970 368 917 728 1675 137 522

Mowse score

160 914 123 917 96,222 97 316 118 931 107 697 106 908 109 335 106 347 98 705 96 222 96 222 105 842 94 382 82 500 79 634 69 478 68 599 67 839 73 528 70 826 68 668 61 162 68 272 71 210 64 687 64 426 60 817 58 179 60 088 56 678 59 388 60 952 51 590 51 590 56 549 55 482 55 780 59 337 64 944 51 939 55 037 59 830 53 162 53 296 47 111

MW [Da]

Table 1. Identified Protein Spots of Differentially Expressed Proteins Following Exposure to B[a]Pa

5.78 6.24 6.41 5.39 5.43 5.41 5.67 5.75 6.15 7.23 6.41 6.41 6.57 5.75 7.22 9.04 5.83 5.84 6.22 6.21 9.52 7.74 6.28 7.23 5.42 6.3 6.87 5.44 5.57 5.44 6.21 8.08 5.82 4.96 4.96 7.14 7.49 7.28 7.16 6.03 5.54 7.91 9.22 5.43 7.61 6.81

pI

18 36 32 50 29 36 45 34 33 35 35 43 30 26 15 47 25 18 37 40 28 15 51 26 37 39 40 50 42 57 53 48 51 79 56 45 46 35 37 52 22 38 32 48 19

sequence coverage [%]

34 49 33 81 42 40 51 17 66 47 47 132 33 24 12 76 15 12 21 22 17 8 28 13 21 64 19 23 87 23 25 18 23 45 27 20 22 15 19 26 8 16 12 15 9 18

peptide hits

Q8BL66 Q9EPU0 P58252 Q61699 Q02053 Q8BGQ7 Q8BGQ7 Q8BHN3 Q8CGK3 P28271 P58252 P58252 Q8CGK3 P48722 P10107 Q3U0V1 P26040 P26043 P26041 P07901 P29341 Q91WJ8 Q3U4U6 P40142 Q99K51 Q9CWJ9 P06801 Q6A0F1 Q8CIN4 P42932 P27773 P63038 P11984 P20152 P20152 Q9CPY7 O70475 P24547 Q3TFC7 Q8BIP0/Q922B2 P30416 P24549 Q03265 Q9D1A2 P47738 P17182

Swiss-Prot ID

research articles Dautel et al.

Enolase 3, beta muscle isoform 1 Actin-like protein 6A Enolase 3, beta muscle isoform 1 Alpha-enolase DnaJ (Hsp40) homologue, subfamily A, member 1 Fumarate hydratase 1 NSFL1 cofactor p47 Methylosome protein 50 Adenylosuccinate synthetase Isocitrate dehydrogenase 1 (NADP+), soluble Calcium-binding mitochondrial carrier protein SCaMC-1 Glyoxalase domain-containing protein 4 Chloride intracellular channel protein 1 Sorbitol dehydrogenase Vimentin Ribosomal protein, mitochondrial, S22 Proteasom, 26S subunit, non-ATPase, 7 Histidine triad protein member 5 COP9 signalosome subunit 5 Uroporphyrinogen decarboxylase Serine/threonine-protein phosphatase PP1-alpha catalytic subunit Transaldolase 1 Heterogeneous nuclear ribonucleoprotein A3 Nucleophosmin 1 Aldose reductase Small glutamine-rich tetratricopeptide repeat (TPR) containing protein Delta-aminolevulinic acid dehydratase Eukaryotic translation initiation factor 3, subunit J Capping protein (actin filament) muscle Z-line, alpha 2 Annexin A1 Inorganic pyrophosphatase 2, mitochondrial Eukaryotic translation elongation factor 1 delta isoform b Thioredoxin-related protein unnamed protein product Inorganic pyrophosphatase 2, mitochondrial Chromatin modifying protein 4B Biliverdin reductase A Clathrin, light polypeptide A isoform d Esterase D/formylglutathione hydrolase Ts translation elongation factor, mitochondrial Esterase D/formylglutathione hydrolase Guanine nucleotide-binding protein subunit beta-2-like 1 Guanine nucleotide-binding protein subunit beta-2-like 1 Haloacid dehalogenase-like hydrolase domain containing 2 isoform 1

