Proteome Analysis of Bronchoalveolar Lavage in Individuals from

Dec 23, 2008 - Department of Pneumonology, Medical School, University of ... Medicine and Immunological Sciences, University of Siena, Siena, Italy...
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Proteome Analysis of Bronchoalveolar Lavage in Individuals from Metsovo, Nonoccupationally Exposed to Asbestos Dimitra T. Archimandriti,† Yotanna A. Dalavanga,‡ Riccardo Cianti,§ Laura Bianchi,§ Carmen Manda-Stachouli,† Alessandro Armini,§ Anna-I. I. Koukkou,⊥ Paola Rottoli,| Stavros H. Constantopoulos,† and Luca Bini*,§ Department of Pneumonology, Medical School, University of Ioannina, Ioannina, Greece, Laboratory of Anatomy, Medical School, University of Ioannina, Ioannina, Greece, Functional Proteomics Laboratory, Department of Molecular Biology, University of Siena, Siena, Italy, Laboratory of Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece, and Respiratory Diseases, Department of Clinical Medicine and Immunological Sciences, University of Siena, Siena, Italy Received May 21, 2008

Inhabitants of Metsovo, NW Greece, have been exposed to an asbestos whitewash, resulting in malignant pleural mesothelioma (MPM) and pleural calcifications (PCs). Interestingly, those with PCs (PC+) are less prone to MPM. They also have lymphocytic alveolitis, and differences in bronchoalveolar lavage (BAL) proteins, compared with those without pleural calcifications (PC-). This may mean a different response to the fiber leading to different susceptibility to neoplasia. To further evaluate this, a proteomic analysis of BAL proteins was performed. Proteomic analysis (2D-electrophoresis/Mass Spectrometry) of BAL in Metsovites nonoccupationally exposed to asbestos revealed increased albumin fragments, R1-antitrypsin, S100-A9 and HSP27, suggesting ongoing inflammation. In those without pleural calcifications, increased expression of acid ceramidase, glutathione-S-transferase and presence of calcyphosin, all involved in cell cycle regulation and death as well as in the detoxification of mutagenic and toxic agents, lend further support to our thesis of possible “protection against neoplasia” in Metsovites with pleural calcifications. Keywords: Metsovo lung • asbestos • tremolite • malignant mesothelioma • pleural calcifications • bronchoalveolar lavage • proteomics • two-dimensional electrophoresis • acid ceramidase

Introduction Inhabitants of the Metsovo area in Northwestern Greece (population 5000), have been exposed since childhood to asbestos, through the use of a tremolite containing whitewash.1,2 The material (“luto”) was taken from local soil, crushed into fine powder, boiled and applied to walls. Crushing “luto” released a very high amount of fibers (>200/cm3).3 “Luto” whitewash was used by practically all households until the 1950s when it was gradually substituted by modern materials. Presently, it is completely out of use.3 The result of this exposure was a very high incidence of pleural calcifications (PCs) (47% of the adult population),1 and very high prevalence of malignant pleural mesothelioma (MPM) (300 times higher than expected in the general population) between 1981 and 1985.4 Since “luto” is out of use, we are observing a decline of * To whom correspondence should be addressed. Prof. Luca Bini, Functional Proteomics Laboratory, Department of Molecular Biology, University of Siena, Via Fiorentina 1, 53100 Siena, Italy. E-mail: [email protected]. For all Greek authors, please contact: [email protected]. † Department of Pneumonology, Medical School, University of Ioannina. ‡ Laboratory of Anatomy, Medical School, University of Ioannina. § Department of Molecular Biology, University of Siena. ⊥ Department of Chemistry, University of Ioannina. | Department of Clinical Medicine and Immunological Sciences, University of Siena.

