Aquatic Toxicology 101 (2011) 109–116
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Differential protein expression in two bivalve species; Mytilus galloprovincialis and Corbicula fluminea; exposed to Cylindrospermopsis raciborskii cells Maria Puerto a,1 , Alexandre Campos b,∗,1 , Ana Prieto a , Ana Cameán a , André Martinho de Almeida c,d , Ana Varela Coelho d,e , Vitor Vasconcelos b,f a
Area of Toxicology, Faculty of Pharmacy, University of Seville, Seville, Spain Centro Interdisciplinar de Investigac¸ão Marinha e Ambiental, CIIMAR/CIMAR, Rua dos Bragas 289, 4050-123, Porto, Portugal Instituto de Investigac¸ão Científica Tropical, Lisboa, Portugal & CIISA-Centro Interdisciplinar de Investigac¸ão em Sanidade Animal Lisboa 1300, Portugal d Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal e Departamento de Química, Universidade de Évora, Évora, Portugal f Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Porto, Portugal b c
a r t i c l e
i n f o
Article history: Received 30 June 2010 Received in revised form 10 September 2010 Accepted 18 September 2010 Keywords: Mytilus galloprovincialis Corbicula fluminea Cylindrospermopsis raciborskii Cylindrospermopsin Proteomics
a b s t r a c t The cyanobacteria Cylindrospermopsis raciborskii is considered a threat to aquatic organisms due to the production of the toxin cylindrospermopsin (CYN). Despite the numerous reports evidencing the toxic effects of C. raciborskii cells and CYN in different species, not much is known regarding the toxicity mechanisms associated with this toxin and the cyanobacteria. In this work, a proteomics approach based in the two-dimensional gel electrophoresis and mass spectrometry was used to study the effects of the exposure of two bivalve species, Mytilus galloprovincialis and Corbicula fluminea, to CYN producing (CYN+) and non-producing (CYN−) C. raciborskii cells. Additionally the activities of glutathione S-transferase (GST) and glutathione peroxidase (GPx) were determined. Alterations in actin and tubulin isoforms were detected in gills of both bivalve species and digestive gland of M. galloprovincialis when exposed to CYN− and CYN+ cells. Moreover, GST and GPx activities changed in gills and digestive tract of bivalves exposed to both C. raciborskii freeze dried cells, in comparison to control animals exposed to the green alga Chlorella vulgaris. These results suggest the induction of physiological stress and tissue injury in bivalves by C. raciborskii. This condition is supported by the changes observed in GPx and GST activities which indicate alterations in the oxidative stress defense mechanisms. The results also evidence the capacity of CYN non-producing C. raciborskii to induce biochemical responses and therefore its toxicity potential to bivalves. The heat shock protein 60 (HSP60), extrapallial (EP) fluid protein and triosephosphate isomerase homologous proteins from gills of M. galloprovincialis were down-regulated specifically with the presence of CYN+ C. raciborskii cells. The presence of CYN may lead to additional toxic effects in M. galloprovincialis. This work demonstrates that proteomics is a powerful approach to characterize the biochemical effects of C. raciborskii and to investigate the physiological condition of the exposed organisms. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Cylindrospermopsis raciborskii (C. raciborskii) is a freshwater filamentous cyanobacterium from the Nostocales order. This species has a worldwide distribution (Fastner et al., 2007) being considered toxic due to the production of the tricyclic alkaloid Cylindrospermopsin (CYN). Outbreaks of human poisoning and cattle mortality
Abbreviations: CYN, cylindrospermopsin; CYN+, CYN producing C. raciborskii strain; CYN−, CYN non-producing C. raciborskii strain; GST, glutathione S-transferase; GPx, glutathione peroxidase; DPE, differential protein expression. ∗ Corresponding author.Tel.: +351 223401800; fax: +351 223390608. E-mail address:
[email protected] (A. Campos). 1 These authors contributed equally to this work. 0166-445X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2010.09.009
have been related with the proliferation of this cyanobacterium and the accumulation of CYN (Saker et al., 1999). The species is therefore of major concern regarding aquatic ecosystems and water quality (Saker et al., 2003). CYN is a sulphated and methylated tricyclic guanidine linked to the hydromethyluracil group (Ohtani et al., 1992). The biosynthesis of the toxin involves the activity of an amidinotransferase, as well as the non-ribosomal polypeptide synthetase (NRPS) and polyketide synthase (PKS) enzymes (AoaA, AoaB, and AoaC genes, respectively) (Mihali et al., 2008; Schembri et al., 2001; Shalev-Alon et al., 2002). Toxic effects of CYN in mammals include centrilobular necrosis in the liver and proximal tubular necrosis in the kidney (Griffiths and Saker, 2003; Hawkins et al., 1985). Hemorrhages in the lungs and in the heart have equally been reported (Hawkins et al., 1985). CYN may induce fetal toxicity in mouse females exposed to sub-
