Proteomic Response to Sublethal Cadmium Exposure in a Sentinel Fish Species, Cottus gobio Jennifer Dorts,*,† Patrick Kestemont,† Marc Dieu,‡ Martine Raes,‡ and Fre´de´ric Silvestre† Unite´ de Recherche en Biologie des Organismes (URBO), The University of Namur (FUNDP), Rue de Bruxelles 61, B-5000, Namur, Belgium, and Unite´ de Recherche en Biologie Cellulaire (URBC), The University of Namur (FUNDP), Rue de Bruxelles 61, B-5000, Namur, Belgium Received June 24, 2010
The present study aimed at evaluating the toxicity of short-term cadmium (Cd) exposure in the European bullhead Cottus gobio, a candidate sentinel species. Several enzymatic activity assays (citrate synthase, cytochrome c oxidase, and lactate dehydrogenase) were carried out in liver and gills of fish exposed to 0.01, 0.05, 0.25, and 1 mg Cd/L for 4 days. Exposure to high Cd concentrations significantly altered the activity of these enzymes either in liver and/or in gills. Second, 2D-DIGE technique was used to identify proteins differentially expressed in tissues of fish exposed to either 0.01 or 1 mg Cd/L. Fiftyfour hepatic protein spots and 37 branchial protein spots displayed significant changes in abundance in response to Cd exposure. A total of 26 and 12 different proteins were identified using nano LC-MS/ MS in liver and gills, respectively. The identified differentially expressed proteins can be categorized into diverse functional classes, related to metabolic process, general stress response, protein fate, and cell structure for instance. This work provides new insights into the biochemical and molecular events in Cd-induced toxicity in fish and suggests that further studies on the identified proteins could provide crucial information to better understand the mechanisms of Cd toxicity in fish. Keywords: cadmium • proteomics • metabolic enzymes • sentinel fish • Cottus gobio
Introduction Cadmium (Cd), a nonessential element, commonly detected in aquatic and terrestrial environments, is released both from natural sources and anthropogenic activities.1 It is a toxic metal with no known biological function in animals and can interfere with various physiological processes in organisms from invertebrates to mammals.2 Cd, as well as mercury, lead and arsenic, exerts its toxicity by multiple mechanisms due to its high affinity for sulfhydryl groups (SH) that play an important role in redox balance of the cell and in structure and function of many enzymes.3 Following Cd treatment, evidence suggests an increase production of reactive oxygen species (ROS). This resulting change in the redox state of the cell is believed to be associated with oxidation of macromolecules, altered calcium homeostasis, as well as disturbances in the antioxidant defense system.4 In order to gain a more detailed toxicological comprehension, “-omics” approaches are useful to complement data acquired at higher levels of biological organization.5 Proteomic analysis, providing global protein information, is one of the possible strategies to provide insight into the underlying mechanisms of chemically induced toxicity. This approach has * To whom correspondence should be addressed. Unite´ de Recherche en Biologie des Organismes (URBO), The University of Namur (FUNDP), Rue de Bruxelles 61, B-5000, Namur, Belgium. Tel.: +32(0)81/724285. Fax: +32(0)81 /724362. E-mail:
[email protected]. † Unite´ de Recherche en Biologie des Organismes. ‡ Unite´ de Recherche en Biologie Cellulaire.
