Study of the plasma proteome of Atlantic cod (Gadus morhua): Effect of exposure to two PAHs and their corresponding diols

Chemosphere 183 (2017) 294e304

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Study of the plasma proteome of Atlantic cod (Gadus morhua): Effect of exposure to two PAHs and their corresponding diols Karianne Skogland Enerstvedt a, b, Magne O. Sydnes b, Daniela M. Pampanin a, b, * a b

International Research Institute of Stavanger (IRIS) e Environmental Department, Mekjarvik 12, NO-4070 Randaberg, Norway Faculty of Science and Technology, Department of Mathematics and Natural Science, University of Stavanger, NO-4036 Stavanger, Norway

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Initial characterization of female Atlantic cod plasma proteome was obtained.
Identification of 369 plasma proteins was done by shotgun mass spectrometry.
The high abundant protein (HAP) profile of Atlantic cod plasma was characterized.
Twelve proteins were proposed as biomarker candidates of PAH exposure.
PAH exposure triggered an immune system related protein response in cod plasma.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 April 2017 Received in revised form 16 May 2017 Accepted 18 May 2017 Available online 19 May 2017

Occurrence of polycyclic aromatic hydrocarbon (PAH) contamination in the marine environment represents a risk to marine life and humans. In this study, plasma samples from Atlantic cod (Gadus morhua) were analysed by shotgun mass spectrometry to investigate the plasma proteome in response to exposure to single PAHs (naphthalene or chrysene) and their corresponding metabolites (dihydrodiols). In total, 369 proteins were identified and ranked according to their relative abundance. The levels of 12 proteins were found significantly altered in PAH exposed fish and are proposed as new biomarker candidates. Eleven proteins were upregulated, primarily immunoglobulin components, and one protein was downregulated (antifreeze protein type IV.) The uniformity of the upregulated proteins suggests a triggered immune response in the exposed fish. Overall, the results provide valuable knowledge for future studies of the Atlantic cod plasma proteome and generate grounds for establishing new plasma protein biomarkers for environmental monitoring of PAH related exposure. © 2017 Elsevier Ltd. All rights reserved.

Handling Editor: David Volz Keywords: Polycyclic aromatic hydrocarbon Atlantic cod Fish toxicology Plasma proteins Biomarker Proteomics

1. Introduction

* Corresponding author. E-mail address: [email protected] (D.M. Pampanin). http://dx.doi.org/10.1016/j.chemosphere.2017.05.111 0045-6535/© 2017 Elsevier Ltd. All rights reserved.

Occurrence of PAH contamination in the marine environment represents a direct risk to marine life (Pampanin and Sydnes, 2013). Indirectly, it also represents a risk to humans upon seafood consumption, as PAHs have the potential to accumulate in fatty tissues

K. Skogland Enerstvedt et al. / Chemosphere 183 (2017) 294e304

and transcend trophic levels (Wang et al., 2010; Dhananjayan and Muralidharan, 2012; Karl et al., 2016). Evaluation of effects of PAH contamination is therefore considered of great importance (Dhananjayan and Muralidharan, 2012; Pampanin, 2016; Karl et al., 2016). Adverse effects from PAHs are dependent upon the type of PAH, its metabolic reactive state, the dose and route of exposure and the exposed organism involved (Beyer et al., 2010; Collier et al., 2013; Cousin and Cachot, 2014). Various marine species have been used for investigating PAH related effects, covering most of the trophic levels (e.g. plankton, coral, mussels, shrimp, fish and whale) (Baussant et al., 2009; Bechmann et al., 2010; Godard-Codding et al., 2011; Huang et al., 2011; Woo et al., 2014; Sanni et al., 2017). In fish, the PAH uptake is primarily via water as it passes through the gills or by consumption of PAH contaminated food (Beyer et al., 2010; Collier et al., 2013). Atlantic cod (Gadus morhua) has been commonly used both in laboratory exposure studies and environmental monitoring surveys, including investigation of PAH exposure effects by using biological markers (Johansen et al., 2009; Ruppert et al., 2009; Brooks et al., 2013; Hernandez et al., 2013; Isomaa et al., 2013; Pampanin, 2016; Sanni et al., 2017). Atlantic cod is an important species to study for multiple reasons as: i) it is a key species of the North Atlantic Ocean; ii) it is a globally important food product (both from wild catch and aquaculture); and iii) it inhabits and spawns in areas of high contamination risk due to industrial activities. Responses to environmental contaminants have been studied at various levels of biological organization, including the “omics”, (Holt et al., 2010, 2014; Brooks et al., 2013; €rundsdo  ttir et al., 2014; Pampanin et al., 2014, 2016; Karl et al., Jo 2016). Application of proteomics as a tool for ecotoxicological investigations has grown over the last 20 years, providing valuable knowledge for both mechanistic understanding and biomarker discovery studies (Dowling and Sheehan, 2006; Monsinjon and Knigge, 2007; Sanchez et al., 2011; Pampanin et al., 2014; Qiao et al., 2016; Song et al., 2016). The development of this research activity has benefitted from the advances in instrumentation and techniques. The currently available mass spectrometry (MS) techniques serve as powerful and sensitive tools for protein detection (Savaryn et al., 2016). Liquid chromatography (LC) coupled with tandem MS (i.e. LC-MS/MS) is now applied as a high throughput strategy for identifying, quantifying and/or characterizing proteins from a large variety of biological samples (Savaryn et al., 2016). Different MS strategies can be used depending on the research aim, as reviewed by Domon and Aebersold (2010). The strategy chosen in this study is the shotgun MS, which is especially suited for open discovery experiments aiming to identify large sets of proteins in complex matrices (Domon and Aebersold, 2010). This approach does not include sample fractionation prior to analysis, thus allowing initial proteome characterization of the sample material and identification of its high abundant proteins (HAPs). A great advantage when conducting proteomic investigations of Atlantic cod is that the full genome has been sequenced (Johansen et al., 2009), hence its full estimated proteome is available (Flicek et al., 2012). Proteomics studies have been conducted on various tissues of this fish species, such as liver, bile, brain, skin and blood plasma (Larsen et al., 2006; Berg et al., 2010; Rajan et al., 2011; Pampanin et al., 2014; Yadetie et al., 2016). Proteome discovery investigations of blood plasma in fish are of increasing interest, being able to provide valuable data for general fish biology, disease characteristics and environmental contaminant effects (Braceland et al., 2013; Li et al., 2016; Chupani et al., 2017). To date, proteomic studies of Atlantic cod plasma has involved investigation of the €m et al., 2005) and fish immune system (as reviewed in Pilstro detection of the effect of exposure to different estrogenic mimics (e.g. nonyl-phenol, bisphenol, alkylphenol) (Larsen et al., 2006;