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lethal doses (Rogers et al., 2007). Moreover the toxin has been shown to induce cytotoxicity and genotoxicity both in vivo and in vitro (Bain et al., 2007; Bazin et al., 2010). The process may be mediated by cytochrome P-450-generated metabolites (Humpage et al., 2005). Nevertheless, it is established that CYN may act trough the inhibition of glutathione and protein synthesis (Froscio et al., 2001; Terao et al., 1994). Furthermore, Froscio et al. (2008) suggested that CYN may directly interact with the eukaryotic translation system. Regarding plants, CYN inhibits the growth of mustard (Sinapsis alba) seedlings (Vasas et al., 2002) and the germination of tobacco (Nicotiana tabacum) pollen (Metcalf et al., 2004). It induces alterations in root histology and microtubule organization in the common reed (Phragmites australis) plantlets cultured in vitro (Beyer et al., 2009). Cylindrospermopsin toxicity has been described in varied aquatic organisms. Decreases in relative growth rate, mortality and injuries in multiple organs of adults and tadpoles of cane toad (Bufo marinus) have been registered in result of animal exposure to freeze-thawed C. raciborskii whole cell extracts and live C. raciborskii cells, in which CYN varied between 107 g/l and 400 g/l (Kinnear et al., 2007; White et al., 2007). Exposure of Daphnia magna juveniles to CYN-producing (CYN+) and CYN-non-producing (CYN−) C. raciborskii cultures led to a significant decrease of survival and growth rates of this planktonic crustacean, with the most severe decreases associated with the toxic strain (Nogueira et al., 2004). D. magna exposure for 24 h to both C. raciborskii cultures led to an increase in GST enzyme activities (Nogueira et al., 2004). The toxicity of this molecule might be associated with its accumulation in animal tissues. In the freshwater gastropod Melanoides tuberculata and B. marinus tadpoles, exposure to CYN+ C. raciborskii cell extracts or live cells led to the bioaccumulation of the toxin (White et al., 2006, 2007). The process was shown to be dependent on the toxin concentration, time of toxin exposure and toxin availability (White et al., 2006, 2007). The highest CYN concentrations in the tissues were obtained when animals were exposed to live CYN+ C. raciborskii cells and not to the free toxin from the whole cell extracts (White et al., 2006, 2007), indicating that the grazing habits of these animals are most likely the major route of CYN uptake. CYN accumulation was described in the Redclaw crayfish Cherax quadricarinatus from aquaculture pond infested by a outbreak of C. raciborskii (Saker and Eaglesham, 1999) and in the freshwater mussel Anodonta cygnea to concentrations up to 2.52 g/g tissue dry weight when exposed to CYN-producing C. raciborskii (Saker et al., 2004). CYN accumulation has shown to be distinct in the several organs/tissues of these animals, reaching higher levels in the hepatopancreas (4.3 g/g freeze dried tissue) relatively to the muscle tissue (0.9 g/g freeze dried tissue) in the redclaw crayfish and higher levels in the haemolymph (68.1%) followed by viscera (23.3%), foot and gonad (7.7%) and mantle (0.9%) in A. cygnea. This toxin accumulation may represent a considerable health threat to aquatic species with potential strong negative impacts to the environment, economy and consumer safety. Likewise molecular biology and biochemistry approaches are fundamental to identify genes/proteins with roles in the process of toxicity and therefore to better understand those impacts. This work aims to characterize the differential protein expression in the Mediterranean mussel Mytilus galloprovincialis and the freshwater bivalve Corbicula fluminea to C. raciborskii cells through proteomics and to gather a better understanding on the biochemical pathways affected by the presence of the cyanobacteria and the toxin. Therefore bivalves were exposed to freeze-dried CYN+ and CYN− C. raciborkii cells. The control groups were exposed to the green algae Chlorella vulgaris. This microalga is a primary producer and food source in the fresh water aquatic ecosystems. Contrary to C. raciborskii no toxic effects have been reported for C. vulgaris. Two-dimensional gel electrophoresis of proteins (2DE) and mass spectrometry methodologies were employed to con-
duct the proteomics analysis in the gills and digestive gland of M. galloprovincialis and C. fluminea, as a result of bivalve’s exposure to CYN+ and CYN− C. raciborskii cells. Furthermore the activities of glutathione S-transferase (GST) and glutathione peroxidase (GPx), enzymes involved in the oxidative stress and toxin biotransformation processes, were measured in the organs of the animals in order to assess their physiological condition. 