860 Journal of Proteome Research 2009, 8, 860–869 Published on Web 12/23/2008

both PCs and MPM in the last 20 years.3-5 In more recent ongoing studies, we have seen no mesotheliomas after 2001 and no pleural calcifications (in chest CT) or asbestos bodies (in bronchoalveolar lavage, BAL) among Metsovites born after 1967 (S. H. Constantopoulos, unpublished data). Already from the first years of our work we had noticed that most mesotheliomas occurred in Metsovites without PCs.4 Combining this finding with the appearance of lymphocytic alveolitis only among those with PCs, we suggested that PCs and lymphocytic alveolitis may be an indirect evidence of protection against neoplasia.6 The aim of the present study was to further test this hypothesis, using a proteomic approach to perform a complete protein analysis of BAL fluid. BAL is a safe, reproducible, minimally invasive procedure, through which material from the surface of alveoli can be obtained.6,7 Cells and alveolar lining fluid analysis offers a picture of the in situ activity at a specific time frame. There is considerable diversity of the proteins present in BAL fluid and a wide variety of origin of each one of them.8 Even so, the knowledge of the protein composition of the microenvironment in different diseases of the lung can be of great help in the elucidation of pathogenetic mechanisms. Proteomics is a key procedure to evaluate the cell function through the analysis of the presence, the levels, and the post10.1021/pr800370n CCC: $40.75

 2009 American Chemical Society

Proteome Analysis of Bronchoalveolar Lavage translational modifications of a large number of proteins simultaneously, thus, studying the molecular background of a disease or several states of the same disease.9,10 Therefore, we performed a proteomics analysis, consisting of 2D electrophoresis, image analysis and mass spectrometry, in the BAL fluid of Metsovites nonoccupationally exposed to asbestos. Differentially expressed polypeptides could be of extreme importance to evaluate proteins which can be involved in the reaction to fiber exposure and to define the two different types of reactions.

Experimental Procedures Study Population. After written consent, 8 Metsovites and 3 healthy individuals (all males, mean age 55 ( 4.6 nonsmokers: control group) underwent BAL. The study was approved by the ethics committee of the Hospital of the University of Ioannina (subcommittee of the scientific council). None had known pulmonary disease or acute infection and they were all nonsmokers. All Metsovites had used “luto” for decades. Metsovites were divided in two groups. The PC+ group (samples from 4 individuals with pleural calcifications in the chest roentgenogram,1 male and 3 female mean age 54 ( 8) and the PC- group (4 samples without pleural calcifications, 3 male and 1 female mean age 52 ( 19). There were no significant differences between them in years of “luto” use. Bronchoalveolar Lavage (BAL). BAL was performed after informed consent as previously described.11 In brief, after complete endoscopic examination, the fiberoptic bronchoscope (Olympus BF P10) was wedged in a segmental bronchus of the right middle lobe. Six 20 mL aliquots of sterile normal saline buffer were instilled through the biopsy channel, and the fluid was subsequently recovered by gentle aspiration. The first aliquot was kept separately and not analyzed (as considered “bronchial”),6 whereas the other five were pooled and centrifuged and the supernatants were stored at -80 °C until analyzed. Two-Dimensional Electrophoresis. Just before 2D electrophoresis, BAL samples were thawed, dialyzed against several changes in distilled water at 4 °C, lyophilized and then dissolved in lysis buffer (8 M urea, 4% CHAPS, 40 mM Tris base, 65 mM dithioerythritol DTE and trace amounts of bromophenol blue). Protein concentration was determined by Bio-Rad assay according to Bradford. Two-dimensional gel electrophoresis was performed as previously described.12-14 IEF separation was carried out on nonlinear wide-rage immobilized pH gradient (pH 3.5-10), 18 cm long IPG strips (GE Healthcare, Uppsala, Sweden). Sample load was 60 µg of total proteins per strip in analytical runs, and 1000 µg per strip in preparative gels. Runs were carried out using the Ettan IPGphor system (GE Healthcare) and performed at 16 °C, until reaching a total of 80 000 V/h for analytical and 110 000 V/h for preparative gels. For analytical runs, sample was applied directly on the rehydration solution. For preparative gels, strips were first swollen overnight in 8 M urea, 2% (w/v) CHAPS, 10 mM DTE and 0.8% carrier ampholytes (Resolyte pH 4-8; BDH, Poole, U.K.) and trace of bromophenol blue. IEF was performed by applying the samples with the sample cup system at the cathodic side of IPG strips. After electrophoresis, IPG strips were equilibrated for 12 min (in 6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS, 0.05 M TrisHCl, pH 6.8, and 2.0% (w/v) DTE), and then for 5 min in the same urea/SDS/Tris buffer solution except that DTE was replaced with 2.5% (w/v) iodoacetamide. The second-dimensional run was carried out on 9-16% polyacryamide linear gradient gels (18 cm × 20 cm × 1.5 mm) at 40 mA/gel constant