44 45 46

70 71 72 74 75 76 77 78 79 80 81

64 65 66 67 68 69

60 61 62 63

58 59

53 54 55 56 57

52

50 51

47 48 49


ID

Table 1. Continued

36 456 29 440 33 118 38 734 38 114 31 387 32 630 33 101 38 115 24 921 33 525 23 521 31 870 35 711 33 720 35 511 35 453 28 883

37 534 39 652 32 711 36 052 34 529

302 623 276 153 105 201 104 1096 542 339 970 1201 1347 550 172 901 100 180 893 230 238 449 480

47 337 47 448 47 337 47 453 44 868 54 564 40 710 36 943 49 990 47 044 53 096 33 317 27 072 38 795 51 590 41 281 36 574 38 988 37 753 40 951 38 257

MW [Da]

1264 634 1234 2348 679 122 562 356 290 598 389 1452 533 67 1081 818 904 663 811 111 480

Mowse score

6.32 4.69 5.57 7.38 6.98 4.96 4.9 5.49 6.98 4.76 7.02 4.43 6.7 6.62 8.27 7.6 8.08 5,7

6.57 9.02 4.62 6.71 4.99

6.73 5.61 6.73 6.37 7.08 9.12 5.14 5.27 6.39 6.73 7.02 5.28 5.09 6.56 4.96 8.63 5.91 6.48 6.54 6.21 5.94

pI

45 24 44 38 41 46 55 58 60 33 36 27 17 55 28 38 52 29

39 31 32 29 12

12 40 23 50 56 7 48 37 34 38 34 64 54 21 57 44 51 57 42 29 37


16 6 46 15 22 28 36 78 30 6 64 6 4 35 8 9 11 25

21 19 11 7 3

4 43 6 10 37 3 40 19 14 11 10 59 22 6 65 32 46 24 37 11 17

peptide hits

P10518 Q3UGC7 P47754 P10107 Q91VM9 P57776 Q8CDN6 Q3UA53 Q91VM9 Q9D8B3 Q9CY64 B1AWE0 Q9R0P3 Q9CZR8 Q9R0P3 P68040 P68040 Q3UGR5

Q93092 Q8BG05 Q61937 P45376 Q8BJU0

P21550 Q9Z2N8 P21550 P17182 P63037-1 P97807 Q9CZ44 Q99J09 P46664 Q5HZJ8 Q8BMD8 Q9CPV4 Q3TIP8 Q64442 P20152 Q9CXW2 P26516 Q9DAR7 O35864 P70697 P62137

Swiss-Prot ID


research articles

Journal of Proteome Research • Vol. 10, No. 2, 2011 385

386


a

NmrA-like family domain containing 1 Glyoxalase domain-containing protein 4 Cyclin-dependent kinase 4 Glyoxalase domain containing 4 Purine-nucleoside phosphorylase 1 Nit protein 2 Uridine-cytidine kinase 2, isoform CRA_b Proteasome (prosome, macropain) 28 subunit, alpha Platelet-activating factor acetylhydrolase IB subunit beta Protein FADD Phosphoglycerate mutase 1 Phosphoglycerate mutase 1 Calcyclin binding protein Proteasome subunit alpha type-4 14-3-3 protein zeta/delta Calcyclin binding protein Calcyclin binding protein Phosphoglycerate mutase 1 Phosphoglycerate mutase 1 Phosphomannomutase 2 Peroxiredoxin-6 Pre-mRNA-splicing factor SPF27 Peroxiredoxin-6 Ribose 5-phosphate isomerase GTPase Ran Glutathione S-transferase, pi 1 Glyoxalase 1 Sod2 protein Heat shock protein HSP 90-beta Actin, aortic smooth muscle Peroxiredoxin-6 Chromobox homologue 3-modifier 2 Myosin light chain, regulatory B-like Heat shock protein HSP 90-beta Actin, aortic smooth muscle Peptidylprolyl isomerase A Eukaryotic translation initiation factor 5A Stathmin Heat shock protein HSP 90-beta Calmodulin Eukaryotic translation elongation factor 2

82 83 84 85 86 87

Spot numbers 1, 5, 73, 107, 111, 117, 119, and 120 were not identified.