2. Materials and methods 2.1. Biological material Bivalves from the species C. fluminea (25–30 mm) were collected in the Minho River (Valenc¸a, North Portugal) in May 2009. At the time, there was no record of cyanobacterial blooms in the area. The species M. galloprovincialis (dimensions between 50 and 60 mm) was collected in the Memória Beach (Porto, North Portugal) in the same month. One hundred and fifty animals were placed in 30 L aquaria with dechlorinated tap water (C. fluminea) or natural sea water (M. galloprovincialis) and acclimatized to laboratory conditions based on the procedure previously described (Amorim and Vasconcelos, 1999). Water temperature was 16 ± 1 ◦ C, animals were fed twice a week with 1 × 105 cells/ml of the green algae C. vulgaris. Water was renewed every two days. CYN-producing (AQS) and CYN-non producing (LJ) C. raciborskii strains (Valério et al., 2005) were grown as described by Saker et al. (2003) in bulk cultures, harvested by filtration and freeze-dried. 2.2. Quantification of CYN in C. raciborskii freeze dried cells The toxin CYN was extracted from C. raciborskii cells and quantified based on the methods described by Welker et al. (2002). Briefly, freeze-dried cells (0.7 g) were sonicated for 15 min in 5 ml distilled water. The homogenate was stirred for 1 h at room temperature, centrifuged and the supernatant collected. Five microliters of TFA (0.1%, v/v) was added to the supernatant, stirred for 1 h and centrifuged a second time. The supernatant was analyzed by HPLC-PDA (Welker et al., 2002). 2.3. Exposure experiment Three aquaria with 150 animals each were used to set up the exposure experiments in both bivalve species. Animal treatment consisted in the exposure to freeze-dried CYN-producing (treatment 1) and CYN-non producing (treatment 2) C. raciborskii cells in a concentration of 5 × 105 cells/ml. Natural C. raciborskii blooms have been registered in Portuguese fresh waters, with cell densities up to 3 × 106 cells/ml (Saker et al., 2003). The toxin concentration in treatment 1 was equivalent to 0.072 g CYN/l provided by the freeze dried C. racibroskii cells. A control group, consisting of animal exposure to 5 × 105 cells/ml live C. vulgaris was also used. The animals were exposed for 6 days with the renewal of the water and inoculums (C. raciborskii and C. vulgaris cells) after the second and fourth day. No animal mortality was registered during the experiment, including treatments. In the end of the exposure experiment pools of 4 animals were collected randomly. Three pools (replicates) were collected for each treatment and the control group. Gills, digestive gland from M. galloprovincialis and digestive tract from C. fluminea were dissected in ice, frozen in liquid nitrogen and stored at −80 ◦ C until further analysis. 2.4. Enzyme activity measurements The tissues were homogenized in phosphate buffer (100 mM) pH 6.5 in a ratio of 1 g of tissue per 10 ml of buffer, with an UltraTurrax on ice. The homogenates were centrifuged at 4000 rpm, for
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20 min at 4 ◦ C. The supernatant was collected, its protein concentration determined with the method of Bradford (1976), and stored at −80 ◦ C until further analysis. The GST activity was determined according to the method of Habig et al. (1974) in microplates. The GPx activity was determined with the method of Lawrence and Burk (1976) in microplates. Both reactions were performed with 0.5 mg prot/ml from gill samples and 0.1 mg prot/ml from digestive gland samples of M. galloprovincialis or digestive tract samples from C. fluminea.
2.5. Sample preparation for 2DE Bivalve organs were ground in liquid nitrogen with mortar and pestle. The ground tissue powder (0.1 g) was homogenized for 1 h in 250 l of urea (8 M), thiourea (2 M), 3[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) (4%, w/v), dithiothreitol (65 mM) and ampholytes, pH 4–7 (0.8%, v/v) (protein solubilization buffer, SB). The homogenate was centrifuged at 16,000 × g, for 20 min at 22 ◦ C. The supernatant was collected and proteins quantified with the method of Bradford (1976). Protein samples were stored at −70 ◦ C.
2.6. Two-dimensional electrophoresis (2DE) The two-dimensional electrophoresis was based in the procedure described by Campos et al. (2009). Protein samples with 400 g of protein were diluted to 300 l in SB buffer. The protein samples were loaded in 17 cm, pH 4–7 IEF gel strips (Bio-Rad, Hercules, CA, USA) and proteins separated by isoelectric focusing (IEF) in a Protean IEF Cell (Bio-Rad, Hercules, CA, USA) with the following program: 16 h at 50 V (strip rehydration); step 1, 15 min at 250 V; step 2, 3 h voltage gradient to 10,000 V (linear ramp); step 3, 10,000 V until achieving 60,000 V/h (linear ramp). Wet paper strips were used in the electrodes to remove the excess of salts from the samples. After the first dimension IEF gel strips were stored at −20 ◦ C until performing the second-dimension SDS-PAGE. IEF Gel strips were equilibrated as described by Campos et al. (2009) using 10 mg/ml dithiothreitol and 25 mg/ml iodoacetamide in urea (6 M), glycerol (30%, v/v), SDS (2%, w/v). Subsequently IEF gel strips were placed on top of 12% (w/v) acrylamide SDS-PAGE slab gels (20 cm × 20 cm × 1 cm) and proteins separated by SDS-PAGE in a Protean Xi Cell (Bio-Rad, Hercules, CA, USA) at 24 mA per gel. In this procedure one 2DE gel was run per replicate. Gel staining and protein visualization was performed as described by Neuhoff et al. (1988).
