Changes in protein expression of pacific oyster Crassostrea gigas ...

Environ Sci Pollut Res DOI 10.1007/s11356-014-3821-8

MOLECULAR AND CELLULAR EFFECTS OF CONTAMINATION IN AQUATIC ECOSYSTEMS

Changes in protein expression of pacific oyster Crassostrea gigas exposed in situ to urban sewage Fabrício Flores-Nunes & Tânia Gomes & Rui Company & Roberta R. M. Moraes & Silvio T. Sasaki & Satie Taniguchi & Márcia C. Bicego & Cláudio M. R. Melo & Afonso C. D. Bainy & Maria J. Bebianno

Received: 15 August 2014 / Accepted: 4 November 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract The composition and concentration of substances in urban effluents are complex and difficult to measure. These contaminants elicit biological responses in the exposed organisms. Proteomic analysis is a powerful tool in environmental toxicology by evidencing alterations in protein expression due to exposure to contaminants and by providing a useful framework for the development of new potential biomarkers. The aim of this study was to determine changes in protein expression signatures (PES) in the digestive gland of oysters Crassostrea gigas transplanted to two farming areas (LIS and RIB) and to one area contaminated by sanitary sewage (BUC) after 14 days of exposure. This species is one of the most cultivated molluscs in the world. The identified proteins are related to the cytoskeleton (CKAP5 and ACT2), ubiquitination pathway conjugation (UBE3C), G proteincoupled receptor and signal transduction (SVEP1), and cell cycle/division (CCNB3). CKAP5 showed higher expression Responsible editor: Philippe Garrigues Electronic supplementary material The online version of this article (doi:10.1007/s11356-014-3821-8) contains supplementary material, which is available to authorized users. F. Flores-Nunes : R. R. M. Moraes : A. C. D. Bainy Laboratory for Biomarkers of Aquatic Contamination and Immunochemistry, Federal University Santa Catarina, Florianópolis, Brazil T. Gomes : R. Company : M. J. Bebianno (*) CIMA, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal e-mail: [email protected] S. T. Sasaki : S. Taniguchi : M. C. Bicego Laboratory of Marine Organic Chemistry, Oceanographic Institute, University of São Paulo, São Paulo, Brazil C. M. R. Melo Laboratory of Marine Molluscs, Federal University of Santa Catarina, Florianópolis, Brazil

in oysters kept at BUC in comparison with those kept at the farming areas, while ACT2, UBE3C, SVEP1, and CCNB3 were suppressed. The results suggest that these changes might lead to DNA damage, apoptosis, and interference with the immune system in oyster C. gigas exposed to sewage and give initial information on PES of C. gigas exposed to sanitary sewage, which can subsequently be useful in the development of more sensitive tools for biomonitoring coastal areas, particularly those devoted mainly to oyster farming activities. Keywords Urban sewage . Crassostrea gigas . Proteomic analysis . Two-dimensional gel electrophoresis

Introduction Urban sewage is one of the major sources of contaminants into the marine and estuarine ecosystems, causing adverse effects in target tissues of exposed organisms, which may reflect changes in the population and community structures (Abessa et al. 2005; Kennish 1992; Martins et al. 2008). Periodically, complex mixtures of solids, nutrients, hydrocarbons, metals, pesticides, pharmaceuticals, and personal care products from sanitary, hospital, gas stations, small industries, and runoffs are discharged in these areas (Abessa et al. 2005; Bolong et al. 2009; USEPA 2009). Many ecotoxicological studies in marine and estuarine environments use bivalve molluscs as sentinel organisms since they are sessile, euryhaline, show wide geographical distribution, and possess filter feeding habits with great capacity to accumulate contaminants (Cajaraville et al. 2000; Beeby 2001). Among bivalve molluscs, the oyster Crassostrea gigas holds a prominent position for being the most cultivated, consumed, and studied marine mollusc in the world (Mao et al. 2006; Saavedra and Bachère 2006). In southern Brazil, as well as in other countries around the world,