research articles current and 10 °C, until the dye front reached the bottom of the gel. For analytical purpose, gels were stained with ammoniacal silver nitrate, as described.15,16 Electropherogram images were obtained with a computing densitometer (Molecular Dynamics 300S) and processed with Melanie 4 (GeneBio, Geneva Switzerland). Relative spot volumes (%V) (V ) integration of OD over the spot area; %V ) V single spot/V total spots) were used for quantitative analysis in order to decrease experimental errors. The normalized spot volume of all gels from the two groups and controls was averaged and SD was calculated. Reported pI and Mr (Da) values were experimentally determined by comigration with human serum as the internal standard.14 Statistical analysis of protein variation was carried out with the t test of the GraphPad software. All the protein variations reported had a statistical significance less than 0.05 (p < 0,05). Mass-Spectrometry Protein Identification. Protein identification was mainly carried out by peptide mass fingerprinting as previously described.17,18 Electrophoretic spots, obtained from preparative 2D gels run with 1 mg of BAL fluid total proteins, were visualized by MS-compatible silver staining procedure,19,20 manually excised, destained and acetonitrile (ACN) dehydrated. They were then rehydrated in trypsin solution, and in-gel protein digestion was performed by overnight incubation at 37 °C. Each protein digest (0.75 µL) was spotted onto the MALDI target and allowed to air-dry. Then 0.75 µL of matrix solution (saturated solution of R-cyano-4-hydroxycinnamic acid in 50% (v/v) ACN and 0.5% (v/v) TFA) was applied to the dried sample, and dried again. MS analysis was performed using an Ettan MALDI-ToF Pro mass spectrometer (GE Healthcare) in reflectron mode with an accelerating voltage of 20 kV. Mass spectra were acquired automatically using the Ettan MALDI Evaluation software (GE Healthcare). Spectra were internally calibrated using the autoproteolysis peptides of trypsin (842.51 and 2211.10 Da). All the resulting mass lists were automatically cleaned up from eventually present contaminant masses, such as those from matrix, autodigestion of trypsin and keratins. Mass fingerprinting searching was carried out in Swiss-Prot/TrEMBL 48.9 and NCBInr 20071013 databases using MASCOT (Matrix Science Ltd., London, U.K., http://www.matrixscience.com) online-available software. The taxonomy was limited to Homo sapiens, a mass tolerance of 100 ppm was allowed and the number of accepted missed cleavage sites was set to one. Alkylation of cysteine by carbamidomethylation was assumed as fixed modification, while oxidation of methionine was considered as a possible modification. The experimental mass values were monoisotopic. No restrictions on protein molecular weight and pI were applied. The criteria used to accept identifications included the extent of sequence coverage, number of matched peptides and probabilistic score sorted by the software, as detailed in Table 1. Tryptic digests that did not produce MALDI-ToF unambiguous identifications were subsequently acidified with 2 µL of a 1% trifluoroacetic acid solution, and then subjected to ESI-Ion trap MS/MS peptide sequencing on a nanospray/LCQ DECA Ion Trap mass spectrometer (Thermo Finnigan, San Jose, CA). With the use of the ZIP-TIP pipet tips for sample preparation (Millipore, Billerica, MA), previously equilibrated in 50% acetonitrile solution and abundantly washed in 0.1% trifluoroacetic acid, acidified samples were enriched. Tryptic peptide elution from the ZIPTIP matrix was achieved with a 70% methanol and 0.5% formic acid solution, and 3 µL of such concentrated sample solutions was then loaded in a distal coated Fused-Silica Picotip emitter to be Journal of Proteome Research • Vol. 8, No. 2, 2009 861

862

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P02768 P06702 gi|149673889 P01009