2.7. Gel image acquisition and protein expression analysis 2DE Gel images were acquired in the GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA, USA) and protein spots detected automatically with the PDQuest 2-D Analysis Software (Bio-Rad, Hercules, CA, USA), reproducing the sensitivity parameters for every gel image. Spot detection and spot matching was manually revised in the software. Protein spot intensities were normalized in terms of the total density in the gel image. For protein expression analysis a master gel was obtained in the software with all the spots detected in the 2DE gel images. The presence/absence of spots and quantitative variations in spot intensities was subsequently analyzed by comparison of the intensity of each protein spot between the experimental groups. In this analysis only spots that were detected in at least two replicate gels were taken in consideration. The quantitative variations were statistically validated using t-Student test (P ≤ 0.05).
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2.8. Protein identification Protein identification was performed based in the method described by Santos et al. (2009). Protein spots were excised from gels and proteins subjected to in-gel digestion using the protease trypsin (Pandey and Mann, 2000). The tryptic digests were desalted and concentrated using reversed phase microcolumns (Gobom et al., 1999). The peptides were eluted directly onto the MALDI plate using the matrix ␣-cyano-4- hydroxycinnamic acid (5 mg/ml) prepared in acetonitrile (70%, v/v) and trifluoroacetic acid (0.1%, v/v). Protein identification was done by MALDI-TOF–TOF with an Applied Biosystem 4800 Proteomics Analyser (Applied Biosystems, Foster City, CA, USA) in MS and MS/MS mode. Each MS spectrum was obtained in an independent acquisition mode with a total of 800 laser shots per spectra and a fixed laser intensity of 3500 V. Spectra were externally calibrated using des-Arg-Bradykinin (904.468 Da), angiotensin 1 (1296.685 Da), Glu-Fibrinopeptide B (1570.677 Da), ACTH (1–17) (2093.087 Da), and ACTH (18–39) (2465.199) (Calibration Mix from Applied Biosystems). Ten s/n best precursors from each MS spectrum were selected for MS/MS analysis. The MS/MS analyses were performed with CID (Collision Induced Dissociation) using a collision energy of 1 kV and a gas pressure of 1 × 106 Torr. The MS/MS spectra were acquired with 2000 laser shots and a laser intensity of 4500 V. The generated mass spectra were used to search the NCBI predicted protein database with the algorithms Paragon, from Protein Pilot software v 2.0 (Applied Biosystems, MDS Sciex), and Mowse, from MASCOT-demon 2.1.0 Software (Matrix-Science). Protein score above 2.0 (P < 0.01) for Paragon and a threshold of 95% (P < 0.05) for Mowse were considered for confident protein identification. In the analysis using Protein Pilot other parameters considered were: enzyme, trypsin; Cys alkylation, iodoacetamide; special factor, urea denaturation; species, none; and ID focus, biological modification. Regarding the Mascot, the analysis of results was performed in the GPS Explorer Software (Version 3.5, Applied Biosystems, Foster City, CA, USA), using the following parameters: missed-cleavage, one; peptide tolerance, 50 ppm; fragment mass. 3. Results 3.1. Two-dimensional gel electrophoresis of proteins Proteins from gills, digestive gland of M. galloprovincialis and digestive tract of C. fluminea were separated in well defined/delimited colloidal Coomassie Blue stained spots by 2DE as shown in Fig. 1. Proteins were separated between the pI 4 and 7 and molecular mass 15 kDa and 80 kDa in the 2DE gels. 3.2. Differential protein expression In this study the expression of individual protein spots resolved by 2DE from the bivalves exposed to the CYN− and CYN+ C. raciborskii cells were compared with the expression of the same protein spots resolved by 2DE from control bivalves, exposed to the green algae C. vulgaris. Differences in protein expression were also studied between animals exposed to the CYN− and CYN+ cells. The differential protein expression (DPE) comprised 31 proteins in C. fluminea and 61 proteins in M. galloprovincialis (results not shown). Twenty six proteins were identified by mass spectrometry analysis, representing 23% and 33% of the total number of spots considered of interest from C. fluminea and M. galloprovincialis, respectively. The differential expression of the identified proteins in the three experimental groups is summarized in Tables 1 and 2. The majority of the
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Fig. 1. Large format two-dimensional gel electrophoresis of proteins from gills, digestive gland and digestive tract of M. galloprovincialis and C. fluminea maintained in control conditions. Gels were loaded with 400 g of protein. Isoelectric focusing was carried out in 17 cm immobiline IEF gel strips, pH range 4–7. The second dimension SDS-PAGE was performed in 12% (w/v) polyacrylamide gels. Gels stained with Colloidal Coomassie G-250. Differentially expressed proteins identified by MALDI-TOF/TOF mass spectrometry and their respective reference number (X).