112 113 114 115 116 118

108 109 110

100 101 102 103 104 105 106

93 94 95 96 97 98 99

90 91 92

88 89


ID

Table 1. Continued

1391 1452 210 1162 150 126 90 97 231 215 752 858 818 781 404 660 287 291 943 462 607 685 2083 749 115 281 102 431 403 281 252 480 493 486 298 103 98 470 292 975 990

Mowse score

34 526 33 581 33 957 33 581 32 527 30 825 26 338 28 826 25 581 22 960 28 928 28 928 26 608 29 737 27 925 28 472 28 472 28 928 28 928 27 981 24 871 26 229 24 871 26 098 24 579 23 765 20 967 24 241 83 325 42 009 24 871 19 911 19 940 83 325 42 009 18 131 12 466 17 274 83 325 16 827 96 222

MW [Da]

6.37 5.31 6.16 5.31 5.78 6.44 6.53 5.73 5.91 6.02 6,67 6,67 7,63 7,59 4.73 7.78 7.78 6,67 6,67 6.01 6.02 5.15 6.02 6.09 7.01 7.68 5.24 8.67 5.04 5.39 6.02 4.96 4.67 5.04 5.39 7.74 5.12 5.97 5.04 4.09 6.41

pI

42 63 38 72 53 26 34 28 26 18 32 39 29 25 41 65 60 61 58 49 75 29 1 38 32 46 44 54 8 14 29 31 51 8 14 52 41 33 8 55 48


56 59 13 48 13 7 7 7 22 10 51 55 66 42 13 35 14 16 31 23 23 6 110 28 9 11 11 23 24 36 14 10 13 33 43 10 4 24 28 28 79

peptide hits

Q8K2T1 Q9CPV4 P30285 Q9CPV4 P23492 Q9JHW2 Q99PM9 Q5HZK3 Q61206 Q61160 Q9DBJ1 Q9DBJ1 Q9CXW3 Q9R1P0 P63101 Q9CXW3 Q9CXW3 Q9DBJ1 Q9DBJ1 Q9Z2M7 O08709 Q9D287 O08709 P47968 P62827 P19157 Q9CPU0 P09671 P11499 P62737 O08709 P23198 Q3THE2 P11499 P62737 P17742 P63242 P54227 P11499 P62204 P58252

Swiss-Prot ID

research articles Dautel et al.

research articles


Table 2. Time- and B[a]P-Concentration-Dependent Protein Regulation of 21 Identified Proteins Grouped to Their Biological Functions that Were Discussed

ID

protein involved in process

benz(a)pyrene exposure 50 nM Fold change regulation

benz(a)pyrene exposure 5 µM Fold change regulation

Swiss-Prot acc. No.

2h

24 h

2h

4h

12 h

24 h

P

FDR

1.30 0.72 0.73 0.92

0.91 0.80 1.48 1.62

0.86 0.96 0.91 0.75

0.92 1.20 1.01 1.12

0.84 1.01 1.53 1.33

0.75 0.80 1.32 1.73

0.0028 0.0030 0.0000 0.0044

0.0343 0.0343 0.0113 0.0406

0.28 0.88 0.52 1.15 3.30 1.19 2.32

0.65 1.54 3.04 1.52 0.66 1.44 1.71

1.31 1.28 1.41 0.77 1.16 1.00 0.88

1.23 1.42 1.10 0.89 0.54 0.94 0.85

0.0000 0.0059 0.0014 0.0021 0.0006 0.0030 0.0021

0.0113 0.0496 0.0260 0.0308 0.0204 0.0343 0.0308

1.51

1.04

2.97

1.09

1.86

0.0001

0.0133

remodeling 1.09 0.90 1.22 0.86 1.05 0.89

1.06 1.26 0.80

0.73 1.06 0.50

1.13 1.36 1.14

0.75 0.69 0.71

0.0018 0.0030 0.0023

0.0289 0.0343 0.0319

85 100 103 105

Glyoxalase domain containing 4 Peroxiredoxin-6 Glutathione S-transferase, pi 1 Sod2 protein

Q9CPV4 O08709 P19157 P09671

25 30 45 61 67 84 113

Plastin-3 60 kDa heat shock protein Actin-like protein 6A Nucleophosmin 1 Annexin A1 Cyclin-dependent kinase 4 Eukaryotic translation initiation factor 5A Calmodulin

Q99K51 P63038 Q9Z2N8 Q61937 P10107 P30285 P63242

116 17 18 19 32 56 88

20 31 93

Ezrin Radixin Moesin

12 h

oxidative stress 1.01 0.80 0.90 1.12 0.89 1.60 0.98 1.40

cell cycle control and apoptosis 0.29 1.03 1.15 1.13 0.91 1.17 1.22 1.36 1.02 4.21 1.37 1.71 1.42 2.44 0.95 0.94 1.97 0.78 1.09 0.89 1.10 1.46 1.10 0.86 2.39 1.24 0.90 0.84