295

Bohne-Kjersem et al., 2009; Meier et al., 2010; Nilsen et al., 2011a; 2011b) and PAH compounds (Bohne-Kjersem et al., 2009). These studies applied plasma fractionation prior to analysis, i.e. specific protein isolation and gel-based separation, leaving the full plasma proteome still undiscovered (Larsen et al., 2006; BohneKjersem et al., 2009; Meier et al., 2010; Nilsen et al., 2011a; 2011b). The aim of our study was to investigate the Atlantic cod plasma protein response to PAHs and provide new exposure biomarker candidates. Two PAH compounds (i.e. naphthalene and chrysene) and their corresponding dihydrodiols (i.e. ()-(1R,2R)-1,2dihydronaphthalene-1,2-diol and ()-(1R,2R)-1,2-dihydrochrysene-1,2-diol) were administered by intramuscular injection. Naphthalene and chrysene are included in the list of 126 priority pollutant by the US Environmental Protection Agency (EPA) (EPA , 2014). They are recognised as potential carcinogenic compounds commonly present in both crude oil and oil related discharges (Pampanin and Sydnes, 2013). The levels and composition of different PAHs vary depending on the natural PAH profile of the oil reservoirs and the treatment strategies applied in the field (Pampanin and Sydnes, 2013). Naphthalene is the most abundant PAH, with average levels of 427 mg/kg in crude oil, while levels of chrysene are about 30 mg/kg (Pampanin and Sydnes, 2013). These PAHs are therefore considered relevant for tracking sources of oil contamination in environmental monitoring activities. In vivo, PAH go through several enzymatic catalyzed oxidation steps forming different metabolites. An initial enzymatic oxidation form epoxides, which undergo ring opening to trans-diols. The further oxidation can lead to reactive metabolites capable of binding proteins and DNA, potentially leading to mutagenic or cancerogenic processes. Therefore, in this study, fish were also exposed to naphthalene and chrysene metabolites in order to study further PAH metabolism in vivo (Lorentzen et al., 2014). Previous findings showed that (1R,2R)-1,2-dihydrochrysene-1,2-diol (i.e. chrysene dihydrodiol) accounts for up to 88% of chrysene metabolites found in Atlantic cod bile and it is known to be the most carcinogenic chrysene metabolite (Jonsson et al., 2004). Species specific characterization of PAHs and their response effects can be of high importance when establishing sensitive and selective protein biomarkers for PAH exposure, and when aiming to track the PAH contamination source. 2. Materials and methods 2.1. Exposure design Wild Atlantic cod (n ¼ 66) were caught in the Idsefjord (Stavanger, Norway) for the exposure experiment. After acclimation, they were exposed by intramuscular injection to single PAHs (i.e. naphthalene and chrysene), and their corresponding dihydrodiol metabolites (i.e. ()-(1R,2R)-1,2-dihydronaphthalene-1,2-diol and ()-(1R,2R)-1,2-dihydrochrysene-1,2-diol) (Fig. 1). A detailed description of the exposure design is reported in Pampanin et al. (2016). In brief, the experiment included the following exposure groups: 1) control, not injected fish, 2) fish injected with the carrier only (DMSO/cod liver oil 1:1, 0,47 mL/kg), 3) fish injected with naphthalene (0.50 and 2.50 mg/kg), 4) fish injected with naphthalene dihydrodiol (0.10 and 0.25 mg/kg), 5) fish injected with chrysene (0.50 and 2.50 mg/kg) and 6) fish injected with chrysene dihydrodiol (0.10 and 0.25 mg/kg) (Table 1). The injected concentrations of the PAH compounds were anticipated to be realistic of exposure events occurring in areas of oil installations, though, as the PAHs were administered by injection of a single dose, the dose was also required to be of non-lethal character in itself. The metabolite exposure compounds (i.e. naphthalene dihydrodiol and

296

K. Skogland Enerstvedt et al. / Chemosphere 183 (2017) 294e304

Fig. 1. Structure of the injected PAH exposure compounds: naphthalene (1), chrysene (2), ()-(1R,2R)-1,2-dihydronaphthalene-1,2-diol (naphthalene dihydrodiol) (3), and ()-(1R,2R)-1,2-dihydrochrysene-1,2-diol (chrysene dihydrodiol) (4).

chrysene dihydrodiol), was considered to be of higher toxicity, therefore the doses were lower for these compounds compared to the other PAH exposure compounds (i.e. naphthalene and chrysene). As the fish were wild-caught, the exposure groups were not homogenous in gender and maturity. Therefore, samples from only female individuals with gonad somatic index (GSI) < 1 were used for the proteomic analysis (n ¼ 51) (Table 1). 2.2. Blood plasma collection One week after the injection, approximately 2 mL of blood was drawn from each fish using heparinized syringes (Na-heparin, 5000 IE/a.e./mL, LEO Pharma). Samples were then centrifuged at 5000 g for 5 min at 4  C and the resulting plasma fraction was transferred into cryotubes, snap-frozen in liquid nitrogen and stored at 80  C until further analysis. 2.3. Blood plasma sample preparation Firstly, the most suitable method to prepare plasma protein samples for MS analysis was investigated. Three techniques were tested, following manufacturer's instructions: 1) ZipTipC4 (ZipTip™, Millipore), a carbon-coated pipette tip designed for affinitybased purification and concentration of proteins; this method is considered suitable for the isolation of low-intermediate molecular weight (MW) proteins (25e100 kDa); 2) C18 Spin Column (Pierce), a carbon-coated resin column for purification and concentration of proteins by reverse-phase chromatography; suitable for low MW proteins, particularly of hydrophobic character; and 3) 3 kDa cut-off