identified proteins have structural functions (actin and tubulin isoforms). Another group of proteins was identified which is involved in energy production (ATPase beta subunit and triosephosphate isomerase), calcium-binding and metal transport (EP protein) as well as a stress related protein (mitochondrial 60 kDa heat shock protein). Variations in the structural proteins tubulin and actin were associated to the CYN− C. raciborskii cells (spots 2106, 2201, 5209, 5711, 3715, 6305, 2258) and CYN+ C. raciborskii cells (spots 4601CF, 5302, 2119, 2307, 4222, 5314, 4218, 4447, 4510). Moreover variations were found between treatments (spots 4601CF, 5314, 6305, 2258, 3715, 4218, 2304, 2402, 3410, 5501). Only one protein (spot 4601MG) displayed similar expression in the treat-
ments with the cyanobacteria cells in comparison with the control group. The ATPase beta subunit in the digestive tract of C. fluminea (spot 2506) and digestive gland of M. galloprovincialis (spot 3701) was up-regulated by C. raciborskii exposure. Higher expression being verified in C. fluminea exposed to CYN+ in comparison to C. fluminea exposed to CYN− C. raciborskii cells. The mitochondrial 60 kDa heat shock protein (spot 4504) was down-regulated in gills of M. galloprovincialis exposed to CYN+ C. raciborskii cells, in comparison with the control mussels. Moreover the EP protein precursor (spot 4322) and triosephosphate isomerase (spot 8204) were differentially expressed between treatments.
Table 1 Variations in protein expression of C. fluminea exposed to C. raciborskii cells. Normalized intensity and standard deviation (in parenthesis) of each identified protein in each experimental group, Control bivalves (C), bivalves exposed to CYN non-producing (CYN−) and CYN producing (CYN+) C. raciborskii cells is presented. Details regarding the identification of proteins by MALDI-TOF/TOF analysis are shown in the supplementary table. C. fluminea organ
Spot #
Protein identification
C
CYN−
CYN+
Gills
2106 2201 4601CF 5209 5302 5711
Alpha-tubulin Actin Tubulin alpha-1 chain Cytoplasmic actin actin Cytoplasmic actin type
3475.2 (1126.0) 2026.1 (220.8) 354.5 (140.0) 1165.2 (242.4) 373.7 (55.7) 348.6 (137.3)
502.8 (131.2)* 1367.2 (157.2)* 163.7 (46.7)a 2048.4 (188.5)a 211.3 (169.5) 768.9 (119.2)*
1909.2 (878.2) 1681.3 (1331.6) 812.0 (220.9)*a 1064.4 (629.1) 188.1 (60.6)* 1570.3 (1612.7)
Digestive tract
2506
ATP synthase beta subunit
355.3 (170.5)
503.7 (36.8)*a
836.9 (124.7)*a
Not detected (–). ** Significant differences in respect to control for P < 0.01. a Significant differences detected between treatments (P < 0.05). * Significant differences in respect to control for P < 0.05.