P00738

P04792 P01009 P02768 P01859

Q96IU4 P02768 P02768 P02768

P02768 P02768 P02768 Q13510 P02768 P09211

Q13938 P02787

P02768 P02768

22 21 16 8

5

13 7 2 4

15 12 6 10

19 11 9 23 14 20

17 18

3 1

Serum Albumin Serum Albumin

Calcyphosin Serotransferin

Serum Albumin Serum Albumin Serum Albumin Acid ceramidase Serum Albumin Glutathione-S-transferaseP

Abhydrolase domain(AB14B) Serum Albumin Serum Albumin Serum Albumin

Heat shock protein β-1 Alpha-1-antitrypsin Serum Albumin Ig gamma-2 chain

Haptoglobin

Serum Albumin Protein S100-A9 immunoglobulin light chain Alpha-1-antitrypsin

protein name

5.92/71317 5.92/71317

4.74/21068 6.81/79280

5.92/71317 5.92/71317 5.92/71317 7.52/45077 5.92/71317 5.44/23438

5.94/22446 5.92/71317 5.92/71317 5.92/71317

5.98/22826 5.37/46878 5.92/71317 7.66/36489

6.13/45861

5.92/71317 5.71/13291 6.97/23800 5.37/46878

theorical pI/Mr (Da)

35 6 27 18

Cluster 2 5.36/37453 15 5.17/49936 10

10 17 13 22 16 40

39 19 32 15

Cluster 1c 5.88/31834 6 6.21/27283 10 5.44/39950 16 5.50/36178 7 Cluster 1d 5.23/22354 7 5.42/30586 11 5.36/37453 7 5.47/9713 10 5.60/24462 8 5.24/13192 5 PPYTVVYFPVRc 4.46/19224 7 7.20/16182 3

41 17 16 21

27

9 77 36 13

sequence coverage%

Cluster 1b 5.13/41665 12 VTSIQDWVQKc 5.67/26566 8 4.87/38058 7 5.61/50707 8 6.50/54226 5

Cluster 1a 6.15/11068 6 5.78/12166 10 7.35/22644 6 4.93/36950 6

measured pI/Mr (Da)

MASCOT search results no. of matched peptides

171 132

111 43

80 116 91 117 94 63

90 101 198 87

92 95 104 61

106

54 106 89 91

score

0.037 ( 0.02 0.025 ( 0.02

0.00 0.011 ( 0.088

0.055 ( 0.034 0.018 ( 0.012 0.011 ( 0.001 0.047 ( 0.028 0.0127 ( 0.01 0.002 ( 0.001

0.007 ( 0.002** 0.030 ( 0.017** 0.064 ( 0.030* 0.014 ( 0.007*

0.092 ( 0.075* 0.031 ( 0.008** 0.117 ( 0.058* 0.115 ( 0.075**

0.240 ( 0.1**

0.018 ( 0.020 0.064 ( 0.073 0.059 ( 0.035* 0.019 ( 0.008**

%V ( SD PC+

0.051 ( 0.022 0.083 ( 0.042

0.00 0.005 ( 0.005

0.0531 ( 0.04** 0.046 ( 0.018* 0.007 ( 0.007* 0.025 ( 0.009**

0.027 ( 0.023 0.041 ( 0.013 0.0137 ( 0.007 0.055 ( 0.026 0.015 ( 0.006 0.009 ( 0.004

0.014 ( 0.008 0.082 ( 0.011 0.219 ( 0.033 0.067 ( 0.0154

0.023 ( 0.015 0.006 ( 0.004 0.033 ( 0.007 0.026 ( 0.016

0.070 ( 0.030

0.0086 ( 0.007 0.010 ( 0.004 0.025 ( 0.004 0.005 ( 0.02

%V ( SD control

0.113 ( 0.031* 0.090 ( 0.042* 0.029 ( 0.006** 0.160 ( 0.032** 0.051 ( 0.005** 0.072 ( 0.027*

0.016 ( 0.004 0.092 ( 0.023 0.1735 ( 0.06 0.054 ( 0.024

0.051 ( 0.027 0.0136 ( 0.0097 0.021 ( 0.018 0.03 ( 0.015

0.132 ( 0.084

0.018 ( 0.013** 0.054 ( 0.032** 0.048 ( 0.039 0.0136 ( 0.016

%V ( SD PC-

a Spot number match those reported in the BAL reference map of PC+, PC- and control groups, shown in Figure 2. b Accession number in SwissProt/Tremble or NCBInr. c Peptide corresponding to MS/MS analysis. Date are shown for all identified spots that significantly changed in one condition (* p < 0.05, ** p < 0.01). Protein spot are clustered according to their expression profile in the three different conditions.

