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Alterations in the Atlantic COD (Gadus morhua) Hepatic Thiol-Proteome After Methylmercury Exposure a

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a

O. A. Karlsen , D. Sheehan & A. Goksøyr a

Department of Biology, University of Bergen, Bergen, Norway

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Proteomics Research Group, Department of Biochemistry, University College Cork, Cork, Ireland Published online: 22 Apr 2014.

Click for updates To cite this article: O. A. Karlsen, D. Sheehan & A. Goksøyr (2014) Alterations in the Atlantic COD (Gadus morhua) Hepatic Thiol-Proteome After Methylmercury Exposure, Journal of Toxicology and Environmental Health, Part A: Current Issues, 77:9-11, 650-662, DOI: 10.1080/15287394.2014.887427 To link to this article: http://dx.doi.org/10.1080/15287394.2014.887427

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Journal of Toxicology and Environmental Health, Part A, 77:650–662, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287394.2014.887427

ALTERATIONS IN THE ATLANTIC COD (Gadus morhua) HEPATIC THIOL-PROTEOME AFTER METHYLMERCURY EXPOSURE O. A. Karlsen1, D. Sheehan2, A. Goksøyr1 1

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Department of Biology, University of Bergen, Bergen, Norway Proteomics Research Group, Department of Biochemistry, University College Cork, Cork, Ireland

Proteomic studies in general have demonstrated that the most effective and thorough analysis of biological samples requires subfractionation and/or enrichment prior to downstream processing. In the present study, Atlantic cod (Gadus morhua) liver samples were fractionated using activated thiol sepharose to isolate hepatic proteins containing free/reactive cysteines. This subset of proteins is of special interest when studying the physiological effects attributed to methylmercury (MeHg) exposure. Methylmercury is a persistent environmental contaminant that has a potent affinity toward thiol groups, and can directly bind proteins via available cysteine residues. Further, alterations in the cod thiol-proteome following MeHg exposure (2 mg/kg body weight) were explored with two-dimensional gel electrophoresis combined with downstream mass spectrometry analyses for protein identifications. Thirty-five protein spots were found to respond to MeHg exposure, and 13 of these were identified when searching cod-specific databases with acquired mass spectrometry data. Among the identified thiol-containing proteins, some are known to respond to MeHg treatment, including constituents of the cytoskeleton, and proteins involved in oxidative stress responses, protein synthesis, protein folding, and energy metabolism. Methylmercury also appeared to affect cod heme metabolism/turnover, producing significantly altered levels of hemoglobin and hemopexin in liver following metal exposure. The latter finding suggests that MeHg may also affect the hematological system in Atlantic cod.

The molecular mechanisms underlying MeHg-induced toxicity have been extensively studied in mammals, and equivalent studies in fish are rapidly emerging (Berg et al., 2010; Cambier et al., 2012; Gonzalez et al., 2005; Nostbakken et al., 2012; Yadetie et al., 2013; Olsvik et al., 2011). The neurological system is considered the main target of MeHg, and effects on the Atlantic cod (Gadus morhua) brain proteome and the Atlantic salmon (Salmo salar) neurological system were recently published (Berg et al., 2010; Berntssen et al, 2003). These analyses revealed that MeHg induced oxidative stress, cell structural degeneration, and altered calcium homeostasis and energy metabolism. These observations are in agree-

Methylmercury (MeHg) is a persistent environmental contaminant that is widely distributed in nature. It originates from elemental mercury (Hg), which is released into the environment from both natural sources and anthropogenic activities, such as burning of fossil fuels, gold mining, and Hg ores (Clarkson, 1997). Inorganic Hg is metabolized to MeHg by sulfate-reducing bacteria, resulting in one of the most toxic forms of organic Hg (Jensen and Jernelov, 1969). Further, the chemical properties of MeHg enable this compound to bioaccumulate and biomagnify in marine food chains, where it eventually poses a threat to the higher trophic levels of fish and mammals through dietary exposure (Bloom, 1992).