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Table 2 Variations in protein expression of M. galloprovincialis exposed to C. raciborskii cells. Normalized intensity and standard deviation (in parenthesis) of each identified protein in each experimental group, Control bivalves (C), bivalves exposed to CYN non-producing (CYN−) and CYN producing (CYN+) C. raciborskii cells is presented. Details regarding the identification of proteins by MALDI-TOF/TOF analysis are shown in the supplementary table. M. galloprovincialis organ
Spot #
Protein identification
C
CYN−
Gills
2119 2307 4222 4322 4504 5314 6305 8204
Beta-tubulin Cytoplasmic actin type Tubulin alpha chain EP protein precursor Mitochondrial 60 kDa heat shock protein Alpha-tubulin Cytoplasmic actin type Triosephosphate isomerase
408.9 (181.1) 442.9 (31.9) 986.5 (401.6) 1210.5 (417.6) 545.2 (263.3) 886.0 (134.7) 615.3 (153.8) 262.4 (77.9)
158.6 (79.5) 1421.2 (1682.3) 529.2 (192.1) 1342.9 (237.7)a 636.5 (267.3) 1504.4 (27.1)a 1173.4 (308.6)*a 339.6 (46.5)a
CYN+ 121.2 (50.7)* 120.8 (46.7)** 327.5 (73.7)* 559.3 (33.0)a 81.7 (9.0)* 329.0 (185.9)*a 424.9 (225.2)a 171.7 (27.6)a
Digestive gland
2258 3701 3715 4601MG 4218 4447 4510 2304 2402 3410 5501
Actin E2 ATP synthase beta subunit Beta tubulin actin 1 Actin Actin Beta-actin Actin Actin Beta-actin Beta-actin
8659.7 (1339.5) – – 197.2 (60.8) 370.7 (206.0) 971.6 (42.5) 3036.6 (1034.4) 1118.6 (661.1) 2536.2 (1223.9) 1303.5 (575.8) 711.0 (387.9)
2437.7 (896.3)**a 165.4 (40.1)** 205.6 (49.6)**a 1974.8 (685.2)a 280.2 (142.4)a 1339.9 (869.5) 3423.8 (1182.9) 288.3 (219.9)a 1201.5 (657.8)a 745.0 (520.9)a 366.4 (158.8)a
5702.6 (1877.4)a 167.8 (12.0)** –a 1164.8 (623.3)a 779.5 (107.5)*a 1916.6 (453.9)* 5335.9 (450.0)* 1158.6 (444.1)a 2438.8 (280.1)a 2143.7 (326.1)a 961.5 (220.5)a
Not detected (–). a Significant differences detected between treatments (P < 0.05). * Significant differences in respect to control for P < 0.05. ** Significant differences in respect to control for P < 0.01.
3.3. Enzyme activity The activities of glutathione S-transferase (GST) and glutathione peroxidase (GPx) were measured in this study to evaluate the toxic effects of M. galloprovincialis and C. fluminea exposure to C. raciborskii cells. Species and organ specific GST and GPx activities were registered (Figs. 2 and 3). GST activity decreased in gills of C. fluminea exposed to CYN− and CYN+ C. raciborskii cells, never-
theless, increased in gills of M. galloprovincialis exposed to CYN− C. raciborskii cells (Fig. 2a). In contrast, the activity of this enzyme increased in C. fluminea digestive tract after animal exposure to both cyanobacteria strains and decreased in the digestive gland of M. galloprovincialis exposed to the CYN− strain (Fig. 3a). A decrease in GPx activity was detected in gills of M. galloprovincialis exposed to CYN+ strain (Fig. 2b). Nevertheless, the activity of this enzyme was not significantly altered in the digestive gland of the mussel
Fig. 2. Glutathione S-transferase (a) and glutathione peroxidase (b) activities in gills of M. galloprovincialis (white bars) and C. fluminea (grey bars) exposed to CYN− and CYN+ C. raciborskii cells. Bars with different superscript letters represent significant differences (*P < 0.05; **P < 0.01). Standard deviation values are shown for each experimental group.
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Fig. 3. Glutathione S-transferase (a) and glutathione peroxidase (b) activities in digestive gland of M. galloprovincialis (white bars) and digestive tract of C. fluminea (grey bars) exposed to CYN− and CYN+ C. raciborskii cells. Bars with different superscript letters represent significant differences (*P < 0.05; **P < 0.01). Standard deviation values are shown for each experimental group.
species exposed to C. raciborskii cells (Fig. 3b). On the other hand an increase in GPx was observed in the digestive tract of C. fluminea exposed to C. raciborskii cells (Fig. 3b) but no changes were detected in the gills (Fig. 2b). 4. Discussion This work concerns the analysis of the effects in protein expression of two bivalve species, the Mediterranean mussel M. galloprovincialis and the Asian clam C. fluminea, from the exposure to C. raciborskii cells. The effects were evaluated by comparing the 2DE protein profiles of control bivalves, exposed to the non toxic green algae C. vulgaris, with the protein profiles of bivalves exposed to CYN− and CYN+ C. raciborskii cells. The 2DE followed by MALDITOF/TOF analysis allowed characterizing protein variations in gills, digestive tract and digestive gland of the two bivalve species when exposed to the cyanobacteria cells. It is worth mentioning that no animal mortality was registered during the exposures indicating the use of a low toxic cell density. The proteomics and biochemical approaches followed were therefore particularly important for the sensitivity in reporting the bivalves’ physiological condition. The majority of the differentially expressed proteins identified belong to the group of structural proteins (essentially actin and tubulin isoforms). Moreover, the variations were observed in the organs of C. fluminea and M. galloprovincialis after animal exposure to CYN− and CYN+ cells suggesting similar biochemical responses. Alterations in actin cytoskeleton and microfilaments have long been reported in situations of cellular stress and apoptosis (Alvarez and Sztul, 1999; Bursch et al., 2000; Kanlaya et al., 2009) and recently Gácsi et al. (2009) described alterations in cytoskeletal structures and apoptosis in Chinese hamster ovary cells (CHO-K1) mediated by CYN. At the proteomic level correlations were found between the changes in the expression of the cytoskeletal proteins