P62204

0.93

P26040 P26043 P26041

cytoskeleton 0.98 0.84 1.12 1.15 0.94 0.60

proteolytic maintenance machinery Vimentin P20152 Proteasom, 26S subunit, P26516 non-ATPase, 7 Proteasome (prosome, macropain) Q5HZK3 28 subunit, alpha Heat shock protein HSP 90-alpha T-complex protein 1 subunit alpha A 14-3-3 protein zeta/delta

4h

2.67

1.10

1.03 0.52

2.22 1.47

0.80 1.08

0.85 0.72

1.04 0.51

2.77 1.77

0.82 0.94

0.96 0.41

0.0028 0.0035

0.0343 0.0369

1.02

0.92

1.16

1.02

0.91

0.93

1.20

0.91

0.0002

0.0133

P07901 P11984

Ran/Raf signaling pathway 0.86 1.11 1.05 1.15 0.89 1.23 1.06 0.77

0.75 0.85

1.08 1.17

1.05 1.10

1.15 0.87

0.0014 0.0042

0.0260 0.0404

P63101

0.84

0.85

0.92

1.15

0.94

0.0045

0.0414

0.90

2). In mammalian cells, the cyclin D-cdk4/6 complex is activated by the mitogenic signals that release cells from the quiescent or G0 arrested state and thus appears specifically involved in the G0 to G1 phase transition.42 The role of CaM, a principal Ca2+-sensor in eukaryotes, as a regulator of cell cycle progression has also been recognized.43 The discovery of a functional protein-protein interaction between the late G1active cyclin E1 and the major calcium signal-transduction factor CaM supports a direct role for CaM in mediating Ca2+sensitive cyclin E/CDK2 activity and G1 to S phase transitions in vascular smooth muscle cells.44 CaM levels increased by 2.97and 2.67-fold for 5 µM and 50 nM at 4 h incubation, respectively (Figure 3D, Table 2). B[a]P at toxic concentration induces an accumulation of cells in the S-phase (Figure 4), which was also shown by Solhaug et al. for Hepa1c1c7 cells at 0.5, 2.5, and 10 µM of B[a]P for 25 h.29 This effect may be caused by a lack of cell cycle arrest in the G1 phase.45 BPDE, a metabolite of B[a]P, forms a covalent bond with DNA, RNA or proteins.46,47 As a consequence, cells may replicate with DNA adducts resulting in a reduced DNA synthesis rate and an accumulation of cells in the S-phase.29 Our results on the protein level support these previous observations indicating an enhanced accumulation of cells in G1 and S-phase. To confirm the influence of B[a]P on the cell cycle in our model, fluorescence-activated cell sorting (FACS) of propidium iodine stained cells was performed (Figure 4). The analysis of the progression of Hepa1c1c7-cells under B[a]P-exposure

1.24

0.94

through the cell cycle showed no effect for the subacute concentration of B[a]P (50 nM). However, 5 uM B[a]P clearly led to an accumulation of cells in the S-phase (24 h: 117%, 48 h: 197% of control), while the G0/G1 as well as the G2/M population decreased (24 h, 90%/83%; 48 h, 64%/45% of control) compared to DMSO control measurements. These results point to an S-phase arrest and thus a lack of S to G2 transition. 3.2.3. B[a]P Decreases Antiapoptotic and Increases Proapoptotic Responses. The detected changes indicated an enrichment of proteins that affect apoptosis in a 2-fold way, first by a decrease of antiapoptotic and second by an increase of proteins with proapoptotic activities. Several proapoptotic proteins were detected with increased expression levels such as Nucleophosmin 1 (Figure 3E), Annexin A1 (Figure 3F) and eukaryotic translation initiation factor 5A. Nucleophosmin 1 (NPM) reached its maximum expression level increase after 4 h treatment with 5 µM (1.52) and 50 nM (2.44) B[a]P (Figure 3E, Table. 2). NPM was also found to be significantly up-regulated in B[a]P-treated Jurkat T-cells.9 This protein plays a role in the antiapoptotic effects of protooncogenes (ras), it interacts with the ADP-ribosylation factor (ARF) tumor suppressor protein, and inhibits cell cycle progression.48 Increased annexin A1 (ANXA1) levels (3.30 for 5 µM and 1.97 for 50 nM B[a]P) were observed for 2 h B[a]Pincubation (Figure 3F). ANXA1 was first described as a steroidregulated protein and thus implicated in some of the beneficial actions of glucocorticoids, including inhibition of cell proliferaJournal of Proteome Research • Vol. 10, No. 2, 2011 387

research articles

Dautel et al.