Address correspondence to O. A. Karlsen, Department of Biology, University of Bergen, PO Box 7803, Bergen, N-5020, Norway. E-mail: [email protected] 650

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ment with physiological effects of MeHg exposure demonstrated in similar studies on mammals (Atchison and Hare, 1994; Belletti et al., 2002; Dreiem et al., 2005; Dreiem and Seegal, 2007; Miura et al., 1999; Ni et al., 2012). However, the adverse responses to MeHg are not limited to the neurological system. Both proteomic and transcriptomic analyses noted adverse effects in other tissues as well, such as the liver, kidneys, and gonads of several different species, including zebrafish, fathead minnow, and Atlantic cod (Klaper et al., 2008; Cambier et al., 2009; Nostbakken et al., 2012; Yadetie et al., 2013; Olsvik et al., 2011). One important aspect of MeHg-induced toxicity is the strong affinity of this compound toward protein thiols (-SH) (Hughes, 1957). It has been shown that MeHg is able to reversibly bind exposed cysteine residues in a range of different proteins including both abundant structural proteins and key metabolic enzymes (Miura et al., 1999; Eriksson and Svenson, 1978). The high affinity for thiol groups makes all cysteine-containing proteins potential targets, regardless of their biological function and cellular process involvement. This chemistry may partly explain the multifactorial and complex situation of effects observed upon MeHg exposure, as no single molecular mechanism alone can fully explain the diversity of cellular responses triggered by this compound. Fractionation using activated thiol sepharose (ATS) was introduced as a feasible and robust method to capture proteins containing accessible and reactive thiols in complex biological mixtures (Hu et al., 2010a). ATS recently demonstrated its usefulness in redox proteomics for studying the role of protein cysteines in adaption to changes in cellular redox status (Hu et al., 2010b, 2010c; Company et al., 2012). Further, the advantages of ATS fractionation are twofold. First, this technique provides a one-step enrichment tool for thiol-containing proteins, including low-abundance proteins that in general are not recovered and quantified in traditional proteomic separations. Second, this method reveals important variation in the thiol status between samples. Although cysteines are relatively rare in proteins (1–2%), this residue

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is one of the most chemically reactive amino acids (Miseta and Csutora, 2000). Importantly, modified or oxidized cysteines are no longer able to bind to the ATS resin, and changes in the thiol-proteome may therefore be traced and mapped in downstream analyses. In the present study, ATS fractionation for isolation of the Atlantic cod hepatic thiol-proteome and two-dimensional (2-D) gel electrophoresis were combined to assess the effects of MeHg on this subset of proteins in cod liver. Thirteen of the 35 spots, initially identified to differ between treatments, were subsequently analyzed by downstream matrix-associated laser desorption time-of-flight (MALDI–ToF) mass spectrometry for protein identification and functional annotation

MATERIALS AND METHODS MeHg Exposure and Sampling MeHg exposure and tissue sampling of Atlantic cod have been described in detail elsewhere (Berg et al., 2010; Yadetie et al., 2013). Briefly, approximately 1.5-yr-old penreared juvenile Atlantic cod (260–530 g body weight [BW]) of mixed gender were purchased and maintained at the Industrial and Aquatic Labarotary (ILAB) in Bergen, Norway, in 500L tanks supplied with continuously flowing seawater at 10◦ C and 34‰ salinity. A 12-h light/dark cycle was used and fish were fed daily with commercial pellets. After acclimation for 6 d, fish were divided into 4 groups (n = 10/group) in separate tanks and injected intraperitoneally (ip) with vehicle (20% acetone and 80% soybean), 0.5, 2, or 8 mg/kg BW MeHg chloride. The doses were administered in 2 aliquots with a 1-wk interval. After 14 d of first injection, fish were sacrificed, and tissues were extracted, frozen in liquid nitrogen, and stored at −80◦ C until use. MeHg exposure levels and dose-dependent distribution to the brain were confirmed by chemical analyses and spans from levels found in the environment (0.5–1 mg/kg being regulatory limits for Hg in fish: Codex Alimentarius, http://www.codexalimentarius.net). Liver tissue harvested from 9 individuals from control and

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9 individuals from the 2 mg/kg body weight MeHg-exposed group were used in the present study. Proteomic and transcriptomic analyses of brain and the liver, respectively, obtained from the same individuals were recently published (Berg et al., 2010; Yadetie et al., 2013).