filter (Ultracel® 3K Membrane, Amicon® Ultra Centrifugal Filters, Merck Millipore), commonly used for desalting and purification of protein samples, as well as for concentrating proteins with MW above 3 kDa. Two random plasma samples were used for comparing sample work-up techniques. Volumes equivalent to 40 mg of plasma proteins were applied in procedure 1 and 2, while 80 mg of plasma proteins were used in procedure 3, due to different technical capacities of the methods. Results from the three methods combined identified a total of 309 different proteins. Procedure 1, 2, and 3 could identify 23%, 35% and 81% of the proteins, respectively (results not shown). Therefore, technique 3 was chosen to be used for the preparation of all samples. This procedure was also found to be less time and reagent consuming. Sample total protein concentrations were measured by the Bradford method (Bradford, 1976). 2.4. LC-MS/MS analysis Two ml of trypsin (4.29 mM in 0.01% formic acid, Trypsin Gold, Promega) was added to 60 mg of prepared plasma sample. Samples were then incubated overnight at 37  C. The plasma protein analysis was then performed using LC-MS/MS, as described in Pampanin et al. (2014). In brief, ultrahigh pressure liquid chromatography (UHPLC) coupled to LTQ Orbitrap XL™ Hybrid Ion TrapOrbitrap Mass spectrometer (Thermo Scientific) was applied with total run-time of 250 min followed by a 90 min washing step and re-equilibration of the column. The mass spectrometer was calibrated (SUPELCO ProteoMass LTQ/FT-Hybrid ESI Pos. Mode Cal Mix MSCAL5-1EA, Thermo Scientific) prior to analysis. 2.5. Bioinformatics The raw data files from the LC-MS/MS analysis were analysed by Proteome Discoverer 2.0 using the Sequest HT search engine against the Gadus morhua translated EST database (downloaded from e!Ensemble 1st November 2012, containing 77,408 sequences). Trypsin was set as a digestion enzyme allowing for 2 missed cleavages. Precursor ion tolerance was set to 10 ppm and fragment ion mass tolerance to 0.6 ppm. Oxidation of methionine was set as dynamic modification. Peptide confidence filter was set to be at least medium, with the following combination of charge (z) and X correlation: high significance: 1.2 (z ¼ 1), 1.9 (z ¼ 2) and 2.3 (z ¼ 3) and 2.6 (z  4); medium significance 0.7 (z ¼ 1), 0.8 (z ¼ 2), 1 (z ¼ 3) and 1.2 (z  4). Minimum peptide length was set to 6 amino acids. Furthermore, a decoy database search was performed with target false discovery rate, strict and relaxed parameters set to 0.01 and 0.05, respectively. Protein blast searches (BLASTP 2.5.1þ, NCBI) were performed on identified proteins searching for homologs in the non-redundant

Table 1 Overview of experimental groups and their respective exposure type, concentrations and number of individuals. Group name

Exposure type

Injected concentration

Number of individuals

Control Carrier control Naphthalene low dose Naphthalene high dose Naphthalene dihydrodiol low dose Naphthalene dihydrodiol high dose Chrysene low dose Chrysene high dose Chrysene dihydrodiol. low dosea Chrysene dihydrodiol. high dose

none DMSO/cod liver oil Naphthalene Naphthalene ()-(1R,2R)-1,2-dihydronaphthalene-1,2-diol ()-(1R,2R)-1,2-dihydronaphthalene-1,2-diol Chrysene Chrysene ()-(1R,2R)-1,2-dihydrochrysene-1,2-diol ()-(1R,2R)-1,2-dihydrochrysene-1,2-diol

e 0.47 0.50 2.50 0.10 0.25 0.50 2.50 0.10 0.25

4 5 7 6 5 7 5 5 2 5

a

Group with low number of fish due to limited amount of ()-(1R,2R)-1,2-dihydrochrysene-1,2-diol.

mL/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg

K. Skogland Enerstvedt et al. / Chemosphere 183 (2017) 294e304

database (All non-redundant,GenBank,CDS,translations þ PDB þ SwissProt þ PIR þ PRF). To find protein homologs with accession numbers connected to gene ontology (GO) information, a protein blast search (BLASTP 2.5.1þ, NCBI) was performed on specific proteins of interest against the UniProt/SwissProt database (Non-redundant UniProtKB/SwissProt sequences). The search was performed both with and without the organism taxonomic identification of Teleostei (taxid: 32443). The homolog outcome from the Teleostei search that satisfied the chosen conditions (i.e. query coverage >50%; identity >25%; Evalue < 0.001) are reported in the result section. If no homolog was found in the Teleostei search, the best fit in the non-tax limited search was reported, still satisfying the above-mentioned criteria. Putative GO information was obtained from the UniProt Knowledgebase (UniProtKB) by entering the UniProt/SwissProt homolog accession numbers into the online application available at http:// www.uniprot.org/. 2.6. Statistical analysis The number of peptide spectrum matches (PSMs) detected between the groups were not significantly different as found by a median test (X2 (9) ¼ 6.678, p ¼ 0.67). The differences detected in further investigations of the data should therefore be mainly due to the PAH exposure. Only proteins detected in at least half of the samples per group were included in the multivariate analysis. The relative protein abundance was calculated according to the normalised spectral abundance factor (NSAF) as described in Zybailov et al., (2006). Since the aim of this study was to investigate the effect of PAHs on the plasma proteome, the two control groups (control and carrier control) were merged into a single control group (n ¼ 9). This also provided a more statistically sound comparison between controls and PAH exposed individuals. Multivariate analysis was performed using Sirius version 8.1 (Pattern Recognition Systems AS), and a description of the software settings applied is given as Supplementary material (Text Box S1). SIRIUS is a chemometric multivariate analysis tool designed for a variety of applications, including exploratory and regression analysis with the aim of identifying responsible factors of variance in complex datasets, such as the differently expressed proteins (i.e.

297

biomarker candidates) in MS data (Kvalheim, 2010). An initial principal component analysis (PCA) was unable to detect significant separation (at significance level 0.05) of control from the exposure groups. Therefore, partial least squares discriminant analysis (PLS-DA) was performed and two PLS components significantly contributed to separation (at significance level 0.05) between control and exposed groups, explaining 23e47% of the total variance. Target projection (TP) was then performed to obtain a single predictive latent variable for this response, followed by a non-parametric discriminating variable (DIVA) test to define the threshold of variability (i.e. by two-sided p-value Mann Whitney). The threshold was defined as 0.25 and corresponds to the mean probability levels of the subsequent selectivity ratio (SR) plot (Supplementary material, Figs. S1 and S2). The SR plot allowed identification of all protein variables with SR values (i.e. the ratio of explained to residual variance of each variable) exceeding the probability-based threshold. The sign of the SR value for each protein variable corresponds to the protein altered expression level in the exposed fish from that of control fish, i.e. positive SR values are equivalent to protein upregulation and negative values are equivalent to protein downregulation (Kvalheim, 2010). PCA was then performed on the dataset of the identified biomarker candidates to verify their biomarker potential by this unsupervised analysis (Fig. 5). 3. Results 3.1. Plasma protein identification and relative abundance A total number of 369 proteins (780 unique peptides) were identified from the analysis of 51 female (GSI < 1) plasma samples. An overview of the plasma profile is shown in Fig. 2, and a complete list of all identified proteins is provided as Supplementary material (Table S1). The top 2 relative abundant proteins are both apolipoproteins (i.e. 14 kDa apolipoproteins and predicted apolipoprotein A-I) and represent 51% of the total amount of identified proteins. Further, the top 10 relative abundant proteins represent 64% of the identified proteins. Due to the shotgun MS approach applied, these identified proteins can be categorized as HAPs of the Atlantic cod (female, GSI < 1) plasma proteome. The top 20 HAPs were ranked

Fig. 2. Overview of the plasma protein profile of female Atlantic cod (gonad somatic index < 1, n ¼ 51) identified by shotgun mass spectrometry analysis. Protein relative abundance was calculated according to Zybailov et al. (2006) and is reported as %. The 10 most abundant proteins are reported individually, while the remaining proteins are grouped as the top 11 to 20 most abundant proteins (top 11e20), the top 21 to 50 most abundant proteins (top 21e50) and the remaining proteins numbered 51 to 369 (other proteins).