Environ Sci Pollut Res

this species was introduced and has been cultivated intensively (EPAGRI 2013), which points to the need of developing tools for monitoring the levels and effects of contaminants in oysters kept in farming zones or other more impacted areas. Contaminants may activate effector cell receptors, which, in turn, interact with specific DNA sequences promoting the transcription of multiple genes. Multiple messenger RNAs (mRNAs) are processed and sent to the cytoplasm for protein synthesis, thus constituting the cellular response (Piña et al. 2007). Therefore, genomic and proteomic techniques are important tools for assessing the global profile of molecular and biological responses in exposed animals and can help in both the development of oyster farming industries (Saavedra and Bachère 2006) and in programs of ecological risk assessment (Kling et al. 2008). Although mRNA sequences could be used to predict the biological processes, proteins are performers of the actual cell response (Rudert et al. 2000). In this context, proteomic analysis reflects the change in protein expression by environmental adaptations induced by pollutants and/or pathological processes (López 2005; Moore 2002) and therefore enables the development of new tools for biomonitoring (Thompson et al. 2012a) to complement transcriptomic studies (López 2005; Pandey and Mann 2000). Several methodologies have been used for proteomic analysis, but the two-dimensional electrophoresis (2DE) on polyacrylamide gels is the most established and used tool (Lemos et al. 2010). The identification of induced and/or inhibited proteins is one way to identify exposure-effect relationships associated to certain stressors in order to propose molecular mechanisms of response (Nesatyy and Suter 2007). “Proteomic maps,” also known as protein expression profiles (PEPs), are groups of specific proteins differentially expressed, reflecting a cellular response elicited by a challenge (Bradley et al. 2002) that can be related to a stressor, as well as recognize potential protein-protein interactions (Rudert et al. 2000) without the need for the identification of proteins (Apraiz et al. 2009; Shepard et al. 2000). Advances in techniques of protein separation and identification have expanded the use of proteomics for application to a large number of sentinel organisms (Veldhoen et al. 2012), providing a better relationship between environmental stress and pollutant toxicity (Campos et al. 2012). This is specially important in the case of bivalve molluscs used as edible resources as oysters C. gigas. The use of proteins as biomarkers has been applied to mollusc farming (López et al. 2002) and for evaluating different environmental impacts such as exposure to toxic algae (Ronzitti et al. 2008), to protozoal infections (Cao et al. 2009), to organic contaminants (Apraiz et al. 2009; Monsinjon et al. 2006; Olsson et al. 2004), to urban effluents (Amelina et al. 2007), to trace metals (Chora et al. 2008; Chora et al. 2009b; Shepard and Bradley 2000; Thompson et al. 2012a), and to nanoparticles (Gomes et al. 2013).

Considering the social and economic importance of bivalve aquaculture in coastal zones near the urban areas, the understanding of the impact of sanitary sewage in such areas is still limited. The aim of this study was to evaluate the PEPs in the digestive gland of C. gigas exposed in the field to urban sanitary sewage discharges.

Materials and methods Experimental design Oysters C. gigas (6–8 cm) were collected in the farming area of the Laboratory of Marine Molluscs (LMM) of the Federal University of Santa Catarina (UFSC) at the Sambaqui beach (SAM; Florianópolis, SC) and transplanted to two oyster farming areas (LIS: Santo Antonio de Lisboa beach, North Bay; RIB: Ribeirão da Ilha beach, South Bay) and to an area impacted by discharges of urban sanitary sewage used as a contaminated site in this study (BUC: Bücheler river mouth) (Fig. 1). After 14 days of exposure, oysters were collected at each site (n=10), digestive glands were dissected, immediately frozen in liquid nitrogen, and stored separately at −80 °C. Analysis of fecal coliforms, detergents, organic matter, oil, and grease in water samples Water samples were collected at 10 cm depth at the beginning (0 h), 24 h, and 14 days of exposure in all sites. Water analysis was performed in the same sampling day by counting coliform bacteria (fecal coliforms, CF), detergents (DTG), organic matter (OM), and oil and grease (O/G) according to standard protocols (Clesceri et al. 1998). Analysis of organic contaminants in sediments and oysters Oysters and sediment samples (one pool of whole oyster tissues, n=8, and 250 g of sediments) were lyophilized for 72 h (Thermo Savant, module D), macerated, and homogenized in mortar with pestle, and stored in glass bottles previously cleaned with solvent. The sediment and tissue samples were freeze-dried and homogenized. An amount of, respectively, 20 and 1 g was Soxhlet-extracted with a 50 % mixture of residue grade nhexane and dichloromethane for 8 h in accordance with UNEP (1992) and McLeod et al. (1986), with some minor modification. Before extraction, PCB 103, PCB 198, n-hexadecene, neicosene, naphthalene-d8, acenaphthene-d10, phenanthrened10, chrysene-d12, perylene-d12, and dodecyl 1-benzene (1C12LAB) were added to all the samples, blanks, and reference material from the National Institute of Standards and Technology (NIST) (SRM 1944 for sediment and SRM

Environ Sci Pollut Res Fig. 1 Sites used for oysters transplantation in the region of Florianópolis. 1 SAM: Sambaqui beach, LMM/UFSC (oysters supplier); 2 LIS: Santo Antonio de Lisboa beach, North bay oyster farming area, North bay; 3 RIB: Ribeirão da Ilha beach, South bay oyster farming area, South bay; 4 BUC: Bücheler river mouth, contaminated site