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Cod Liver Homogenization and Activated Thiol Sepharose Fractionation Thawed cod livers (approximately 250 mg) were washed and further homogenized in 1 ml of phosphate-buffered saline (PBS, pH 7.4) with a FastPrep-24 and Lysing Matrix D for 30 s. Homogenates were subsequently centrifuged at 13,000 × g for 10 min. The supernatant was kept as the crude hepatic proteome, and total protein concentrations were determined with a microplate adaption of the Bradford assay (1976). ATS fractionation for isolation of the hepatic thiol-cotaining subproteome was performed essentially as described by Hu et al. (2010a). Briefly, 2 mg of crude hepatic proteome fraction (obtained as described earlier) was mixed with ATS (20 mg) and 200 μl binding buffer (0.1 M Tris-HCl [pH 7.5], 0.5 M NaCl, 1 mM ethylene diamine tetraacetic acid [EDTA]), followed by incubation on ice with shaking for 1.5 h. Samples were centrifuged (13,000 × g, 3 min) and washed with 500 μl of binding buffer 8 times (centrifuged after each wash and the supernatant discarded). One hundred microliters elution buffer (binding buffer added 25 mM dithiothreitol [DTT]) was added to the ATS beads after the final washing step, and subsequently incubated for 20 min on a rotating wheel at 4◦ C to release thiol-containing proteins. The beads were centrifuged at 13,000 × g, 3 min, and supernatant was collected as the hepatic thiol-containing subproteome. The Bradford (1976) assay was used for protein concentration measurement of resulting thiol-containing protein samples. Two-Dimensional (2-D) Gel Electrophoresis Twenty-five micrograms protein from each of the hepatic thiol proteome samples was

precipitated with 10% trichloroacetic acid (TCA) and resuspended in 120 μl twodimensional electrophoresis (2DE) rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mM dithiotreitol [DTT], 0.5% Triton X-100, 0.5% ampholine 3.5–10, and trace bromphenol blue). Two-dimensional gel electrophoresis was further carried out as described previously, using 7 cm Immobiline DryStrips, pH 4–7 (linear), for isoelectric focusing, and 12.5% polyacrylamide gels in the second dimension electrophoresis (Berg et al., 2010). Image Analyses and Statistics The 2DE image analysis was performed using the Delta2D (4.0) software (Decodon), as described previously (Berg et al., 2010). Statistical significance was evaluated using Student’s t-test (two-tailed) and including all biological replicates (individuals). Significance was defined as p < .05. Multivariate statistics, including principal component analyses (PCA) and hierarchial clustering, were performed with the JMP 10 software (SAS Institute, Inc.). MALDI-ToF Mass Spectrometry and Database Searches Protein spots excised from the 2DE gels were washed and trypsinated, followed by desalting and concentration of proteolytic peptides, as described previously (Berg et al., 2010). Samples were subsequently spotted onto a MALDI target with a matrix solution consisting of 6 mg/ml α-cyano-4-hydroxycinnamic acid (CHCA) dissolved in 60% acetonitrile (ACN), 15% methanol, and 0.1% trifluoroacetic acid (TFA). Mass spectra were acquired on an Ultraflex 2 ToF/ToF mass spectrometer (Bruker Daltonics) in positive reflector mode, using the FlexControl and FlexAnalysis software (Bruker Daltonics). Mass spectrometry (MS) spectra were externally calibrated. When possible, the most intense peaks of the MS spectra were selected for MS/MS fragmentation. The Mascot interface was used for database searches with the acquired MS and MS/MS data. Two different databases containing Atlantic cod specific

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sequences were used, one expressed sequence tag (EST) assembly and one genome assembly. The EST assembly (GmE100215) was provided by the Computational Biology Unit (Uni Research, Bergen, Norway) in collaboration with Institute of Marine Research (Bergen, Norway), Norwegian Institute of Research on Nutrition and Seafood (Bergen, Norway), and groups affiliated with the National Research Council, Canada. The genomic cod data were provided by the Cod Genome project (http:// www.codgenome.no). A database of protein sequences (GmA100602) predicted by the ENSEMBL annotation pipeline, based on the GmG100427 Atlantic cod genome assembly, was used.

Functional Analyses of Proteins Annotation of identified proteins and assignment of human orthologs were performed manually using BLASTp (NCBI) and the human proteome database provided by SWISSPROT. Functional annotation, network, and pathway analyses were carried out using the Ingenuity Pathway Analysis (IPA) software. IPA performs a search with the queried proteins in the Ingenuity Pathways knowledge database and predicts possible networks of which the proteins may interact. IPA calculates a network score and links the assigned proteins to other possible interaction partners. This calculation is based on the hypergeometric distribution, and is calculated using a right-tailed Fisher’s exact test. For more details on IPA see www.ingenuity. com.