accession numberb

spot numbera

Table 1. Mass Spectrometry Identified Proteins

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Figure 1. Haematoxylin and Eosin staining of (a) normal BAL, (b) BAL with lymphocytic (in circle) alveolitis. (c) Asbestos body (arrow) in BAL (magnification ×40 in all).

nanosprayed in the mass-spectrometer. The collision energy was set based on the mass of the precursor ions, that are doubly charged, and spectra were acquired using Excalibur software (Thermo). MS/MS database searching was performed by TurboSEQUEST (Thermo) and Mascot MS/MS ion search software (http://www.matrixscience.com) in the Swiss-Prot/TrEMBL 48.9 or NCBInr 20071013 databases. The following criteria were applied: MS accuracy ( 1.2 Da, MS/MS mass accuracy ( 0.6 Da, Peptide precursor charge 2+, monoisotopic experimental mass values, trypsin digestion with one allowed missed cleavage, fixed carbamidomethylation of cysteine, and variable oxidation of methionine. Cluster Analysis. Cluster analysis was carried out using the EPCLUST tool for clustering and analysis of gene-expression data within Expression Profiler, a set of tools for microarray analysis developed at the European Bioinformatics Institute (EBI)21 and available online at the following Web site: http:// ep.ebi.ac.uk/. The data were clustered and visualized as linear correlation based distance (Pearson, centered) and average linkage (average distance UPGMA).

Results BAL Cell Morphology (Haematoxylin & Eosin Staining). Asbestos bodies were recognized in the BAL fluid of all Metsovites (both PC+ and PC-, Figure 1c) but not in the control samples (Figure 1a). The percentage of lymphocytes was higher in the PC+ group (Figure 1b) in accordance to our previous findings6 (data not shown). High Resolution 2-DE, Image analysis and MS. To investigate the protein profile of these three groups, the samples were resolved using 2-D electrophoresis. Four gels were produced for each of the PC+ and PC- samples and three gels for the control group. All the gels were elaborated by MELANIE 4 software. The spots were identified using MALDI-TOF MS

and ESI-IT MS. Protein identification process was carried out in SwissProt/TrEMBL and NCBInr databases. Typical electropherograms from the three groups are shown in Figure 2. More than 1500 spots were detected in each gel. According to the silver staining sensitivity, 60 quantitative and 5 qualitative significant variations were found by the software between the three groups. We considered significant all the quantitative differences with variations of at least two times in spot relative volume %V (Vol ) integration of OD over the spot area: %Vol ) Vol of the single spot divided by the total spot Vol) and with the t test statistical probability less than 0.05 (p < 0.05). Twenty-two quantitative and 1 qualitative variations have been successfully identified by mass spectrometry. They are numbered, and pointed out on the gels reported in Figure 2 as circles. The figure represents the typical protein composition of BAL fluid from PC+, PC- and Control individuals. Cluster Analysis of Quantitative and Qualitative Variations. To highlight the expression profiles of the 23 identified qualitative and quantitative differences found between the three groups (PC+, PC- and control), we performed a cluster analysis based on algorithms developed for DNA microarray expression data analysis. EPCLUST software was used and the results are reported in Figure 3, where the mean of normalized relate intensity (%V) of each difference is plotted against the three tested groups of samples. Two main clusters have been recognized and between them cluster 1 can be subdivided in 4 subclusters. Each of the 5 clusters of proteins revealed by the hierarchical clustering classification is composed of proteins with the same pattern of change among the 3 groups of samples. These clusters represent three main protein expression profiles. Cluster 1a includes 4 protein spots whose expression profiles are similar in both groups of Metsovites (with and without Journal of Proteome Research • Vol. 8, No. 2, 2009 863

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Figure 2

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Figure 2. Silver stained electropherograms of BAL fluid from individuals (A) with pleural calcifications, (B) without pleural calcifications and (C) control. Circles highlight the 23 variations that have been identified by mass spectrometry and reported in Table 1.