Figure 3. Time curves of 14 selected regulated proteins which functionally belong to the 7 discussed pathways that were found to be cellular response mechanisms (Table 2). The log2 ratio (treated/control) for both concentrations (50 nM and 5 µM) for the 5 different time points is plotted for each of the triplicate measurements. Error bars indicate the 5th and 95th percentiles of bootstrapped, interpolated time courses (1000 bootstrap replicates). All proteins had a significant (FDR < 0.05) time effect in a 2-way ANOVA model with time and B[a]P concentration as factors.

tion, anti-inflammatory effects, the regulation of cell differentiation, and membrane trafficking.49,50 It is now known to have an antiproliferative effect by a mechanism that involves the induction of aberrant cytoskeletal organization along with a down-regulation of cyclin D1 protein expression.51 The eukaryotic translation initiation factor 5A (eIF5A) showed increased expression levels at the earliest time point with 2.32 for 5 µM and 2.39 for 50 nM B[a]P (Table. 2). It was originally described as a translation initiation factor; however, it has been demonstrated to be involved in cell proliferation and apoptosis (through a direct up-regulation of p53).52 Thus, increased levels of eIF5A could point to an indirect proapoptotic signal initiated by cellular B[a]P-exposure. One reason for the multifactorial processes might lie in the direct interaction between the Ahr and the RB/E2F complex. RB is phosphorylated upon CDK2 activity for cells to enter the S-phase. This RB-phosphorylation in G1-phase is blocked through a direct complex formation of Ahr with the hypophosphorylated RB protein.53,54 Another explanation may be the induction of p53 accumulation by reactive B[a]P-metabolites, which was translocated to the nucleus. Furthermore, proapoptotic signals such as the translocation of the protein Bax to the mitochondria, the reduction of antiapoptotic Bcl-xl and activation of proapoptotic caspase-3 were observed.27 Bcl-2 proteins recruit mitochondria into the apoptotic pathway. Treatment with B[a]P, B[a]P-7,8-DHD or BPDE resulted in a down-regulation of Bcl-xl, thus leading to apoptosis.55 However, it has been demonstrated in earlier studies, that B[a]P as well as its metabolites appear to induce both apoptotic 388


as well as antiapoptotic signals in Hepa1c1c7 cells.29 Reduced plastin-3 (T-plastin) levels were found at 2 h B[a]P-exposure (5 µM: 0.28, 50 nM: 0.29 fold change) (Figure 3G, Table. 2). Plastins belong to a well conserved class of actin-bundling proteins. Besides their role in actin-binding, plastins are involved in the activation of hematopoietic cells, in cytoskeletal rearrangements during bacterial invasion, in the cellular response to DNA-damaging agents and toxins and cancerogenic growth.56 Reduced levels of T-plastin, as observed B[a]Pdependent in this study, were associated with a decrease in radiation-induced G2 arrest in CHO-cells indicating a correlation between T-plastin expression and G2/M-cell cycle control.57 Furthermore, it has been demonstrated that upon TCDDdependent Ahr-activation, the transcription of plastin genes is diminished.36 In addition, a large up-regulation was found for the Actin-like protein 6A (BAF53a) with 3.04 (5 µM) and 4.21 (50 nM) fold change at 4 h B[a]P-treatment together with the 60 kDa heat shock protein (Hspd1) (1.54 for 5 µM and 1.17 for 50 nM B[a]P) (Figure 3H, Table 2). BAF53, a component of chromatin remodeling and histone acetyltransferase complexes, functions on p53-mediated transcription through a direct interaction of BAF53 and p53 which lead to the decrease of p53-binding ability to p53-response elements (PREs) and thus inhibits cellular apoptosis.58 Hspd1 (or Hsp60) belongs to a heterogeneous group of heat shock proteins (Hsp) with a variety of functions, many of which are related to stress response and protein folding. In addition, elevated levels of Hsp60 in tumor cells have been linked to the ability to survive apoptotic stimuli, loss of replicative senescence and uncontrolled proliferation.59


research articles

Figure 4. FACS cell cycle analysis. For determination of the cell cycle status, Hepa1c1c7 cells incubated with 50 nM, 5 µM B[a]P or DMSO for either 24 or 48 h were stained PI and analyzed with FACS. No effect for 50 nM B[a]P is detectable whereas 5 µM B[a]P led to an accumulation of cells in the S-phase pointing to an S-phase arrest and thus a lack of S to G2 transition.