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Bradford assay, and corresponded to approximately 2–3% of the initial amount of hepatic proteins mixed with the resin (2000 μg→ 20–30 μg). This recovery of thiol-containing proteins was in agreement with previously reported yields obtained with this procedure (Hu et al., 2010a, 2010b; Company et al., 2012). The protein profiles of the crude hepatic proteome (prior to ATS fractionation) and the thiol-proteome were assessed and compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 1). The resulting gels revealed significant differences in protein profiles of crude homogenates and corresponding ATS-enriched samples, suggesting that thiol-containing proteins were successfully enriched. Further, hepatic thiol-containing subproteomes obtained from nine control and nine MeHg-exposed individuals were resolved with 2DE gel electrophoresis to reveal alterations in the thiol-protein profiles (Figure 2). The Delta2D software (Decodon) was used for quantitative analyses, and, in total, 436 spots were identified and quantified in these 2DE gels. Thirty-five of the protein spots (passing visual inspection) were considered as differentially expressed between control and

RESULTS Two-Dimensional Gel Electrophoresis of the Cod Hepatic Thiol-Proteome Atlantic cod liver samples were homogenized and further subjected to ATS fractionation for capturing the subset of proteins containing accessible and reactive cysteine residues. The quantities of –SH-containing proteins retained on the ATS beads were determined with the

FIGURE 1. Comparative SDS-PAGE analysis of the crude hepatic proteome and the hepatic thiol-proteome. Lanes 1–3: representative samples of the crude hepatic proteome isolated from liver tissue obtained from three different individuals of the control group. Lanes 4–6: representative samples of the hepatic thiolproteome obtained by ATS fractionation of the hepatic proteome of the corresponding individuals shown in Lanes 1–3. The polyacrylamide gel was stained with Coomassie brilliant blue G250. Molecular weight markers are indicated.

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algorithm produced two main clusters, each harboring either all control or all exposed samples (Figure 4B). Although sample variation exists within our data set (which is to be expected when analyzing individual animals), the consistent responses in thiol-containing subproteomes to MeHg treatment suggest that our data are robust and reflect consistent physiological effects in cod liver.

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Protein Identifications by Mass Spectrometry

FIGURE 2. Two-dimensional gel electrophoresis of the hepatic thiol-proteome. A representative 2DE gel of the hepatic thiolcontaining subproteome obtained from a control Atlantic cod is shown. Twenty-five micrograms of the ATS-enriched thiolproteome fraction was resolved with a 7-cm IPG strip (pH 4–7) in the first dimension isoelectric focusing, followed by the second dimension electrophoresis in a 12.5% polyacrylamide gel. Statistically significant differentially expressed proteins between control and MeHg-exposed cod are encircled and numbered according to Delta2D. Proteins identified by MALDI-ToF mass spectrometry are indicated in white. The 2DE gel was stained with colloidal Coomassie. Molecular mass markers are indicated.

exposed samples using Student’s t-test statistics and a fold change of at least 1.2 (0.8). Twenty-three of these proteins were more abundant in the thiol-proteome after MeHg exposure, while 12 proteins decreased their abundance (Figure 3). Since the majority of the differentially expressed spots were identified in elevated amounts, data suggest that ATS fractionation functioned as a one-step enrichment procedure that also revealed thiolproteins responding to MeHg treatment without binding directly to this compound. Multivariate Statistics The 2DE data were further evaluated by applying multivariate statistics including PCA and hierarchical clustering. The threedimensional PCA plot demonstrated clearly that cod liver thiol-proteome samples clustered into two distinct groups, distinguishing between control and MeHg exposure (Figure 4A). In agreement with the PCA plot, pattern analyses using a two-way hierarchical clustering

The most abundant spots representing differentially expressed proteins were excised from our gels and analyzed with MALDI-ToF mass spectrometry (MS). Both MS and MS/MS spectra were recorded when possible and further queried for protein identification in the inhouse cod-specific EST and genomic databases. The majority of the differentially expressed spots were of low abundance and could not be recovered in MS analyses. However, it was possible to identify 13 spots representing 10 different protein species (Table 1). Functional Annotation and Pathway Analyses Although the number of proteins identified in this study is limited, IPA was used to explore their known role in cellular pathways and diseases, their biological functions, and protein-protein interactions. The differentially expressed proteins could all be included in a single network (Figure S1), visualizing interactions between identified MeHg-responding proteins and proteins in the databases known to interact with the queried proteins. The physiological functions associated with this network were cancer, hematological disease, and cellular assembly and organization. Other biological functions related to the differentially regulated proteins are summarized in Table 2. DISCUSSION The hepatic proteome and hepatic thiolcontaining subproteome were isolated from