pleural calcifications) but different in controls. Nine proteins belong to clusters 1b and 1c with an expression profile similar in the PC- and control groups, with consistent variation in the PC+ patients. Finally cluster 1d represents 8 spot variations that are significantly different in the PC- group compared with the two other groups (PC+ and controls). Protein Identification by Mass Spectrometry. Forty spots, corresponding to major protein variations, were cut from preparative gels, destained, digested with trypsin and subjected to peptide mass fingerprinting and peptide sequencing analyses followed by database searching. MALDI-TOF MS and ESI-Ion Trap MS/MS results allowed the unambiguous protein identification of 23 protein variations (Table 1). Table 1 summarizes all the information obtained on protein identification and quantification. The proteins are specified by numbers, corresponding to that reported in Figure 2. SwissProt accession number and protein name are also included. The comparison between theoretical and measured molecular weight and pI values contribute to confirm the MASCOT search results or to assess the presence of some protein fragments, such as for all the serum albumin spots (spots 1-3, 6, 9-12, 14, 19, 22), acid ceramidase (spot 23) and serotransferin (spot 18). The MASCOT search results are reported in the table, showing the number of experimentally measured peptide masses matching the theoretical ones from Swiss-Prot/TrEMBL or NCBInr entries, the percentage of the protein sequence covered by the matching peptides (sequence coverage), and the probabilistic score. For spot 5 and 20, the table also contains the peptide corresponding to MS/MS analysis. As quantitative

informations, Table 1reports mean and SD of relative volume values from PC+, PC- and control gels, to visualize the different protein expression profiles existing between the three samples. Finally, protein variations are classified in different clusters, as reported in Figure 3, according to their expression profiles in the three groups. Comparison of Results between Metsovites and Controls. Several proteins and protein fragments were detected in increased amounts in all samples of Metsovites (PC+ and PC-), compared with the controls. Of these, Albumin, Protein S100A9 and R1-Antitrypsin were increased to a statistically significant level (Figure 3, cluster 1a). Differential Findings between the Two Groups of Metsovites (PC+, PC-). In the PC- samples, we observed increased amounts of Albumin fragments, Acid Ceramidase fragment, Serotransferin fragment, Glutathione S Transferase P, and calcyphosin, compared to both PC+ and controls (Figure 3, cluster 1d). In this latter case, the PC+ and the control groups did not show any significant intergroup differences. One protein, calcyphosin, was detected only in the PC- group. Interestingly, several protein fragments have been differentially detected in the three groups considered. In particular, it is evident, as shown in clusters 1c and 1d, that PC- individuals have an abundance of albumin proteolysis in comparison to PC+ individuals. Journal of Proteome Research • Vol. 8, No. 2, 2009 865

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Figure 3. Cluster analysis of the identified protein variations in PC+, PC- and controls. Left: Normalized %V mean values of identified qualitative and quantitative differences obtained by %V individually computed in PC+, PC- and control. Each reported %V value is proportional to the intensity of the color, and its corresponding spot identity is annotated on the right of the diagram near the pearson dist aver cluster. Right: For each cluster, normalized %Vol mean values plotted against the three different groups are reported, as well as the total number of spots/cluster (size).

Discussion The inhabitants of Metsovo, on the mountains of North Western Greece, have been nonoccupationally exposed to asbestos through the use of a tremolite-containing whitewash. The type of exposure was rather unique. It was a brief exposure to a very high concentration of fibers (>200/cm3) two or three times a year. The result of this exposure was the presence of pleural calcifications in half of the adult population and a 300fold higher (than expected in nonasbestos exposed population) prevalence of malignant pleural mesothelioma in the area during the 1980s.4 As we mentioned previously, mesotheliomas were much more frequent among Metsovites without PCs.3 This prompted further studies with BAL where a T-lymphocytic alveolitis was observed in the PC+ group.6 A preliminary protein analysis revealed some nonspecific differences between the two groups (PC+ and PC-).22 All these findings suggested a different way of reaction to the asbestos fibers between the two groups, resulting in different susceptibility to neoplasia. 866