In conclusion, the results of time-dependent B[a]P-altered protein expression demonstrate B[a]P-induced cell cycle alterations and contradictory pro- and antiapoptotic signaling. The maximum expression level changes on the protein level occur between 2 and 4 h of B[a]P-treatment, indicating an immediate response. Thus, our results support previous results obtained with other techniques such as Western blotting, PCR, and flow cytometry.27,29,36 3.2.4. HPRD Network Analysis Reveals Further Cellular Pathways Altered Due to B[a]P Exposure. The previous interpretation of our data was largely based on the enrichment of functional categories among those proteins that changed their levels significantly. Such analysis, however, does not take into account interactions among the proteins, that is, binding of responding proteins is not completely captured in functional annotations such as the Gene Ontology. Thus, a network analysis was performed to gain further insight into the relation of the identified B[a]P-regulated proteins. Protein-protein binding information was taken from the Human Protein

Figure 5. Human protein reference database (HPRD) network analysis. (A) Observed and random distributions of shortest paths between proteins in the HPRD physical interaction network. A significant deviation between the regulated distance distribution and the overall distance distribution was observed. (B) Regulated proteins (colored in gray) and not observed proteins (white) that were experimentally demonstrated to be involved in physical protein-protein interactions were included. One component with 38 nodes (17 of which were regulated proteins) was observed if only a single not regulated protein in between the regulated proteins was allowed. The connected proteins functionally belong to cytoskeleton remodeling (1), proteolytic maintenance machinery (2), and Ran/Raf signaling pathway (3). The nodes contain the human gen name and the identification number (only for regulated proteins).

Reference Database (HPRD) reporting experimentally validated interactions.16 Mouse genes of the identified proteins were mapped to their human orthologs, including proteins with Journal of Proteome Research • Vol. 10, No. 2, 2011 389

research articles multiple identifications for a maximum input. We hypothesized that proteins responding to B[a]P exposure should act in a concerted way, that is, they should be part of small subnetworks that serve specific functions for dealing with B[a]P induced stress.60 To confirm this notion, we compared the distributions of distances between all regulated proteins versus an identically sized random sample from the network. Applying the Kolmogorov-Smirnov test, a significant deviation between the two distance distributions was found, concluding that the regulated proteins tend to be closer in the network than one would expect by chance (Figure 5A). Next, subnetworks responding to B[a]P were extracted while allowing for at most one intermediate protein between two significantly regulated proteins. This procedure identified one particularly large connected component with 38 nodes (17 of which were regulated proteins) and 64 interactions (Figure 5B). Analysis of the genes’ functions participating in this component immediately began to give further insight into the relationships among the regulated proteins. 3.2.5. B[a]P Induces Cytoskeleton Remodeling. First, the proteins ezrin (Ezr), moesin (Msn) and radixin (Rdx) are known as the ERM proteins and were down-regulated as a response to B[a]P. Ezrin was down-regulated at the latest B[a]P-incubation time point of 24 h by 0.75 (5 µM) and 0.90 (50 nM) fold change, while moesin expression was reduced already at 4 h (0.50 for 5 µM and 0.60 for 50 nM B[a]P) (Figure 3I, Table 2). The maximum of radixin expression was reached at 12 h (1.36 for 5 µM and 1.22 for 50 nM), but decreased at 24 h to 0.69 (5 µM) and 0.86 (50 nM) (Figure 3J, Table 2). These proteins link the actin cytoskeleton to the cell membrane, and their dynamics affects cell morphology, motility, trafficking, and signal transduction.61 In the HRPD network, they are connected through a group of cell adhesion proteins such as the intracellular adhesion molecules 1 and 2 (ICAM-1, ICAM-2), Lselectin (Sell), leukosalin (Spn), vascular cell adhesion protein 1 (Vcam1), neutral cell adhesion molecule L1 (L1cam), and the Na+/H+ exchange regulatory cofactor NHE-RF1 and 2 (Slc9a3r1, Slc9a3r1) (Figure 5B). Following exposure to many microbial and/or xenobiotic toxicants, the collective interaction between cells is mediated, in part, by different families of adhesion molecules. Although the role of the Ahr signaling pathway in altering cell morphology and cytoskeleton remodeling has been widely described,62-64 the involvement of the ERM proteins has not been discussed. Treatment of Hepa-1 cells with the Ahr-ligand β-naphthoflavone lead to an increase in microfilament density, possibly caused by an up-regulation of GAPDH-levels,64 resulting in stronger rigidity of the cells. However, Diry et al. postulated that cytoskeleton remodeling triggered in epithelial cells by dioxin or related pollutants leads to an increased interaction with the extracellular matrix while loosening cell-cell contacts and enhancing motility.62 A mechanism involving the Ahrdependent activation of the Jun NH2-terminal kinase (JNK) was proposed. Other studies have demonstrated a modulation of the cytoskeleton by B[a]P- or BPDE treatment at the protein level. Several keratins have been reported to be modulated in human amniotic FL cells as a response to BPDE.11 Furthermore, in B[a]P-treated Jurkat T-cells the expression of two cytoskeleton proteins, (actin-related protein (actr1b) and capping protein (capzb)), was reduced.9 3.3. B[a]P Leads to an Increased Activity of the Proteolytic Maintenance Machinery. In addition to ERM-proteins connected to cellular adhesion proteins within the identified 390