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FIGURE 3. Expression profiles (fold change) of differentially expressed proteins in the hepatic thiol-containing subproteome. The vertical axis corresponds to the log2 -transformed average ratio (exposed vs. control) of expression. Values above 0 indicate upregulated proteins, while values below 0 indicate downregulated proteins. In the horizontal axis of the graph, the upregulated proteins are organized with the highest fold change shown at the left side, while the downregulated proteins are visualized with the highest fold change on the right side. Spot numbers correspond to those given in Figure 2.

FIGURE 4. Multivariate statistical analysis of the proteome changes in the hepatic thiol-proteome after exposure to MeHg. (A) Threedimensional (3D) PCA of spot-volume normalized data of differentially expressed proteins. Individual samples are indicated according to the group to which they belong; blue circles correspond to controls, while red triangles correspond to exposed samples. (B) Twoway hierarchal clustering analysis of normalized data. Individual samples (left axis) are labeled according to group (C_(1–9) control individuals; 2_(1–9) 2 mg/kg body weight MeHg-exposed individuals). Lower axis indicates spot numbers. Multivariate statistical analyses were performed with the JMP 10 software.

Atlantic cod, and their respective protein profiles were compared using SDS-PAGE. The protein profile of the ATS-fractionated thiolcontaining subproteome was clearly distinct from the crude hepatic proteome, suggesting that a significant enrichment of proteins containing accessible and reactive cysteine residues was achieved. In addition, the majority of the protein bands (SDS-PAGE) resolved from thiolcontaining proteins was not easily observed in the crude hepatic protein fractions, suggesting that many of these proteins are of

low abundance and were successfully recovered by the one-step fractionation procedure. ATS fractionation was further used to capture the thiol-containing subproteomes from juvenile Atlantic cod injected ip with MeHg. The thiol-containing subproteomes were analyzed by 2DE and compared to thiol-containing subproteomes obtained from unexposed individuals. Multivariate statistical analyses of 2DE data showed evident clustering that clearly distinguished controls and exposed samples, indicating that the acquired data set represents

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TABLE 1. Differentially Expressed Hepatic Thiol-Proteins Identified With MALDI-ToF MS and MS/MS

Spot number

Fold change

p Value (t-test)

Number of peptides, MS (MS/MS)a

Mascot score

Coverage (%)b

Protein identity

51 53 66 75 141 179 313 328 341 343 345 346 373

0.61 0.44 1.35 1.48 1.90 4.09 1.43 0.35 2.12 2.16 1.51 1.28 4.53

0.0123 0.0018 0.0097 0.0009 0.0331 0.0112 0.0037 0.0215 0.0019 0.0003 0.0016 0.0235 1.9E-6

10 (2) 5 (2) 11 (1) 7 (1) 18 (1) 6 (1) 25 (3) 5 (1) 24 (3) 18 (1) 10 (2) 14 (3) 8

207 100 92 62 127 80 293 127 232 213 128 305 65

73 40 18 23 38 28 39 25 51 47 36 43 21

Hemoglobin, subunit β Hemoglobin, subunit β T-complex protein, subunit β Hydroxyphenylpyruvate dioxygenase Asparginyl-tRNA synthetase Hydroxypyruvate isomerase Heat shock protein 70 kD (HSP70) Hemopexin Tubulin, β-chain Tubulin, β-chain Tubulin, a-chain Tubulin, a-chain DnaJ homolog (HSP40)

a Number

of tryptic peptides and MS/MS spectra (in parentheses) matching the amino acid sequence. coverage.

b Sequence

TABLE 2. Summary of the IPA-Tox Analysis of the Differentially Expressed Thiol-Proteins Top Network Cancer, hematological disease, cellular assembly and organization