470 Journal of Proteome Research 2011, 10, 470–478 Published on Web 11/12/2010
been recently applied in ecotoxicology to gain a better understanding of toxicity and of the mechanisms of action of several toxicants, as for instance perfluorooctanoic acid in rare minnow (Gobiocypris rarus),6 perfluorooctane sulfonate in zebrafish embryos (Danio rerio),7 polychlorinated biphenyls (PCBs) mixture Aroclor 1254 in African clawed frogs (Xenopus laevis),8 microcystin in medaka (Oryzias latipes),9 or tetrabromobisphenol-A in zebrafish liver.10 Nevertheless, the use of proteomics in environmental toxicology is still in its infancy due to a number of drawbacks such as the limited number of organisms fully covered in sequence databases.11,12 The toxicity of Cd to animals including fish has been extensively studied. Most studies have focused on the biological impact of Cd and their effects on living organisms both in nature and in the laboratory. The alteration of protein expression in aquatic organisms exposed to Cd has also been explored in different species,13-17 but it is still in its infancy in fish.18-21 To extend our understanding with respect to the toxic effects and modes of action of Cd in fish, the effects of short-term sublethal Cd exposure in liver and gill tissues of the European bullhead Cottus gobio were investigated by monitoring the response of some enzymes (citrate synthase CS, cytochrome c oxidase CCO, and lactate dehydrogenase LDH), and by undertaking a proteomic analysis using two-dimensional differential in-gel electrophoresis (2D-DIGE) technique. The European bullhead, a small bottom-dwelling freshwater cottid fish, has become endangered in several areas like Switzerland, Germany and the northern part of Belgium as a result of pollution and 10.1021/pr100650z
2011 American Chemical Society
Sentinel Fish Species, Cottus gobio 22
habitat destruction. Bullheads typically live in well oxygenated streams with rocky bottoms, and commonly co-occur with freshwater species associated to waters of good biological quality, for example, salmonid fish and polluosensitive insects (Ephemeroptera, Plecoptera and Trichoptera).23 In addition, bullheads are nonmigratory and have small home ranges. Because of these characteristics, bullhead has been chosen in our study as a candidate sentinel species reflecting the biodiversity of headwater zones in river networks.24
Material and Methods Animals and Exposure Condition. Adult European bullhead of both genders weighing 9.0 ( 3.4 g were caught by electrofishing in the Samson River (Belgium) in May 2008. Fish were acclimated to laboratory conditions in dechlorinated tap water at 15.3 ( 1.4 °C under a 14:10 h (light/dark) photoperiod for 4 weeks before the experiment. During the acclimation period, fish were fed daily to satiation with chironomid larvae (Chironomus sp.). After acclimation, 90 fish were randomly distributed over 15 tanks filled with 16 L dechlorinated tap water. Fish were exposed to CdCl2 (Sigma C2544) at nominal concentrations of 0.01, 0.05, 0.25, and 1 mg/L during 4 days while the control fish were kept in clean water. Each treatment included three replicate tanks, with 6 fish per tank. After 4 days of exposure, each fish was weighed, and liver and gills were collected on ice, directly snap-frozen in liquid nitrogen and stored at -80 °C until homogenization. Animals were not fed during exposure, and half-water was gently siphoned out, replaced, and recontaminated every day. No mortality was observed during the experiment. In previous studies on related fish species, the 96-h LC50 value for adult Cottus bairdi was found to be 0.176 mg Cd/L;25 while Mebane reported a 96-h LC50 value for Cottus confusus (30-60 mm in length) of 0.00013 mg Cd/L.26 Total Cd concentrations in the exposure water were monitored every other day using a Sector Field Inductively Coupled Plasma Mass Spectrometer (Thermo Finnigan Element 2) and an Atomic Absorption Spectrometer (PU9200X Philips, The Netherlands). Certified reference water samples (Riverine water certified reference material SLRS-4, National Research Council Canada) were also analyzed for Cd during each analytical run; measured Cd concentrations were consistently within the certified range. Cd water concentrations were stable over the course of the experiment; the mean concentrations and standard deviations were 0.0002 ( 0.0002, 0.0093 ( 0.0008, 0.0610 ( 0.0046, 0.2597 ( 0.0147, and 0.9979 ( 0.0430 mg/L, respectively. Metabolic Enzyme Activities. Enzymatic activities were assessed in liver and gills from 6 fish pooled per replicate tank. One unit of fish tissue was homogenized with 10 (liver) or 15 (gills) units of ice-cold phosphate buffer (100 mM, pH 7.4) containing Complete-MiniTM Protease inhibitor cocktail (Roche). The homogenates were centrifuged at 1000× g for 10 min at 4 °C, and the supernatants were kept at -80 °C for enzyme activity assays. Protein contents were determined by the method of Lowry et al.27 using Folin’s reagent and BSA as standard. The experimental conditions for testing the enzymatic activities were as follow: Citrate synthase (CS): 100 mM Tris/HCl, 0.1 mM DTNB, 0.3 mM acetyl CoA, 0.5 mM oxaloacetate, pH 8.1. Cytochrome c oxidase (CCO): 22 µM cytochrome C 90% reduced with sodium hydrosulfite, 1 mM EDTA, 30 mM potassium phosphate, pH 7.4. Lactate dehydrogenase (LDH):