and stress factors in different biological systems including bovine kidney and blood cells (Riedmaier et al., 2009; Zhang et al., 2009), human cell lines (Ou et al., 2008), in the fish sea bream (Ibarz et al., 2010), the fresh water bivalve C. fluminea (Martins et al., 2009) and in rabbits (Almeida et al., 2010). Taking in consideration this knowledge we can infer that the changes in actin and tubulin isoforms observed in this work translate to a condition of physiological stress and cellular injury in both bivalve species exposed to the cyanobacterial cells. Another important aspect in the expression of structural proteins is the presence of low molecular mass (MM) forms (e.g. 14, 30, 38 kDa) as a result of protease activity in apoptosis processes (Bareyre et al., 2001; Brown et al., 1997). In fact, actin and tubulin forms lower than 42 kDa and 50 kDa, the molecular masses of the respective native proteins, were identified evidencing the cellular injury and organ damage in both bivalve species after C. raciborskii exposure. The ATPase beta subunit is a constituent of the ATP synthase complex, responsible for the production of ATP in the mitochondria and for the supply of energy to the cells (Leyva et al., 2003). Interestingly, this protein was up-regulated in the digestive tract and digestive gland of C. fluminea and M. galloprovincialis, respectively demonstrating to be an organ-specific alteration induced by the presence of C. raciborskii cells. The mitochondrial 60 kDa heat shock (HSP60), extrapallial (EP) fluid protein and triosephosphate isomerase were, respectively, downregulated in gills of M. galloprovincialis exposed to CYN+ C. raciborskii cells or differentially expressed between treatments suggesting an organ-specific biochemical response in M. galloprovincialis regarding the two cyanobacteria strains and thereby to the presence of the toxin CYN. The HSP60 is a specific mitochondrial chaperone assisting in the synthesis and transportation of essential mitochondrial proteins by promoting conformational and structural changes (Koll et al., 1992). This family of proteins
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is usually upregulated in situations of stress, individual members are known as stress proteins (Santoro, 2000). The EP protein is a calcium-binding glycoprotein from the extrapallial fluid of the mussel, playing a role in building block of the shell-soluble organic matrix (Hattan et al., 2001). Besides calcium the EP protein has shown affinity to other divalent ions including Cd2+ , Cu2+ , Mn2+ , and Mg2+ suggesting a multiple function, as Ca2+ -transporter, shell matrix constituent and metal detoxification (Yin et al., 2005). A homologous protein is putatively expressed in gills of M. galloprovincialis, likely associated with metal carrier or scavenging functions. Triosephosphate isomerase plays an important role in the glycolysis being an essential enzyme for efficient energy production. In summary specific biochemical pathways putatively affected in M. galloprovincialis gills are the glycolysis, metal transport and mitochondrial protein transport. We further analyzed the activities of GST and GPx, two enzymes involved in the toxin biotransformation and oxidative stress defense pathways, as a complementary approach to proteomics. Changes in activities of both enzymes were registered for C. fluminea and M. galloprovincialis likely affecting the pathways and subsequently the levels of reactive oxigen species (ROS) in the bivalve’s organs. The oxidative stress is known to have a severe impact in cell’s metabolism and viability (Campos and Vasconcelos, 2010). We may consider that this stress mechanism was active in the bivalves exposed to C. raciborskii cells with significant consequences to animal physiology and organ injury. Cytotoxicity and cell apoptosis induced by CYN was recently described in different mammalian cell lines (Bain et al., 2007; Gácsi et al., 2009). Gene expression and immunochemical analyses indicated that the transcription factor p53, a gene that plays a key role in cell cycle and tumor suppression, as well as the gap-junctional intercellular communication gene (GJIC) and the mitogen activated protein kinases ERK1/2, involved in signal transduction, cell proliferation and differentiation, may play a critical role in the regulation of these processes and in the tumor promoting properties of cyanotoxins (Bain et al., 2007; Bláha et al., 2010). Our proteomics approach did not render information in the expression of these proteins, probably due to the limited resolution and sensitivity of the 2DE protocol. Nevertheless we cannot discard the possibility that these proteins may play a role in the cytotoxicity and organ damage induced by C. raciborskii cells in bivalves, similarly as proposed in animal cell systems. Surprisingly the CYN− strain in this work was able to induce alterations in the expression of cytoskeletal proteins and in the activities of GST and GPx concomitant with a condition of physiological stress in the bivalves. The results therefore indicate the toxic properties of these cells lacking CYN, suggesting the presence of unknown bioactive metabolites. The presence of bioactive compounds, other than microcystin and CYN, from Microcystis aeruginosa, Aphanizomenon flos-aquae and water bloom samples dominated by these species have been considered by Bain et al. (2007). Such compounds may significantly affect the expression of biomarkers of tumor promotion in animal cells. Moreover Berry et al. (2009) showed that C. raciborskii cell extracts affect zebrafish embryo development independently of the presence of the toxin CYN. The increasing evidences of the presence of unknown metabolites with major influence in cyanobacteria toxicity states the need of further research regarding their identification and characterization of their mode of action. Both bivalve species showed similar biochemical alterations to the presence of C. raciborskii, suggesting that both species may be equally susceptible despite belonging to distinct aquatic ecosystems and be putatively exposed to different levels of the cyanobacteria. The biochemical alterations observed may represent a general condition of tissue damage and cellular stress in animals induced by C. raciborskii.