Scorea 30

Top Tox List Decreased permeability transition of mitochondria and mitochondrial membrane Positive acute-phase response proteins Acute renal failure panel LXR/RXR activation NRF2-mediated oxidative stress response

p Valueb 3.22E-03 1.92E-02 3.92E-02 7.71E-02 1.40E-01

Ratioc 0.2 0.033 0.016 0.008 0.004

Top canonical pathway Remodeling of epithelial adherens junctions 14-3-3-Mediated signaling Epithelial adherens junction signaling Tyrosine degradation I Aldosterone signaling in epithelial cells

p Value 8.32E-04 2.44E-03 3.71E-03 3.86E-03 4.07E-03

Ratio 0.029 0.017 0.014 0.067 0.012

Molecular and cellular functions Cellular assembly and organization Cellular compromise Cellular function and maintainance Amino acid metabolism Cell morphology

p Value 6.44E-04 to 3.80E-02 6.44E-04 to 3.490E-02 6.44E-04 to 2.80E-02 1.93E-03 1.93E-03 to 9.62E-03

Moleculesd 2 4 3 1 2

a The score is a numerical value calculated by IPA and used to rank networks according to how relevant they are to the proteins in the input dataset. The network score is based on the hypergeometric distribution and is calculated with the right-tailed Fisher’s exact test. b The p values are calculated with the right-tailed Fisher’s exact test. c Ratio is calculated by dividing the number of input proteins mapped to a specific pathway on the number of total proteins associated to this pathway. d Molecules indicate how many proteins from the input data set that are involved in the particular function.

consistent and reliable observations regarding physiological responses to MeHg exposure. In total, 35 spots were considered as differentially regulated, and 13 of these were successfully identified by MS. Functional annotation and pathway analyses using IPA indicated

that MeHg affected proteomic markers in the thiol-containing subproteome involved in various biological processes, including cellular assembly and organization (microtubule), amino acid metabolism, protein translation, stress responses (including oxidative stress), and

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cellular functions related to illness such as hematological diseases and cancer. The central node in the network predicted by IPA was ubiquitin C, which is a polyubiquitin precursor with a critical role in cell signaling including protein degradation. Accordingly, Hwang (2011) reported that the ubiquitin-proteasome system plays a prominent role in mediating protection to cells against adverse effects produced by MeHg. It has previously been demonstrated that MeHg affects the cytoskeletal organization in a range of different tissues and cell lines, including human fibroblasts, neuroblastoma, glioma cells, liver, and kidney (Miura and Imura, 1989; Sager et al., 1983; Prasad et al., 1979; Miura et al., 1984; Yadetie et al., 2013; Mela et al., 2007). Tubulins α and β, which are the constituents of the cellular microtubule, were identified in our analyses as components of the hepatic thiol-containing subproteome responding to MeHg exposure. In vitro studies have previously shown that MeHg possesses high affinity for tubulin sulfhydryl groups, which, upon binding, induce depolymerizing of cerebral microtubules and directly inhibit their assembly (Sager et al., 1983; Vogel et al., 1989). A decrease in tubulin synthesis was noted upon MeHg exposure (e.g., mouse glioma cells), and Miura and Imura (1989) suggested that this could partly be ascribed to an autoregulatory depression owing to an increase in the pool of tubulin subunits released from microtubule depolymerization. However, in contrast to the observations reported from MeHg in vitro exposures, both tubulin α and β were found in elevated amounts in the cod hepatic thiolcontaining subproteome after in vivo exposure. This discrepancy may be explained by the manner in which respective cells and liver tissue are exposed to MeHg. Cultured cell lines are subjected to MeHg via the culture medium, promoting uptake of unconjugated MeHg through the cell membrane and thus allowing a rapid reaction with tubulin sulfhydryl groups. It is more unclear which form of MeHg (most likely to a large extent conjugated) enters cod hepatocytes after an ip injection and whether the strong affinity toward tubulin sulfhydryl groups