research articles 100 mM Tris/HCl, 0.3 mM NADH, 10 mM pyruvate, pH 7.4. Reactions were assayed spectrophotometrically following the reduction of DTNB for CS (at 412 nm), the oxidation of cytochrome C for CCO (at 550 nm), and the oxidation of NADH for LDH (340 nm). Millimolar extinction coefficients used were 13.6 for DTNB, 21.84 for reduced cytochrome C, and 6.22 for NADH. Enzymatic activities were performed in duplicate. They are expressed in milliunit per mg protein. One unit corresponds to the amount of the enzyme that will convert 1 µmol of substrate into product per minute. Statistical Analysis. Results for the enzymatic activities were expressed as the mean (n ) 3) ( SD. Normality analysis of data was assessed by the Shapiro-Wilks W test. Homogeneity of variances was tested by the Bartlett test. Differences between groups were analyzed using one-way analysis of variance followed by a multiple comparison Fisher LSD test at a 5% significant level. All tests were performed using the Statistica 5.5 software (StatSoft, INC. 2000). Protein Extraction and CyDye Labeling. Proteins from liver and gill tissue were extracted from fish exposed to 0, 0.01, and 1 mg Cd/L after 4 days of exposure. There were three replicates per treatment. One unit of fish tissue was homogenized with 10 units of ice-cold RIPA buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% v/v Nonidet P-40, 1% v/v Triton X-100, 1% w/v CHAPS, 2 mM NaF, 2 mM activated Na3VO4) containing Complete-MiniTM Protease inhibitor cocktail (Roche). Each homogenate was maintained for 10 min on ice for protein release. The soluble protein fractions were harvested by centrifugation at 19 000× g for 15 min at 4 °C and the pellet discarded. Supernatants were aliquoted into 1.5 mL siliconized microcentrifuge tubes, and protein concentration was determined using the method of Bradford28 with BSA as a standard. A sample volume containing 300 µg of proteins was then precipitated for 2 h at -30 °C in 4 volumes of precooled 100% acetone/10% TCA. Precipitated proteins were centrifuged at 10 000× g for 10 min at 4 °C, and the pellets were rinsed 4 times in pure acetone. The pellets were left 1 h on ice in acetone during the last round, and air-dried for 1 min. Proteins were resuspended in DLA buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris/HCl, pH 8.5). The pH of the protein extract was adjusted to 8.5 by addition of the appropriate volume of 50 mM NaOH, and protein concentration was measured using the method of Bradford.37 For DIGE minimal labeling, 25 µg of protein were labeled with 200 pmol of fluorescent amine reactive Cyanine dyes freshly dissolved in anhydrous dimethyl formamide following the manufacturer’s recommended protocols (GE Healthcare). Labeling was performed on ice for 30 min in the dark and quenched with 1 mM lysine for 10 min on ice. Cy3 and Cy5 were used to label samples, while a mixed sample composed of equal amounts of proteins from each replicate was minimally labeled with Cy2 and was used as the internal standard. The three labeled mixtures were combined and the total proteins (75 µg) were added v/v to reduction buffer (7 M urea, 2 M thiourea, 2% DTT, 2% CHAPS, 2% IPG 4-7 buffer) for 15 min at room temperature. Separation of Proteins by 2D DIGE. Prior to electrofocusing, IPG strips (24 cm, pH 4-7; GE Healthcare) were passively rehydrated overnight with 450 µL of a standard rehydration solution (7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG 4-7 buffer, 2% DTT). Sample sets containing the labeled mixtures were then cup-loaded onto the IPG strips and isoelectric focusing was performed with an Ettan IPGphor II isoelectric Journal of Proteome Research • Vol. 10, No. 2, 2011 471