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5. Conclusions In this work we investigated the effects of bivalve exposure to the toxic cyanobacteria C. raciborskii. Proteomics and biochemical analysis were conducted to assess the physiological condition of the organisms. Alterations in the activities of GST and GPx enzymes and in the expression of cytoskeleton proteins were reported in the bivalve’s organs in result of animal exposure to C. raciborskii cells, translating a condition of physiological stress. Moreover the toxicity of CYN− C. raciborskii cells was highlighted in this approach. A DPE associated with the presence of CYN was identified in gills of M. galloprovincialis. CYN is likely to affect different biochemical pathways, related with energy production, mitochondrial function and metal transport. Furthermore, the toxin may not be the main factor responsible for the physiological stress induced in the bivalves. The presence of other bioactive molecules synthesized by C. raciborskii cells should be clarified in order to be possible a more rigorous evaluation of the ecological implications of CYN− and CYN+ C. raciborskii blooms in aquatic ecosystems. This work demonstrates the importance of proteomics to assess the biochemical changes and the physiological conditions of the organisms. Moreover it is a promising approach towards the elucidation of the underlying mechanisms of toxicity in bivalves induced by C. raciborskii cells and CYN. Acknowledgements Alexandre Campos and André de Almeida contract work is supported by the Ciência 2007 program of the Ministério da Ciência, Tecnologia e Ensino Superior (MCTES, Lisbon, Portugal). Anabel Prieto and Maria Puerto acknowledge, respectively José Castillejo and Ministério de Ciencia e innovación (MICINN, Madrid, Spain) scholarships. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.aquatox.2010.09.009. References Almeida, A.M., Campos, A., Francisco, R., Harten, S.V., Cardoso, L.A., Coelho, A.V., 2010. Proteomic investigation of the effects of weight loss in the gastrocnemius muscle of wild and NZW rabbits via 2D-electrophoresis and MALDI-TOF MS. Anim. Genet. 41, 260–272. Alvarez, C., Sztul, E.S., 1999. Brefeldin A (BFA) disrupts the organization of the microtubule and the actin cytoskeletons. Eur. J. Cell Biol. 78, 1–14. Amorim, Á., Vasconcelos, V., 1999. Dynamics of microcystins in the mussel Mytilus galloprovincialis. Toxicon 37, 1041–1052. Bain, P., Shaw, G., Patel, B., 2007. Induction of p53-regulated gene expression in human cell lines exposed to the cyanobacterial toxin cylindrospermopsin. J. Toxicol. Environ. Health A 70, 1687–1693. Bareyre, F.M., Raghupathi, R., Saatman, K.E., McIntosh, T.K., 2001. DNaseI disinhibition is predominantly associated with actin hyperpolymerization after traumatic brain injury. J. Neurochem. 77, 173–181. Bazin, E., Mourot, A., Humpage, A.R., Fessard, V., 2010. Genotoxicity of a freshwater cyanotoxin, cylindrospermopsin, in two human cell lines: Caco-2 and HepaRG. Environ. Mol. Mutagen. 51, 251–259. Berry, J.P., Gibbs, P.D.L., Schmale, M.C., Saker, M.L., 2009. Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo. Toxicon 53, 289–299. Beyer, D., Suranyi, G., Vasas, G., Roszik, J., Erdodi, F., M-Hamvas, H., Bacsi, I., Batori, R., Serfozo, Z., Szigeti, Z.M., Vereb, G., Demeter, Z., Gonda, S., Mathe, C., 2009. Cylindrospermopsin induces alterations of root histology and microtubule organization in common reed (Phragmites australis) plantlets cultured in vitro. Toxicon 54, 440–449. Bláha, L., Babica, P., Hilscherová, K., Upham, B.L., 2010. Inhibition of gap-junctional intercellular communication and activation of mitogen-activated protein kinases by cyanobacterial extracts—indications of novel tumor-promoting cyanotoxins? Toxicon 55, 126–134. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
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