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is maintained. Importantly, MeHg is a wellknown inducer of oxidative stress, which is considered one of the most important adverse effects induced by this compound (Clarkson, 1997). Previously it was found that an increase in expression of tubulins occurs during oxidative conditions, and evidence indicated that these proteins play an important role in cellular stress protection (Paron et al., 2004; Ibarz et al., 2010). Such a functional role is in agreement with greater abundance of tubulins α and β in cod thiol-containing subproteome after MeHg exposure, which is also further supported by upregulation of HSP70, an often-used biomarker of oxidative/cellular stress (El Golli-Bennour and Bacha, 2011). Upregulation of the tubulin α transcript was also found in a global transcriptome analysis of the same MeHg-exposed Atlantic cod liver tissue as in the present study (Yadetie et al., 2013). Intoxication rapidly induces expression of heat-shock proteins that are regulated at either the transcriptional or posttranscriptional level (Silver and Noble, 2012). As indicated earlier, HSP70 was identified in increased amounts in the thiol-proteome after MeHg exposure. HSP70 proteins have in general a protective function in cells exposed to thermal and oxidative stress, but also respond to other stimuli that act as cellular stressors (e.g., toxic chemicals) (Kregel, 2002). Increased expression of HSP70 was previously observed in regard to both Hg and MeHg exposure (Reus et al., 2003). Further, HSP70 stress response is a highly sensitive marker of cellular aggression, and its increased abundance probably reflects a cellular strategy to protect and repair other cellular proteins damaged by MeHg. Rand et al. (2008) also suggested that HSP70 might counter the effects of MeHg in disrupting thiol disulfide bond formation in newly-synthesized proteins. DnaJ/HSP40 was also detected in elevated amounts after MeHg exposure. DnaJ/HSP40 is a crucial partner for HSP70, and regulates its activity by stabilizing their interactions with substrate proteins (Qiu et al., 2006). Notably, DnaJ/HSP40 and HSP70 are also important components of

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the ubiquitin-mediated proteolysis of proteins pathway (Esser et al., 2004). A third molecular chaperone, T-complex protein (TCP, subunit β), was also found in increased abundance in the hepatic thiol-proteome after treatment with MeHg. TCP chaperones assist protein folding, and have in humans been identified as components of the BBSome complex. Further, Sternlicht et al. (1993) showed that TCP chaperones play a role in the folding of tubulin and actin, suggesting that elevated abundance of TCP subunit β reflects a cellular response to MeHg-induced cytoskeletal disruption. Asparagine-tRNA ligase (asparaginyl-tRNA synthase) was found to be 1.9-fold upregulated in the thiol-containing subproteome of cod exposed to MeHg. In general, the aminoacyltRNA synthetases ensure that the first step of protein translation is performed quickly and accurately. Further, the protein translation machinery is a known target of MeHginduced toxicity (Brubaker et al., 1971; Omata et al., 1978, 1980, 1981; Kuznetsov et al., 1987a, 1987b). However, the particular molecular mechanisms targeted by MeHg in protein synthesis appear to be diverse, but also dependent on tissue, route of administration, and phase of toxicity. In vivo and in vitro reports from mice and rats suggested that MeHg inhibited protein synthesis (Kuznetsov et al., 1987a, 1987b). In contrast, studies demonstrated that MeHg stimulates protein synthesis in rat liver (Omata et al., 1981; Brubaker et al., 1971). The latter observation is in accordance with the elevated amount of asparagine-tRNA ligase in cod liver, and may indicate that MeHg triggers corresponding responses in cod hepatocytes. Other components of the protein translation machinery, including ribosomal subunits, were previously identified in increased levels in rat liver after MeHg exposure (Brubaker et al., 1971). These findings are also in agreement with the recently reported observations made in the cod hepatic transcriptome after MeHg exposure (Yadetie et al., 2013). The hemoglobin subunit β was identified with decreased abundance in the thiolproteome following MeHg exposure. Previously Giblin and Massaro (1975) demonstrated in

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rainbow trout (Oncorhynchus mykiss) that red blood cells (RBC) rapidly accumulate MeHg when it enters the bloodstream, and that hemoglobin is responsible for MeHg binding. Further, Giblin and Massaro (1975) determined that rainbow trout hemoglobin has four reactive sulfhydryl groups that all are able to reversibly bind MeHg. Most likely, ip injected MeHg becomes tightly bound to cod hemoglobin when entering the bloodstream and is distributed widely throughout the cod body by RBC. Interestingly, early reports found that MeHg binding to hemoglobin sulfhydryl groups exerts an impact on heme– heme interaction between the hemoglobin subunits (Riggs and Wolbach, 1956). This may negatively affect the allosteric interactions and the cooperative oxygenation of hemoglobin, which, in turn, impacts overall oxygen transportation in cod. Eventually, hemoglobin is transported to liver when RBC end their life cycle and degradation of the protein occurs. Thus, hemoglobin becomes a transient component of liver proteome. Importantly, decreased amounts of ATS-binding hemoglobin in cod liver may reflect that a significant amount of these proteins is conjugated to MeHg via their cysteine residues, thereby prevent binding of protein to the ATS resin. Interestingly, the lowered level of hemoglobin present in the hepatic thiol-containing subproteome was accompanied with decreased levels of hemopexin. Hemopexin binds heme with the highest affinity of any known protein, and functions in scavenging of heme released or lost by turnover of heme proteins, such as hemoglobin (Tolosano and Altruda, 2002). Hemopexin is mainly synthesized in liver, and is further released into plasma. Hemopexin possesses an important function in preventing hememediated formation of reactive oxygen species (ROS), and is a marker for oxidative stress (Li et al., 2009). Decreased levels of hemopexin were also previously noted in Atlantic salmon exposed to MeHg (Nøstbakken et al., 2012). The hemopexin–heme complex is transported back to the liver, where heme is catabolized, or reused for synthesis of new hemoproteins. The decreased levels of hemopexin in