2974 for oyster) as surrogates. The organochlorine extract was purified in an alumina chromatographic column. The hydrocarbon extracts were fractionated into F1 (aliphatics and linear alkylbenzenes), F2 (polycyclic aromatic hydrocarbons), and F3 (only for sediment, steroids) by silica gel-alumina column chromatography. Fraction 3 was evaporated to dryness, and steroids were derivatized to form trimethylsilyl ethers using bis(trimethylsilyl) trifluoroacetamide (BSTFA) with 1 % trimethylchlorosilane (TMCS) for 90 min at 65 °C. Organochlorine pesticides were analyzed in an Agilent Technologies gas chromatograph in splitless mode using an electron capture detector (GC-ECD). Aliphatic hydrocarbons and steroids were determined on a gas chromatography with flame ionization detector (GC-FID). PAHs, PCBs, and LABs

were analyzed by an Agilent 6890 gas chromatograph coupled to a 5973N mass spectrometer (GC/MS) in a selected ion mode (SIM). Certified standards at five different concentrations were used for calculations. PAH, PCBs, and LABs identification was based on mass/charge ratio of the individual quantitation ion (m/z) and retention times of certified standards. Proteomic analysis The sample preparation as well as the protocol used for twodimensional electrophoresis is described by Chora et al. (2009a). For each study site (LIS, RIB, and BUC), five pools were prepared using two digestive glands each (n = 5)


suspended in a specific buffer (10 mM HEPES and 250 mM sucrose solution) containing 1 mM DTT, 1 mM EDTA, 1 mM PMSF, 10 % of protease inhibitor (protease inhibitor Cocktail Sigma P8340), and homogenized using an Ultra-Turrax homogenizer IKA-Werke on an ice bath at 4 ° C in a 3:1 (buffer/ tissue). The resulting homogenate was centrifuged at 15,000×g for 2 h (4 °C), and the supernatant was stored at −80 °C for later use. The quantitation of total proteins was carried out according to the Bradford method using bovine serum albumin (BSA) as standard (Bradford, 1976). Aliquots of 150 μg of proteins were suspended in protein precipitating solution (1:9; 10 % trichloroacetic acid in cold acetone containing 20 mM DTT) for 2 h at −20 °C, centrifuged (10,000×g; 30 min; 4 ° C), the supernatant discarded, and the pellet washed three times with cold acetone. The residual acetone was removed by air-drying. Two-dimensional electrophoresis Proteins were first separated by isoelectric focusing (IEF; first dimension), followed by separation by molecular weight by two-dimensional electrophoresis (2-DE). Each sample containing 150 μg of protein was incubated for 30 min in 300 μl of lysis buffer (7 M urea, 2 M thiourea, 4 % CHAPS, 0.8 % Pharmalyte, 65 mM DTT, and traces of bromophenol blue), centrifuged (14,000×g, 10 min, 4 °C), and applied over a GE Immobiline DryStrip, pH 4–7, 18 cm. After 6 h of passive rehydration, followed by 6 h of active rehydration (50 V), the IEF was performed (20 °C and 50 A per strip) in a Protean® IEF Cell (Bio-Rad, CA, USA) following a five step program: 1000 V for 1 h, 4000 V for 1 h, 8000 V, and 8000 V for 1 h for 5 h to reach a total of 50,000 Vh. Before 2-DE, strips were equilibrated in buffer (6 M urea, 75 mM Tris, pH 8.8, 4 % SDS, and 29.3 % glycerol) containing 2 % DTT for 15 min, followed by further immersion for 15 min in buffer containing 2.5 % iodoacetamide. The 2-DE was performed in 10 % polyacrylamide gels using the vertical system of Protean® XL Cell Format Cell (Bio-Rad, CA, USA) at 20 °C in two steps: 90 V for 30 min for a gradual entry of the sample into the top of the gel and 300 V until the sample went through the whole gel (∼5 h). The gels were silver-stained in compliance with a protocol compatible with mass spectrometry analysis (Blum et al. 1987). To ensure reproducibility, four gels were used for image analysis.