298

K. Skogland Enerstvedt et al. / Chemosphere 183 (2017) 294e304

by the NSAF calculated relative abundance and their GO characteristics are reported in Table 2. Biological processes and molecular functions associated with these proteins, together with the cellular component that they derived from, are shown in Fig. 3. Since there is currently no GO information for the 14 kDa apolipoprotein (top 1 HAP), this could not be included in the GO distribution charts.

3.2. PAH exposure effects in Atlantic cod plasma proteome According to the statistical analysis, the levels of 12 proteins were found significantly different in the PAH exposed groups compared to the control groups (control and carrier control). These proteins were listed by their expression regulation (up- or downregulation), and their GO characteristics were reported when available (Table 3). Eleven proteins were found upregulated, mainly

Table 2 Top 20 high abundant proteins identified in plasma of Atlantic cod females (gonad somatic index < 1, n ¼ 51), calculated according to Zybailov et al. (2006). Proteins are ranked according to their relative abundance, and their respective gene ontology (GO) information of biological processes and molecular function, and the cellular component which they are derived from is reported. The accession numbers behind the protein homolog identities and GO information is given as Supplementary material (Table S2). Protein identification

Biological processes

Molecular function

Cellular component

#1 #2

14 kDa apolipoprotein, partial PREDICTED: apolipoprotein A-I

information not available lipid binding

information not available high-density lipoprotein particle

#3

PREDICTED: alpha-2macroglobulin-like, partial

information not available cholesterol metabolic process; lipid transport; lipoprotein metabolic process regulation of endopeptidase activity

extracellular exosome; extracellular space

#4 #5 #6

Type-4 ice-structuring protein Serotransferrin, partial hemopexin-like protein, partial

peptidase inhibitor activity; serinetype endopeptidase inhibitor activity information not available metal ion binding heme transporter activity; metal ion binding

#7

fibrinogen beta chain precursor

#8

PREDICTED: hemopexin-like

#9

angiotensinogen

#10

PREDICTED: alpha-2macroglobulin-like protein 1, partial PREDICTED: hemopexin

#11

response to freezing ion transport; iron ion homeostasis cellular iron ion homeostasis; cellular response to estrogen stimulus platelet activation; protein polymerization; signal transduction cellular iron ion homeostasis; cellular response to estrogen stimulus regulation of systemic arterial blood pressure by reninangiotensin; vasoconstriction regulation of endopeptidase activity cellular iron ion homeostasis; cellular response to estrogen stimulus B cell differentiation; B cell receptor signaling pathway; complement activation, classical pathway; defense response to bacterium; innate immune response; phagocytosis, engulfment; phagocytosis, recognition; positive regulation of B cell activation information not available regulation of endopeptidase activity

#12

immunoglobulin light chain L1 region J-C, partial

#13 #14

No homolog found PREDICTED: alpha-2macroglobulin-like

#15

PREDICTED: alpha-2macroglobulin-like

female pregnancy; negative regulation of complement activation, lectin pathway; stem cell differentiation

#16

hemopexin

#17

PREDICTED: apolipoprotein C-I-like

#18

PREDICTED: complement C1q-like protein 2

#19 #20

PREDICTED: complement C3-like PREDICTED: apolipoprotein A-IVlike

cellular iron ion homeostasis; cellular response to estrogen stimulus lipid catabolic process; lipid transport; lipoprotein metabolic process negative regulation of ERK1 and ERK2 cascade; negative regulation of fat cell differentiation; negative regulation of fibroblast proliferation information not available cholesterol metabolic process; lipid transport; lipoprotein metabolic process

extracellular region extracellular space cell; extracellular region

metal ion binding

fibrinogen complex

heme transporter activity; metal ion binding

cell; extracellular region

information not available

extracellular space

peptidase inhibitor activity; serinetype endopeptidase inhibitor activity heme transporter activity; metal ion binding

extracellular exosome; extracellular space

antigen binding; immunoglobulin receptor binding

blood microparticle; external side of plasma membrane; immunoglobulin complex, circulating; plasma membrane

information not available peptidase inhibitor activity; serinetype endopeptidase inhibitor activity calcium-dependent protein binding; enzyme binding; growth factor binding; interleukin-1/8 binding; peptidase inhibitor activity; protease binding; receptor binding; serine-type endopeptidase inhibitor activity; tumor necrosis factor binding heme transporter activity; metal ion binding