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the thiol-containing subproteome may, as suggested for hemoglobin, be a result of an interaction with MeHg in the bloodstream, resulting in binding to available and reactive cysteines. However, decreased levels of hemopexin are often used as an indicator of high amounts of heme in circulation, a condition associated with various hemolytic anemias (Delanghe and Langlois, 2001). Thus, diminished levels of hemopexin in liver may then reflect elevated levels of heme in plasma resulting from MeHgmediated hemolysis of RBC. Further proteomic and biochemical analyses of cod plasma are necessary for elucidation of effects of MeHg on the cod hematologic system. Two thiol-containing enzymes were found in elevated amounts in the thiol-proteome: 4-hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxypyruvate isomerase (HYI). HPPD is an abundant liver enzyme that catalyzes conversion of 4-hydroxyphenylpyruvate to homogentisate, the second reaction in the catabolism of tyrosine (Moran, 2005). HYI catalyzes conversion of hydroxypyruvate to 2-hydroxy-3-oxopropanoate. This enzyme is not well characterized in eukaryotes, but studies in bacteria suggests that HYI is most likely involved in carbohydrate transport and metabolism (Ashiuchi and Misono, 1999). Interestingly, Liu et al. (2009) reported that HYI was upregulated in epithelial cells after exposure to anti-7,8-dihydroxy-9,10epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), a metabolite of the polycyclic aromatic hydrocarbon benzo[a]pyrene. Further, the comprehensive transcriptome analyses of MeHg-exposed cod liver revealed that several pathways related to energy metabolism were affected by this compound, including amino acid metabolism, which includes amino acid catabolism, glycolysis/gluconeogenesis, acyl-coenyme A (CoA) metabolism, and fatty acid metabolism (Yadetie et al., 2013). This coordinated response observed for many energy pathways indicates that MeHg severely perturbs nutrient metabolism. Our findings on the protein level regarding enzymes that are functioning in cellular processes related to energy metabolism substantiate that the

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findings at the mRNA level are also evident at the translational level. Proteins acting in cellular processes that are well-known targets of MeHg were revealed in these analyses, but also novel proteins that have previously not been reported associated with MeHg-induced toxicity were identified (Table 1). Moreover, pathway/network analyses conducted with IPA, combined with detailed annotation of identified proteins suggest contaminant responses that affect various biological processes, including the cytoskeletal organization, protein translation, oxidative stress, and cellular functions related to severe illness such as cancer and hematological diseases (Table 2). Our results correlate well with previous studies of MeHg-induced toxicity, further emphasizing the adverse effects accumulation of MeHg may exert on wild animals, with possibly severe impacts on fish populations. A quantitative proteomics study using both gel-based and liquid chromatography (LC)–MS/MS-based methodology to reveal alterations in the crude hepatic cod proteome after MeHg exposure is currently in progress to obtain a greater insight into molecular mechanisms and cellular processes triggered by this toxicant.

FUNDING/ACKNOWLEDGEMENT The authors are grateful to the Norwegian Cod Genome Sequencing Consortium (www. codgenome.no) for providing access to the cod EST and genome assemblies ahead of the publication of the cod genome sequence. Silje Bjørneklett (MSc) was important for establishing the activated thiol sepharose methodology in our laboratory (UiB, Bergen). Eirny Katrin Berg contributed to this article with excellent technical assistance. The Research Council of Norway (RCN) supported this work by grant number 192441/I30.

SUPPLEMENTAL DATA Supplemental data for this article can be accessed at http://dx.doi.org/10.1080/ 15287394.2014.887427

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