Dautel et al. HPRD network component, the proteasomal subunits proteasome 26S subunit, non-ATPase 7 (Psmd7) and proteasome (prosome, macropain) 28 subunit, alpha (Psme1) are linked to the rest of the proteins through vimentin (Figure 5B). Vimentin is a protein that makes up intermediate filaments in the cytoskeleton, which function as sustainers for mechanical and nonmechanical stresses and thus provide structural support. In addition, they operate in a tissue-specific manner in cell signaling, stress response and apoptosis.65 Psmd7 expression at 2 h B[a]P-exposure was down-regulated by 0.51 (5 µM) and 0.52 (50 nM), respectively, but increased at 4 h to 1.77 (5 µM) and 1.47 (50 nM) (Figure 3K, Table 2). Psme1 appeared slightly induced at 12 h (1.20 for 5 µM and 1.16 for 50 nM B[a]P) (Figure 3L, Table 2). The expression of vimentin was multiply identified but showed a maximum of induction at 4 h to 2.77 (5 µM) and 2.22 (50 nM). B[a]P-induced oxidative stress and protein-adducts formed by reactive B[a]P-metabolites can cause a continuous formation of (oxidatively) damaged proteins within cells. Besides other functions like transcription regulation, DNA repair or chromatin remodeling, one of the main roles of the proteolytic machinery is the prevention of the accumulation of oxidized cellular proteins and the selective destruction of misfolded or otherwise damaged proteins.66 Aggregates of misfolded proteins from environmental stress are transported and removed from the cytoplasm by dynein motors via the microtubule network to a novel organelle termed aggresome for further processing.67 One of its known components is vimentin.68,69 In contrast to our results, Shen et al. reported the down-regulation of vimentin and the two proteasome 20 S subunits Psmb3 and Psmb4 in human amniotic epithelial FL cells in response to BPDE.10 However, while misfolded ubiquitinated proteins are degraded by the 26 S proteasome, the elimination of oxidatively denatured proteins is mainly carried out by the 20S proteasome,70 which might explain their differently reported regulation in response to B[a]P or BPDE treatment. However, the downregulation of the 20S proteasome can result in cell death as aggregated proteins are toxic,71 which could be another possible reason for the increased cytotoxicity observed with the NR, AB, and LDH-assay. 3.4. B[a]P Treatment Affects the Ran/Raf Signaling Pathway. Finally, the HPRD-network analysis also showed changes in the Ran/Raf- growth signaling cascade. Cross-talk of mitogen-activated protein kinases with the ligand activated Ahr has been reported previously.36 TCDD, another PAH representative and Ahr-ligand, contributes to tumor promotion by stimulating Raf-1 (aka c-Raf). In lung tumorigenesis, the promotion of N-nitrosomethylamine-initiated lung adenocarcinomas in mice by TCDD is chaperoned by a positive role for Raf-1.72 Raf-1 is known to exist as a complex of several chaperone/ adaptor proteins, several of which are regulated proteins (Cct3, Hsp90aa1, Ywhaz).73 T-complex protein 1 (Cct3) expression was increased at 12 h by 1.39 (5 µM) and 1.28 (50 nM) (Figure 3M, Table. 2). Reduced levels of heat shock protein 90-alpha (Hsp90aa1) were observed with 0.75 (5 µM) and 0.86 (50 nM) at 2 h exposure time. In addition, a large increase of the 14-3-3 protein epsilon (Ywhaz) in concentration was observed at 2 h B[a]P-incubation with 5.53 (5 µM) and 7.63 (50 nM) (Figure 3N, Table. 2). 14-3-3 proteins in general are involved in cell signaling, regulation of cell cycle, intracellular trafficking and the organization of the cytoskeleton. Specifically 14-3-3 protein epsilon, together with 14-3-3 protein zeta, play an