information not available extracellular exosome; extracellular space

lipid binding

chylomicron; very-low-density lipoprotein particle

identical protein binding

collagen trimer; extracellular space

information not available lipid binding

information not available high-density lipoprotein particle

cell; extracellular region

blood microparticle; extracellular exosome

cell; extracellular region

K. Skogland Enerstvedt et al. / Chemosphere 183 (2017) 294e304

immunoglobulins (i.e. 10 different immunoglobulin components and one alpha-2-macroglobulin-like protein), while one protein was found downregulated (i.e. antifreeze protein type IV). Only the antifreeze protein type IV is among the top 20 HAPs. The 12 proteins identified can therefore be considered as biomarker candidates of PAH exposure in Atlantic cod plasma. Based on their GO characteristics (Fig. 4), the biological processes most strongly affected by the exposure are the cellular process (13%), singleorganism process (13%), regulation of biological process (13%), response to stimulus (13%) and immune system process (12%). The PCA plot in Fig. 5 shows the separation of the exposure groups from the control groups (encircled) based upon the dataset of the 12 biomarker candidates. The first two components explain 84.5% of the total variance. The groups contributing most strongly to the separation from control group are naphthalene low dose, chrysene high dose and chrysene dihydrodiol high dose. No clear dose-response traits of the exposure compounds were observed. The protein contributing most strongly to the separation in the first component was the antifreeze protein type IV (GENSCAN00000035270), while one of the immunoglobulin light chains (GENSCAN00000007861) was the strongest contributor to the separation in the second component (Table S3). 4. Discussion The main aim of this work was to investigate the potential of the plasma proteome of Atlantic cod to serve as a medium for biomarker discovery for source tracking of PAH contamination. The shotgun MS strategy applied is a method particularly well suited for discovery experiments since no prior proteome knowledge is required (Domon and Aebersold, 2010; Cunningham et al., 2012). It is commonly used qualitatively for identifying proteins in complex samples as initial characterization of the proteome. The main limitation is its bias toward the most abundant proteins of the sample matrix which can dominate the instruments capacity for analysis and detection (Domon and Aebersold, 2010; Zhang et al., 2013). A potential challenge in this type of study is the natural variation of protein levels according to biological factors, such as gender, size, maturity and season, potentially masking or posing as exposure  ttir et al., 1999a; 1999b; Li et al., 2016). To related effects (Magnado ensure consistent and comparable results, homogenisation of the samples was found necessary. Therefore, samples from only female fish with a GSI <1 were considered for proteomics analysis. The gonad maturity cycle of Atlantic cod has been extensively studied and GSI <1 is representative of juvenile or resting females (i.e. females out of spawning) (DIFR , 2002; Vitale, 2008; Yaragina, 2010; de Figueiredo, 2013). 4.1. Plasma protein identification and relative abundance Plasma was chosen as the matrix for proteomic analysis. Due to its role in the organisms, traces of adverse exposure effects originating from various organs and tissues throughout the body can potentially be detected. Furthermore, blood samples are relatively easy to draw, without necessarily sacrificing the individual, giving the opportunity to investigate effects over time (Cunningham et al., 2012). Previous studies conducted on Atlantic cod plasma have been limited to specific proteins and/or certain protein groups (Larsen et al., 2006; Bohne-Kjersem et al., 2009; Nilsen et al., 2011a, 2011b), thereby making this study the first open discovery analysis to map this proteome in a broader extent. Herein, 369 proteins were identified and relative abundance levels were found to span over four orders of magnitude between the lowest and highest abundant proteins detected. Considering the shotgun approach

299

applied, most of the identified proteins are likely to be true high abundant proteins (HAPs) of the Atlantic cod plasma proteome, and such HAP characterization have not previously been reported. This knowledge is valuable for future studies on the Atlantic cod plasma and protein biomarker discovery. In general, one can assume that HAPs are less prone to present significant exposure effects, as the alterations in level needs to be considerable to be evident and fall outside normal ranges. Also, their abundance often exceeds the analytic capacity, hence potential exposure effects can be quenched or lost during sample preparation and analysis (Domon and Aebersold, 2010). The HAP identification performed in this study can therefore be of value for designing future proteomic investigations where HAP depletion prior to analysis is considered favourable. It is important to note that the abundance reported is relative, and over-estimations of some proteins (e.g. lower ranks) and under-estimations of the HAPs are possible limitations of the shotgun approach (Domon and Aebersold, 2010; Zhang et al., 2013). The most dominating HAP was the 14 kDa apolipoprotein, followed by a predicted apolipoprotein A-1. Moreover, the predicted apolipoprotein C-like and the apolipoprotein A-IV-like protein were also present among the top 20 HAPs. In fish, apolipoprotein levels are generally higher than in other vertebrates, and the 14 kDa apolipoprotein and the apolipoprotein A-I are commonly found as major high density lipoproteins (Kondo et al., 2001; Dietrich et al., 2014). In previous studies, reports of apolipoprotein levels in fish varied between 20 and 40% of the total proteome, e.g. 26% in salmon (Salmo saalar) (Sandnes et al., 1988; Kondo et al., 2001), 36% in rainbow trout (Oncorhynchus mykiss) (Babin, 1987), 30% in channel catfish (Ictalurus punctatus) (Smith et al., 1988) and 20% in zebrafish (Danio rerio) (Li et al., 2016). Apolipoproteins are involved in lipid uptake and transport, and since lipids are the major energy source in fish, they are of high importance for homeostatic condition maintenance (Qu et al., 2014). Additionally, the apolipoprotein A-I and the 14 kDa apolipoprotein are also found likely to be involved in the immune system response (Kim et al., 2009; Dietrich et al., 2014, 2015). The 14 kDa apolipoprotein was first described by Kondo et al. (2001) as a novel apolipoprotein specific to fish. Little is known regarding its role and function. However, it has been identified in the blood plasma of various fish species, (e.g. eel (Anguilla japonica), puffer fish (Takifugu rubripes), carp (Cyprinus carpio) and seven species of elasmobranch), and also in other fish tissues, (e.g. liver, brain, heart, epidermis, gills, intestine, reproductive tract and muscle), suggesting a high importance of this protein in fish physiological functions (Kondo et al., 2001, 2005; Metcalf and Gemmel, 2005; Zhou et al., 2005; Xia et al., 2008; Choudhury et al., 2009; Kim et al., 2009; Dietrich et al., 2014). Regarding exposure effects, increased gene expression levels of the 14 kDa apolipoprotein was found in Atlantic cod exposed to simulated produced water containing PAHs and alkylphenols. However, its toxicological relevance remains unknown (Holt et al., 2010). The identified top 20 HAPs were found to be primarily from the extracellular matrix, mainly serving the molecular function of binding, according to GO information. Their involvement in biological processes was diverse, including processes of regulation, localization, metabolism, development, immune system and others. 4.2. Effects on Atlantic cod plasma proteome upon exposure to PAHs In order to confirm the exposure of fish in this study, PAH metabolite were analysed in bile samples by means of gas chromatography-MS (GC-MS), as published in Pampanin et al. (2016). High levels of naphthalene metabolites were found in the bile of naphthalene and naphthalene dihydrodiol exposed fish (Pampanin et al., 2016). These levels were similar to those found in

300

K. Skogland Enerstvedt et al. / Chemosphere 183 (2017) 294e304

Table 3 Biomarker candidates of PAH exposure identified in plasma of Atlantic cod females (gonad somatic index < 1, n ¼ 51). Proteins are listed with their altered expressed regulation (up or down), and their respective gene ontology (GO) information of biological processes and molecular function, and the cellular component which they derive from. The accession numbers of the protein homolog identities and GO information is given as Supplementary material (Table S3). Protein expressed regulation

Protein identification

Biological process

Molecular function

Cellular component

Down Up

Antifreeze protein type IV Alpha-2-macroglobulin-like

response to freezing female pregnancy; negative regulation of complement activation, lectin pathway; stem cell differentiation

extracellular region blood microparticle; extracellular exosome

Up

Immunoglobulin D heavy chain constant region variant b

information not available

information not available calcium-dependent protein binding; enzyme binding; growth factor binding; interleukin-1/8 binding; peptidase inhibitor activity; protease binding; receptor binding; serine-type endopeptidase inhibitor activity; tumor necrosis factor binding antigen binding; immunoglobulin receptor binding