research articles focusing unit (GE Healthcare). The electrophoresis conditions were as follows: 20 °C for a total of 68 000 V-h. Focused IPG strips were reduced (1% DTT) and alkalized (2.5% iodoacetamide) in equilibration buffer (50 mM Tris, 6 M urea, 30% glycerol, 2% SDS, pH 8.8) just before loading onto a 10% 24 cm, 1 mm thick acrylamide gel. The strips were overlaid with 1% agarose in SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) and run in an ETTAN DALTsix electrophoresis unit (GE Healthcare) at constant 2 W/gel at 15 °C until the blue dye front had runoff the bottom of the gels. Image Analysis and Statistics. Labeled CyDye gels were scanned with a Typhoon 9400 scanner (GE Healthcare) at wavelengths specific to the CyDyes (488 nm for Cy2, 532 nm for Cy3, and 633 nm for Cy5). Resolution was of 100 µm. The PMT were set to ensure maximum pixel intensity between 40 000 and 60 000 pixels. Image analysis was performed using the DeCyder BVA 5.0 software (GE Healthcare). Briefly, the Differential In-Gel Analysis (DIA) module codetected and differentially quantified the protein spot intensity in each image using the internal standard sample as a reference to normalize the data. At a second step, the Biological Variation Analysis (BVA) was used to calculate ratios between samples and internal standard abundances by performing a gel-to-gel matching of the internal standard spot maps for each gel. Data was analyzed using one-way analysis of variance (ANOVA). When significant differences between groups were found (p < 0.05), a multiple comparison Fisher LSD test was used to demonstrate the significant differences between means. Mass Spectrometry and Protein Identification. For peptide sequencing and protein identification, preparative gels including 250 µg of proteins of mixed samples were performed following the protocol described above except they were poststained with 10% krypton overnight after twice 30 min of fixation in 40% ethanol, 10% acetic acid. Peptides were analyzed by using nanoflow LC-ESI-MS/MS (Waters) instrument on a CapLC Q-TOF2 mass spectrometer (Waters). Spots were excised from preparative gels using the Ettan Spot Picker (GE Healthcare), and proteins were digested with trypsin by in-gel digestion. The gel pieces were twice washed with distilled water and then shrunk with 100% acetonitrile. The proteolytic digestion was performed by the addition of 3 µL of modified trypsin (Promega) suspended in 100 mM NH4HCO3 cold buffer. Proteolysis was performed overnight at 37 °C. The supernatant was collected and combined with the eluate of a subsequent elution step with 5% formic acid. The eluates were kept at -20 °C prior to analysis. The digests were separated by reverse-phase liquid chromatography using a 75 µm × 150 mm reverse phase NanoEase Column (Waters) in a CapLC (Waters, USA) liquid chromatography system. Mobile phase A was 95% of 0.1% formic acid in water and 5% acetonitrile. Mobile phase B was 0.1% formic acid in acetonitrile. The digest (15 µL) was injected, and the organic content of the mobile phase was increased linearly from 5% B to 40% in 40 min and from 40% B to 100% B in 5 min. The column effluent was connected to a PicoTip emitter (New Objective) inside the Q-TOF source. Peptides were analyzed in the DDA mode on a Q-TOF2 (Waters) instrument. In survey scan, MS spectra were acquired for 1 s in the m/z range between 450 and 1500. When intensity of 2+ or 3+ ions increased above 20 counts/s there was an automatic switch to the MS/MS mode. The collision-induced dissociation (CID) energy was automatically set according to mass to charge (m/ z) ratio and charge state of the precursor ion. Acquisition in 472
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Dorts et al. MS/MS was stopped when intensity fell below 5 counts/s or after 15 s. Q-TOF2 and CapLC were piloted by MassLynx 4.0 (Waters). For the electrospray survey, background was subtracted with a threshold of 35%, polynomial order 5. For smoothing, we used the Savitzky-Golay method with 2 iterations and a window of 3 channels. Finally, we assigned the mass of peaks with 3% of threshold, a minimum peak width of 4 channels and a centroid top method at 80%. For MS/MS raw data, we performed a rigorous deisotoping method with a threshold of 3%. Peak lists were created using ProteinLynx Global Server 2.2.5 (Waters) and saved as PKL file for use with Mascot 2.2 (Matrix Science). Enzyme specificity was set to trypsin, and the maximum number of missed cleavages per peptide was set at one. Carbamidomethylation was allowed as fixed modification and oxidation of methionine as variable modification. Mass tolerance for monoisotopic peptide window and MS/MS tolerance window were set to (0.3 Da. The peak lists were searched against the full NCBInr database (9694989 sequences downloaded on September the 15th 2009). Scaffold (version Scaffold2_06_01, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.2) and X! Tandem (The GPM, thegpm.org; version 2007.01.01.). Peptide identifications were accepted if they could be established at greater than 95% probability as specified by the Peptide Prophet alogarithm.29 Protein identifications were accepted if they could be established at greater than 99% probability and contained at least 1 identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm.30 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principle of parcimony.