Analysis of B[a]P-Exposed Hepatoma Cells active role in the S-phase DNA damage response and in the G2/M cell cycle checkpoint.74,75 Taken together, the network analysis revealed a complex network of identified proteins with B[a]P-affected expression patterns in connection to other proteins not identified in this study (Figure 5B). The recurring themes seem to be cytoskeleton, proteasome, cytotoxicity and signal transduction proteins. However, apart from the selected discussed apertures, many more conclusions on the cellular effects of B[a]P could be drawn with further insight into the displayed network of identified B[a]P-altered proteins and other proteins connected to them.

4. Concluding Remarks In conclusion, we have used the DIGE-technology to investigate the time- and concentration dependent global transformation in protein expression attributable to B[a]P exposure in murine Hepa1c1c7 cells. The results indicate an early response on the protein level within 4 h, without a significant concentration effect. The fact that the early response of the investigated proteins is independent of the B[a]P concentrations implies a robust cellular response to B[a]P exposure. Several proteins were altered that point to B[a]P-induced oxidative stress and alterations in the cell cycle progression and apoptotic behavior of the cells. Additionally, assessing physical binding of proteins revealed a network of connected B[a]Pregulated proteins linking intermediate filaments to the proteasome.

Acknowledgment. We thank Yvonne Kullnick, Antje Thonig, Dan Feng, Anna Lohse, and Kerstin Krist for cooperation and technical assistance. The project was supported by the Helmholtz-Alliance on Systems Biology and by Helmholtz Impulse and Networking Fund through Helmholtz Interdisciplinary Graduate School for Environmental Research (HIGRADE). A.B. acknowledges funding from the Klaus Tschira Foundation. Supporting Information Available: Supplemental figure and table. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Polynuclear aromatic compounds. General remarks on the substances considered. IARC Monogr. Eval. Carcinog. Risk Chem. Hum. 1983, 32, 33-91. (2) Miller, K. P.; Ramos, K. S. Impact of cellular metabolism on the biological effects of benzo[a]pyrene and related hydrocarbons. Drug Metab. Rev. 2001, 33 (1), 1–35. (3) Barhoumi, R.; Mouneimne, Y.; Ramos, K. S.; Safe, S. H.; Phillips, T. D.; Centonze, V. E.; Ainley, C.; Gupta, M. S.; Burghardt, R. C. Analysis of benzo[a]pyrene partitioning and cellular homeostasis in a rat liver cell line. Toxicol. Sci. 2000, 53 (2), 264–70. (4) Weyand, E. H.; Bevan, D. R. Benzo(a)pyrene disposition and metabolism in rats following intratracheal instillation. Cancer Res. 1986, 46 (11), 5655–61. (5) Sun, J. D.; Wolff, R. K.; Kanapilly, G. M. Deposition, retention, and biological fate of inhaled benzo(a)pyrene adsorbed onto ultrafine particles and as a pure aerosol. Toxicol. Appl. Pharmacol. 1982, 65 (2), 231–44. (6) Nebert, D. W.; Roe, A. L.; Dieter, M. Z.; Solis, W. A.; Yang, Y.; Dalton, T. P. Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis. Biochem. Pharmacol. 2000, 59 (1), 65–85. (7) Hankinson, O. The aryl hydrocarbon receptor complex. Annu. Rev. Pharmacol. Toxicol. 1995, 35, 307–40. (8) Gao, Z.; Jin, J.; Yang, J.; Yu, Y. Zinc finger proteins and other transcription regulators as response proteins in benzo[a]pyrene exposed cells. Mutat. Res. 2004, 550 (1-2), 11–24.

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