Up

information not available

antigen binding

Up Up

Immunoglobulin heavy chain variable region Immunoglobulin light chain Immunoglobulin light chain

extracellular region; integral component of membrane; plasma membrane information not available

antigen binding antigen binding

information not available extracellular space

Up

Immunoglobulin light chain

antigen binding; serine-type endopeptidase activity

blood microparticle; extracellular region; plasma membrane

Up

Immunoglobulin light chain

information not available immune response; immunoglobulin production complement activation; complement activation, classical pathway; Fc-epsilon receptor signaling pathway; Fc-gamma receptor signaling pathway involved in phagocytosis; immune response; immunoglobulin production; receptor-mediated endocytosis; regulation of immune response information not available

information not available

Up

Immunoglobulin light chain

antigen binding; serine-type endopeptidase activity

Up

Immunoglobulin light chain

Up

Immunoglobulin light chain

Up

Immunoglobulin light chain

complement activation; complement activation, classical pathway; Fc-epsilon receptor signaling pathway; Fc-gamma receptor signaling pathway involved in phagocytosis; immune response; immunoglobulin production; receptor-mediated endocytosis; regulation of immune response complement activation; complement activation, classical pathway; Fc-epsilon receptor signaling pathway; Fc-gamma receptor signaling pathway involved in phagocytosis; receptor-mediated endocytosis; regulation of immune response complement activation; complement activation, classical pathway; Fc-epsilon receptor signaling pathway; Fc-gamma receptor signaling pathway involved in phagocytosis; immune response; receptormediated endocytosis; regulation of immune response complement activation; complement activation, classical pathway; Fc-epsilon receptor signaling pathway; Fc-gamma receptor signaling pathway involved in phagocytosis; immune response; receptormediated endocytosis; regulation of immune response

extracellular exosome; extracellular region; plasma membrane blood microparticle; extracellular region; plasma membrane

antigen binding; serine-type endopeptidase activity

extracellular exosome; extracellular region; extracellular space; plasma membrane

antigen binding; serine-type endopeptidase activity

extracellular exosome; extracellular region; plasma membrane

antigen binding; serine-type endopeptidase activity

extracellular exosome; extracellular region; plasma membrane

K. Skogland Enerstvedt et al. / Chemosphere 183 (2017) 294e304

301

Fig. 3. Characteristics of the top 20 high abundant proteins in plasma of Atlantic cod females (gonad somatic index < 1, n ¼ 51). Gene ontology distribution according to: biological processes (A), molecular functions (B) and cellular components (C), results based on the UniProt homolog search.

Fig. 4. Characteristics of the 12 potential biomarker candidates of PAH exposure in plasma of Atlantic cod females (gonad somatic index < 1, n ¼ 51). Gene ontology distribution according to: biological processes (A), molecular functions (B) and cellular components (C), results based on the UniProt homolog search.

Fig. 5. Overview of the sample distribution according to the Principal Component Analysis using the dataset of 12 protein biomarker candidates previously identified. Legend: control (circle); carrier control (filled circle); naphthalene low dose (squares); naphthalene high dose (filled squares); naphthalene dihydrodiol low dose (light grey diamonds); naphthalene dihydrodiol high dose (dark grey diamonds); chrysene low dose (triangles); chrysene high dose (filled triangles); chrysene dihydrodiol low dose (down triangles) and chrysene dihydrodiol high dose (down filled triangles).

Atlantic cod caged nearby oil installations in the North Sea during a multi-year monitoring study (Hylland et al., 2008), thus representing realistic low environmental PAH contamination conditions. The biomarker investigation comparing the exposed samples to the controls, found the protein levels of 12 proteins significantly

different due to the PAH exposure. Eleven proteins were upregulated, and 10 of these were further identified as immunoglobulin (Ig) components (i.e. Ig light or heavy chains). This suggests a triggered immune system response caused by the exposure. Previous studies have shown that the immune system of fish is very

302

K. Skogland Enerstvedt et al. / Chemosphere 183 (2017) 294e304

sensitive to PAH pollution, affecting both the non-specific and specific immunity of the individuals (Reynaud and Deschaux, 2006). However, the immunotoxic responses reported varies between different fish species and are dependent on the type and dose of PAHs (Reynaud and Deschaux, 2006; Barron, 2012). The immune system of Atlantic cod is considered unique compared to other teleost, as it presents a minimal antibody response to pathogens, has natural high levels of IgM and natural high numbers of phagocytic neutrophils in blood (Magnadottir et al., 2009; Øverland et al., 2010; Star et al., 2011). Immunoglobulins of Atlantic cod are of € m et al., 2005). The protein homolog two types: IgM and IgD (Pilstro results reported in this study do not give information on the origin of the Ig components detected, i.e. whether they stem from IgM or IgD, with the exception of one of the proteins identified as an IgD heavy chain component. However, most of the remaining Ig components are likely to be IgM components as this is known to be the most abundant Ig in Atlantic cod. Levels of IgM have been reported at about 33% of the total serum protein levels of this species € m et al., 2005; Solem and Stenvik, 2006), and in some in(Pilstro dividuals, as high as 50e70% (Magnadottir et al., 2009). Levels of IgD in Atlantic cod are not reported; however, from the results of a gene expression analysis, it was considered to be of relatively low € m et al., 2005). abundance (Stensvik and Jørgensen, 2000; Pilstro Previous studies have found no specific Ig response in Atlantic cod after immunization, infection or acute phase stimulation (Magnadottir, 2014). The immune system effects of PAH exposure in Atlantic cod do not appear to have been investigated as single events to this date, however, exposure to PAH containing produced water found significantly increased expression levels of IgM light rez-Casanova et al., 2010). The current results may chains (Pe therefore also be of value for future investigations regarding the open questions of the Atlantic cod natural antibody responses. An alpha-2-macroglobulin-like protein was also found upregulated, and as it is a protease inhibitor with important functions of the humoral immune defence (Chuang et al., 2013), this also indicates immune system related effects of the PAH exposure. The only downregulated protein detected was the antifreeze protein type IV, which was also established as one of the 20 HAPs of Atlantic cod plasma in this study. This protein is essential for fish survival during the winter as it keeps it from freezing when the temperature drops below the freezing point of sea water (Goddard and Fletcher, 1994). Down-regulation of antifreeze protein in response to PAH exposure may suggest increased vulnerability to cold-damage at the cellular level (Goddard and Fletcher, 1994). The overall PAH exposure response of the 12 biomarker candidates for each fish is shown in Fig. 5. Here, the groups of naphthalene low dose, chrysene high dose and chrysene dihydrodiol high dose can be seen as the ones contributing to the strongest separation from the controls. This shows that the application of a panel of all 12 biomarker candidates could be representative for both naphthalene and chrysene exposure. However, the use of Ig proteins as biomarkers of exposure needs to take into account the natural variation of these proteins due to both biotic and abiotic €m et al., 2005). Further factors (Cuesta et al., 2004, 2011; Pilstro studies are suggested to gain more knowledge on the Atlantic cod immune system and mapping of its toxicological response characteristics. 5. Conclusions Shotgun MS analysis of plasma from female Atlantic cod resulted in the identification of 369 proteins. The HAP profile was characterized, identifying the 14 kDa apolipoprotein as the most abundant plasma protein. The levels of 12 proteins were found significantly altered due to the PAH exposure. Eleven proteins were