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To further address this, a more in depth protein analysis is carried out in the present study through a proteomic analysis of the BAL fluid. This analysis confirmed that there are important quantitative differences between Metsovites and normal controls and also between the two groups of Metsovites, those with and those without PCs. (a) Differential Findings between Metsovites and Controls. Metsovites differ from controls, expressing in higher amounts proteins involved in inflammation (Albumin and albumin fragments, R1-Antitrypsin, S100-A9), although there is no clinical evidence of such an inflammation. The cytotoxicity and carcinogenicity of asbestos have been recognized and studied thoroughly.23-25 There is increasing evidence that generation of reactive oxygen species (ROS) may underlay the cytotoxic and cell-activation reactions seeing after exposure to asbestos and the consequent inflammatory reaction. These radicals may be generated through redox reactions catalyzed by metals on the surface of the fibers.26 Radicals may also be generated after phagocytosis of the fibers by the alveolar and

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Proteome Analysis of Bronchoalveolar Lavage interstitial macrophages and neutrophils which are attracted subsequently, in the attempt of the tissue to eliminate them.27 In our population, the presence of asbestos bodies in the BAL of Metsovites was a constant finding. Therefore, the hypothesis of an ongoing inflammation was expressed early on, but not proven, especially in the population without visible pleural calcifications in the chest roentgenogram and no alveolitis in BAL.6 The findings of the present analysis strengthen this hypothesis. Albumin is a highly soluble protein (MW: 71 000), comprising about 40% of the protein content of extracellular fluid. It functions regulating osmotic pressure and transport of a variety of substances. Moreover, it possesses specific binding sites for copper ions28,29 and has the ability to adhere to asbestos fibers, sometimes covering their entire surface.32 Metal ions as copper and iron are very important in accelerating free radical reactions. It seems that albumin acts as an antioxidant, offering a “buffer” at the site of ROS generation.31-38 Increased amounts of albumin flooding to inflammatory sites offer protection against oxidative damage and the result is an increase in albumin fragments. Our results offer an example of this mechanism. Albumin fragments were detected in increased amounts (more so in the PC- samples), compared with the controls, indicating a chronic oxidative stress which seems to be effectively controlled. Finally, further evidence of the chronic subclinical inflammation caused by the inhalation of asbestos fibers is offered by the increased presence of S100-A9 protein and of Heat Shock Protein β-1 (HSP 27). The S100 proteins are Ca++ binding proteins, expressed on phagocytes39-42 and have been recognized as very good markers of inflammation, as they are overexpressed at sites of inflammation and can indicate even minimal, undetected, inflammatory activity.43,44 The HSPs are stress proteins protecting cells exposed to a variety of noxious agents. Overexpression of HSP27 enhances the survival of cells exposed to death stimuli as asbestos by inhibiting the processing of procaspase 9,45-48 and also by preventing the cytochrome-c mediated caspase activation.49,50 Thus, the overexpression of HSP27 in Metsovites is similarly indicative of chronic inflammation and activation of protective mechanisms. (b) Differential Findings between the Two Groups of Metsovites (PC+, PC-). The PC- samples reveal (compared to PC+) an increased expression of acid ceramidase (AC) and glutathione-S-tranferase (GST). Another protein, calcyphosin, was identified only (in accordance with the detection limit of the silver-staining procedure used), in the PC- group. All these proteins are involved in the regulation of the cell cycle and death as well as in the detoxification of endogenous and exogenous mutagenic and toxic agents. Acide Ceramidase (AC) is the lysosomal enzyme that degrades ceramide into sphingosine and fatty acids.51 Inherited deficiency of AC activity is the cause of Faber Lipogranulomatosis (a lethal disease characterized by accumulation of ceramide in the lysosomes) while elevated levels of AC are present in Alzheimer’s disease.52 There is ample evidence implicating ceramide in apoptotic cell death,53,54 and there is no doubt that it is an essential inducer of apoptosis in certain tissues, like lung and oocytes.54,55 It appears that the AC activity interferes with cell growth regulation. Increased AC activity enhances cell growth through the reduction of ceramide levels.56 In vitro inhibition of the AC activity increases apoptosis,57,58 while overexpression of AC suppresses apoptotic