the 2-DE maps from the oyster farming areas (LIS and RIB), used as reference sites, were overlaid with the 2-DE map from the contaminated site (BUC). The spot intensity normalization was carried out in order to enable an accurate comparison among the gels, where the volume of each protein spot was divided by the total volume of all detected spots in each image. The normalized volumes were compared with the corresponding values of the reference gels (calibration). The number of protein spots was determined for each valid gel, and the qualitative and quantitative differences in protein patterns between the farming and contaminated sites were determined. After image analysis, the protein spots with a higher or lower expression than those from the reference group were selected for further sequencing. Digestion of spots and protein identification by mass spectrometry Proteins of oysters exposed in situ to urban sanitary sewage were selected and removed manually from silver-stained gels, digested with trypsin (Shevchenko et al. 2006), and submitted to “peptide mass fingerprint” (PMF) for identification of peptides and acquisition of mass spectra using the Ultraflex II MALDI-TOF-TOF (Bruker Daltonics) operating at positive polarity mode. The reflection spectra were acquired in the range m/z 900–3500. A total number of 3000 spectra were obtained at each dot position with a frequency of 50 Hz laser. For the experiments, MS/MS ion peptides with S/N higher than 25 and peak intensity higher than 800 were selected for MS/MS. Laser shots (300 and 1000) were used to acquire the MS and MS/MS, respectively. The laser power was 2 to 5 % above the threshold of ionization. Data acquisition and processing were performed using the software FlexAnalysis 3.0 (Bruker Daltonics) with the peak detection algorithm SNAP. The obtained peptide mass list was sent to the software MASCOT using NCBI database. Searches were conducted using the following parameters: taxonomy, other metazoans; proteolytic enzyme, trypsin peptides tolerance, 100 ppm; fixed modifications, carbamidomethyl (C), variable modification, oxidation (M); peptide charge state, + 1, missed cleavages allowed until 1. The limit of significance was set at 95 %. MS BLAST searches (NCBI / Blastp) were performed on a database of non-redundant protein sequences available for C. gigas (taxid: 29159) using protein-protein BLAST algorithm (default parameters).

Acquisition and image analysis After silver-staining, the gels run with samples from each site were scanned using a densitometer (GS-800, Bio-Rad, CA, USA). Data analysis and statistical tests were performed using PDQuest 8.0 software (Bio-Rad, CA, USA). A master gel was constructed by combining the 2-DE maps (n=4 gels), where

Statistical analysis Differences among the levels of protein expression in digestive gland of oysters from different sites were compared using


nonparametric tests, namely Mann-Whitney U rank. The level of significance was set at 0.05. The results were analyzed using GraphPad Prism ® 5.01 and XLSTAT (Excel Windows) software.

Results Water quality The levels of fecal coliforms, detergents, organic matter, and oil and grease were detected in the water samples of BUC site at the beginning of the exposure and after 24 h and 14 days of exposure evidence that this area is contaminated by sanitary sewage. SAM, RIB, and LIS sites showed undetected or very low levels of these contaminants during the exposure period (Suppl. Fig. 1).

linear alkylbenzenes (ΣLAB) were also significantly higher in BUC than in the other sites (BUC, 108.0; RIB, 12.1; SAM, 1.56 ng g−1; LIS: below detection limit). Lower levels of total PAHs were observed in sediments from RIB (14.5 ng g−1) and higher levels in BUC (112.1 ng g −1), while LIS and SAM showed similar values (96.9 and 88.2 ng.g−1, respectively). Coprostanol, a biomarker for the presence of human fecal matter in the environment, was detected in sediments of BUC, with levels 125-fold higher (2.55 μg g−1) than in the sediments caught at the other sites (SAM, 0.02; RIB, 0.08; LIS, 0.05 μg g−1) (Suppl. Fig. 3a). The ratio between fecal sterols used as indicative of contamination by sanitary sewage (Grimalt et al. 1990; Mudge and Norris, 1997) observed in sediment from BUC was higher than the other sites (Suppl. Fig. 3b). Levels of organic contaminants in oysters

Organic contaminants in sediments Sediment analysis showed that the level of ΣDDT {sum of the p,p′- and o,p-isomers of DDT [1,1,1-trichloro-2,2bis(p-chlorophenyl)ethane], DDE [1,1-dichloro-2,2-bis(pchlorophenyl)ethylene], and DDD [1,1-dichloro-2,2bis(p-chlorophenyl)ethane]} caught at SAM (=11.9 ng g−1) was 3–7 times higher when compared to other sites (RIB, 1.6; LIS, 3.8; BUC, 2.9 ng g−1) (Suppl. Fig. 2). In contrast, the content of total aliphatic hydrocarbons (ΣAH) in sediments sampled at BUC site showed higher levels (52.6 ng g−1) than in the other sites (LIS, 18.4; SAM, 10.0; RIB, 3.67 ng g−1). Total Fig. 2 Levels of organic contaminants in oysters Crassostrea gigas exposed for 14 days at the different sites. SAM Sambaqui beach (oysters supplier); RIB Ribeirão da Ilha beach, South bay oyster farming area; LIS Santo Antônio de Lisboa beach, North bay oyster farming area; BUC Rio Bücheler, contaminated site;