upregulated, primarily immunoglobulin components, and one protein was dowregulated: antifreeze protein type IV. The relative uniformity of the characteristics and functions of identified proteins suggests a triggered immune system response due to the exposure. The exposure groups that showed the strongest response in the plasma proteome were naphthalene low dose, chrysene high dose and chrysene dihydrodiol high dose. However, no clear doseresponse trends were observed. Overall, the obtained results provide valuable knowledge for future studies of the Atlantic cod plasma proteome and generate grounds for establishing new plasma protein biomarkers to be used as sensitive tools for environmental investigations and monitoring of PAH related exposure. Acknowledgments The Research Council of Norway, PETROMAKS 2 (project # 229153), is gratefully acknowledged for financial support, including the PhD grant of Karianne Skogland Enerstvedt. The authors would also like to thank Emily Lyng (IRIS) for the revision of the language, Kjell Birger Øysæd and Eivind Larsen (IRIS) for the technical support in the LC-MS/MS analysis, and IRIS personnel for their help during the exposure study. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2017.05.111. References Babin, P.J., 1987. Plasma lipoprotein and apolipoprotein distribution as a function of density in the rainbow trout (Salmo gairdneri). Biochem. J. 246, 425e429. Barron, M.G., 2012. Ecological impacts of the Deepwater Horizon oil spill: implications for immunotoxicity. Toxicol. Pathol. 40, 315e320. http://dx.doi.org/ 10.1177/0192623311428474. Baussant, T., Bechmann, R.K., Taban, I.C., Tandberg, A.H., Bjørnstad, A., Torgrimsen, S., Nævdal, A., Øysæd, K.B., Jonsson, G., Sanni, S., 2009. Enzymatic and cellular responses in relation to body burden of PAHs in bivalve molluscs: a case study with chronic levels of North Sea and Barents Sea dispersed oil. Mar. Pollut. Bull. 58, 1796e1807. http://dx.doi.org/10.1016/j.marpolbul.2009.08.007. Bechmann, R.K., Larsen, B.K., Taban, I.C., Hellgreb, L.I., Møller, P., Sanni, S., 2010. Chronic exposure of adults and embryos of Pandalus borealis to oil causes PAH accumulation, initiation of biomarker responses and an increase in larval mortality. Mar. Pollut. Bull. 60, 2087e2098. http://dx.doi.org/10.1016/ j.marpolbul.2010.07.010. Berg, K., Puntervoll, P., Valdersnes, S., Goksøyr, A., 2010. Responses in the brain proteome of Atlantic cod (Gadus morhua) exposed to methylmercury. Aquat. Toxicol. 100, 51e65. http://dx.doi.org/10.1016/j.aquatox.2010.07.008. Beyer, J., Jonsson, G., Porte, C., Krahn, M.M., Ariese, F., 2010. Analytical methods for determining metabolites of polycyclic aromatic hydrocarbon (PAH) pollutants in fish bile: a review. Environ. Toxicol. Pharmacol. 30, 224e244. http:// dx.doi.org/10.1016/j.etap.2010.08.004. Bohne-Kjersem, A., Skadsheim, A., Goksøyr, A., Grøsvik, B.E., 2009. Candidate biomarker discovery in plasma of juvenile cod (Gadus morhua) exposed to crude North Sea oil, alkyl phenols and polycyclic aromatic hydrocarbons (PAHs). Mar. Environ. Res. 68, 268e277. http://dx.doi.org/10.1016/j.marenvres. 2009.06.016. Braceland, M., Bickerdike, R., Tinsley, J., Cockerill, D., Mcloughlin, M.F., Graham, D.A., Burchmore, R.J., Weir, W., Wallace, C., Eckersall, P.D., 2013. The serum proteome of Atlantic salmon, Salmo salar, during pancreas disease (PD) following infection with salmonid alphavirus subtype 3 (SAV3). J. Proteom. 94, 423e436. http:// dx.doi.org/10.1016/j.jprot.2013.10.016. Bradford, M.M., 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. http://dx.doi.org/10.1016/0003-2697(76)90527-3. Brooks, S., Pampanin, D., Harman, C., Dunaevskava, E., 2013. The Water Column Monitoring Programme 2013: Determining the Biological Effects of Two Offshore Platforms on Local Fish Populations. Oslo, p. 61. https://brage.bibsys. no/xmlui/bitstream/handle/11250/274093/6595-2015_200dpi.pdf?sequence ¼4&isAllowed¼y (Accessed 16 December 2016). Choudhury, M., Yamada, S., Komatsu, M., Kishimura, H., Ando, S., 2009. Homologue of mammalian apolipoprotein A-II in non-mammalian vertebrates. Acta Biochim. Biophys. Sin. 370e378. http://dx.doi.org/10.1093/abbs/gmp.015. Chuang, W.-H., Lee, K.K., Liu, P.-C., 2013. Characterization of alpha-2-macroglobulin