57

cell death. Thus, enhanced cell growth through increased expression of AC could be a mechanism of increased susceptibility to mesothelioma among Metsovites expressing higher levels, namely, the PC- group. The Glutathione-S-Trasferases (GSTs) are a family of enzymes widely expressed in mammalian tissues and involved in detoxification reactions.58 The different isoenzymes have a broad substrate specificity and catalyze the conjugation of reduced glutathione (GSH) with products of oxidative stress, electrophiles and DNA-reactive intermediates.59 GSTµ class (GSTM) has been shown to be involved in the detoxification of carcinogenic diolepoxide derivatives of PAHs present in tobacco smoke.60 In Finland, an increased risk of mesothelioma has been observed among asbestos-exposed workers lacking the GSTM1 gene.61,62 The GSTP isoenzyme, widely expressed in different human epithelia (including the lung epithelium), has been found to be overexpressed in many tumors and it may be involved in acquired resistance to some anticancer drugs.63,64 GSTs seem to be involved in the regulation of the cell death and apoptosis through the regulation of intracellular glutathione levels. It has been reported that increased levels of GSH mediated in vitro by albumin resulted in down-regulation of nuclear factor kappa b (NF-kB), following stimulation with TNF-R.65 Thus, we consider that the increased expression of GSTP1 observed of the PC- group could result in increased risk of mesothelioma through mechanisms that possibly permit the survival of damaged cells. Calcyphosin is a highly conserved protein involved in cell survival, differentiation and transformation. It is a calciumbinding protein. Calcium ion is an intracellular messenger in many signal transducing pathways related (among other functions) to development and differentiation, to fertilization and to cell death. One property of Ca++ that makes it a highly suitable intracellular messenger is that it can bind tightly to proteins. The EF-hand proteins are calcium-binding proteins. The binding of Ca++ results in conformational changes that enable the protein to stimulate a variety of enzymes, integrating Ca++ signals into specific cellular responses.66-68 Calcyphosin is one of these EF-hand proteins initially identified as a marker of differentiation and a substrate of cAMP dependent protein kinase, in dog thyroid cells.69,70 It has been shown that it is expressed in most tissues and more intensively in lung, colon, testis, placenta and brain.71 Human calciphosin is a highly conserved protein. It shares homology to another ancient conserved EF-hand protein, centrin of green algae, which is a ubiquitous cytoskeletal protein crucial for proper cell division.72,73 Calcyphosin seems to be involved in the crosstalk of cyclicAMP-mediated and IP3 (phosphatidyl inositol)mediated signal transduction pathways.74 The expression of this protein could be involved in mechanisms which enhance cell division and perhaps expansion of even transformed cells, leading to creation of substantial numbers of neoplastic cells. These actions of GSTP1 and AC, which have as result the impairment of detoxification of asbestos fiber and the enhancement of cell survival, may be creating a microenvironment favoring carcinogenicity. In our study, this is expressed as increased susceptibility to mesothelioma preferentially in Metsovites without pleural calcifications.

Concluding Remarks The proteomic analysis of the BAL fluid of Metsovites, nonoccupationally exposed to asbestos, revealed several proteins indicative of an ever going inflammation in the lung Journal of Proteome Research • Vol. 8, No. 2, 2009 867

research articles parenchyma. Proteomic analysis proved that asbestos fibers, as long as they are present (and that is forever), trigger the tissue to react, resulting in an subclinical inflammation. More importantly, the proteomic analysis strengthened our hypothesis of two different types of reaction to the asbestos fiber, by revealing a different protein profile in those with PCs/ alveolitis and those without PCs/alveolitis. The overexpressed proteins in the PC- group are related to regulatory mechanisms which may enhance carcinogenesis. And that is in accordance with the fact that most of mesotheliomas come from that “quiet” group. As perspectives, it would be of interest to investigate if these differences in BAL proteins are expressed in the blood as well. Indeed, we are conducting such a study with Glutathione-STrasferases and Acide Ceramidase. If such studies lead to results similar to the present study, they could offer the identification of possible biomarkers and certainly serum is much easier to obtain than BAL fluid. Another interesting field could be a similar study of BAL and/ or blood in Metsovites with mesothelioma and other malignancies, in order to figure out which of these proteins are indeed crucial for carcinogenesis.

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