Results and Discussion Metabolic Enzyme Activities. Cd is a hazardous environmental pollutant known to cause a wide spectrum of toxic effects on aquatic fauna and flora. One of the main routes by which Cd exerts its toxic actions is by altering enzyme activities. The activities of metabolic enzymes measured in control and exposed bullhead are depicted in Table 1. First of all, the aerobic capacity of liver and gill tissues was estimated by the activity of citrate synthase (CS), the first enzyme of the Krebs cycle located within the mitochondria, and by the activity of the cytochrome c oxidase (CCO), the terminal enzyme of the electron transport system located in the inner membrane of the mitochondria. In the liver, exposure to 0.25 mg Cd/L weakly increased the activity of CS by 15%, while a 24% significant decrease was observed in fish exposed to 1 mg Cd/L. No significant changes occurred in hepatic CCO activity after 4 days of Cd exposure. In gills, the activity of CCO increased by 40% after exposure to 1 mg Cd/L, while no significant changes occurred at lower Cd concentrations. Moreover, no significant change occurred in branchial CS activity after Cd exposure. Experimental evidence indicates that mitochondria are likely to be an early, if not the primary target for Cd-induced cytotoxicity.31,32 Previous studies have examined the in vitro impact of Cd on key mitochondrial enzymes activity.33,34 Ivanina et al.33 have shown that Cd exposure resulted in a decline of mitochondrial enzyme activities in gills and hepatopancreas of the eastern oyster Crassostrea virginica. Similarly in vivo inhibition of CS correlated with Cd accumulation was
research articles
Sentinel Fish Species, Cottus gobio
Table 1. Activities (mU/mg protein) of Metabolic Enzymes (mean ( S.D.) Measured in Liver and Gill Tissues of C. gobio Exposed for 4 Days to Different Sublethal Cd Concentrationsa tissues
parameters
0 mg Cd/L
0.01 mg Cd/L
0.05 mg Cd/L
0.25 mg Cd/L
1 mg Cd/L
Liver
CS CCO LDH CS CCO LDH
23.8 ( 1.6b 60.9 ( 10.6 23.8 ( 4.6a 50.8 ( 2.7 12.4 ( 0.4b 161.5 ( 20.6ab
25.8 ( 2.2ab 59.1 ( 8.3 25.1 ( 4.5a 56.0 ( 3.4 10.5 ( 1.8b 142.3 ( 6.1a
24.7 ( 1.7ab 48.1 ( 5.0 11.5 ( 1.5c 57.4 ( 9.0 10.9 ( 0.40b 111.2 ( 10.9b
27.3 ( 0.2a 50.3 ( 17.0 17.2 ( 3.8b 52.2 ( 1.8 13.6 ( 2.3b 87.4 ( 12.6c
18.2 ( 1.5c 65.0 ( 13.5 13.7 ( 2.3bc 59.3 ( 0.2 21.1 ( 1.1a 95.1 ( 14.9c
Gills
a CS, citrate synthase; CCO, cytochrome c oxidase; LDH, lactate dehydrogenase. Different letters (a, b, and c) mean significant (p1 indicate up-regulation and 1 indicate up-regulation and