K. Skogland Enerstvedt et al. / Chemosphere 183 (2017) 294e304 from groupers. Fish. Shellfish Immunol. 35, 389e398. http://dx.doi.org/10.1016/ j.fsi.2013.04.050. , E., Niksirat, H., Pana  Chupani, L., Zuskova cek, A., Lünsmann, V., Haange, S.-B., Bergen, M., Jehmlich, N., 2017. Effects of chronic dietary exposure of zinc oxide nanoparticles on the serum protein profile of juvenile common carp (Cyprinus carpio L.). Sci. Total Environ. 579, 1504e1511. http://dx.doi.org/10.1016/ j.scitotenv.2016.11.154. Collier, T.K., Anulacion, B.F., Arkoosh, M.R., Dietrich, J.P., Incardona, J.P., Johnson, L.L., Ylitalo, G.M., Myers, M.S., 2013. Effects on fish of polycyclic aromatic hydrocarbons (PAH) and naphthenic acid exposures. Org. Chem. Toxicol. Fish. 33, 195e255. http://dx.doi.org/10.1016/B978-0-12-398254-4.00004-2. Cousin, X., Cachot, J., 2014. PAHs and fish - exposure monitoring and adverse effects - from molecular to individual level. Environ. Sci. Pollut. Res. 21, 13685e13688. http://dx.doi.org/10.1007/s11356-014-3161-8. Cuesta, A., Meseguer, J., Esteban, M.A., 2004. Total serum immunoglobulin M levels are affected by immunomodulators in seabream (Sparus aurata L.). Vet. Immunol. Immunopathol. 101, 203e210. http://dx.doi.org/10.1016/j.vetimm. 2004.04.021. Cuesta, A., Meseguer, J., Esteban, M.A., 2011. Immunotoxicological effects of environmental contaminants in teleost fish reared for aquaculture. In: Stoytcheva, M. (Ed.), Pesticides in the Modern World e Risks and Benefits. InTech, Rijeka, pp. 241e266. Cunningham, R., Ma, D., Li, L., 2012. Mass spectrometry-based proteomics and peptidomics for systems biology and biomarker discovery. Front. Biol. 7, 313e335. http://dx.doi.org/10.1007/s11515-012-1218-y. DIFR (Danish Institute for Fisheries Research), 2002. Manual to Determine Gonadal Maturity of Baltic Cod. Ministry of Food, Agriculture and Fisheries, Denmark. http://orbit.dtu.dk/files/6618782/116-02_manual_to_determine_gonadal_matu rity.pdf (Accessed 02 February 2017). de Figueiredo, F.A.M., 2013. Control of Sexual Maturation and Growth in Atlantic Cod (Gadus morhua) y Use of Cold Cathode Light Technology (Master Thesis). lar University College. http://skemman.is/stream/get/1946/16873/39137/1/ Ho MSc_Thesis_Filipe_Figueiredo_Final_version_23-05-3.pdf (Accessed 02 January 2017). Dhananjayan, V., Muralidharan, S., 2012. Polycyclic aromatic hydrocarbons in various species of fishes from Mumbai Harbour, India, and their dietary intake dose to human. Int. J. Oceans Oceanogr. 2012, 1e6. http://dx.doi.org/10.1155/ 2012/645178.  ska, B., Hejmej, A., Steinhagen, D., Ciereszko, A., Dietrich, M.A., Adamek, M., Bilin 2014. Characterization, expression and antibacterial properties of apolipoproteins A from carp (Cyprinus carpio L.) seminal plasma. Fish. Shellfish Immunol. 41, 389e401. http://dx.doi.org/10.1016/j.fsi.2014.09.020. Dietrich, M.A., Nynca, J., Adamek, M., Steinhagen, D., Karol, H., Ciereszko, A., 2015. Expression of apolipoprotein A-I and A-II in rainbow trout reproductive tract and their possible role in antibacterial defence. Fish. Shellfish Immunol. 45, 750e756. http://dx.doi.org/10.1016/j.fsi.2015.05.048. Domon, B., Aebersold, R., 2010. Options and considerations when selecting a quantitative proteomics strategy. Nat. Biotechnol. 28, 710e721. http:// dx.doi.org/10.1038/nbt.1661. Dowling, V.A., Sheehan, D., 2006. Proteomics as a route to identification of toxicity targets in environmental toxicology. Proteomics 2006 (6), 5597e5604. http:// dx.doi.org/10.1002/pmic.200600274. EPA (Environmental Protection Agency), 2014. Priority Pollutant List. United States. https://www.epa.gov/sites/production/files/2015-09/documents/priority-pollu tant-list-epa.pdf (Accessed 14 May 2017). Flicek, P., Amode, M.R., Barrell, D., Beal, K., Brent, S., Carvalho-Silva, D., Clapham, P., Coates, G., Fairley, S., Fitzgerald, S., Gil, L., Gordon, L., Hendrix, M., Hourlier, T., €h € Johnson, N., Ka ari, A.K., Keefe, D., Keenan, S., Kinsella, R., Komorowska, M., Koscielny, G., Kulesha, E., Larsson, P., Longden, I., McLaren, W., Muffato, M., Overduin, B., Pignatelli, M., Pritchard, B., Riat, H.S., Ritchie, G.R.S., Ruffier, M., Schuster, M., Sobral, D., Tang, Y.A., Taylor, K., Trevanion, S., Vandrovcova, J., White, S., Wilson, M., Wilder, S.P., Aken, B.L., Birney, E., Cunningham, F., Dunham, I., Durbin, R., Fern andez-Suarez, X.M., Harrow, J., Herrero, J., Hubbard, T.J.P., Parker, A., Proctor, G., Spudich, G., Vogel, J., Yates, A., Searle, S.M.J., 2012. Ensembl 2012. Nucleic Acids Res. 40, D84eD90. http:// dx.doi.org/10.1093/nar/gkr991. Godard-Codding, C.A.J., Clark, R., Fossi, M.C., Marsili, L., Maltese, S., West, A.G., Valenzuela, L., Rowntree, V., Polyak, I., Cannon, J.C., Pinkerton, K., RubioCisneros, N., Mesnic, S.L., Cox, S.B., Kerr, I., Payne, R., Stegeman, J.J., 2011. Pacific Oceanewide profile of CYP1A1 expression, stable carbon and nitrogen isotope ratios, and organic contaminant burden in Sperm Whale skin biopsies. Environ. Health Perspect. 119, 337e343. http://dx.doi.org/10.1289/ehp.0901809. Goddard, S.V., Fletcher, G.L., 1994. Antifreeze proteins: their role in cod survival and distribution from egg to adult. ICES Mar. Sci. Symp. 198, 676e683. Hernandez, K.M., Risch, D., Cholewiak, D.M., Dean, M.J., Hatch, L.T., Hoffman, W.S., Rice, A.N., Zemeckis, D., Parijs, S.M.V., 2013. Acoustic monitoring of Atlantic cod (Gadus morhua) in Massachusetts Bay: implications for management and conservation. ICES J. Mar. Sci. Adv. http://dx.doi.org/10.1093/icesjms/fst003. Holt, T.F., Thorsen, A., Olsvik, P.Å., Hylland, K., 2010. Long-term exposure of Atlantic cod (Gadus morhua) to components of produced water: condition, gonad maturation, and gene expression. Can. J. Fish. Aquat. Sci. 67, 1685e1698. http:// dx.doi.org/10.1139/F10-089.  mez, C., Holt, T.F., Eidsvoll, D.P., Farmen, E., Samnders, M.B., Martínez-Go Budzinski, H., Burgeot, T., Guilhermino, L., Hylland, K., 2014. Effects of water accommodated fractions of crude oils and diesel on a suite of biomarkers in

303

Atlantic cod (Gadus morhua). Aquat. Toxicol. 154, 240e252. http://dx.doi.org/ 10.1016/j.aquatox.2014.05.013. Huang, Y.-J., Jiang, Z.-B., Zeng, J.-N., Chen, Q.-Z., Zhao, Y-q., Liao, Y-b., Xu, X-q, 2011. The chronic effects of oil pollution on marine phytoplankton in a subtropical bay, China. Environ. Monit. Assess. 176, 517e530. http://dx.doi.org/10.1007/ s10661-010-1601-6. Hylland, K., Tollefsen, K.-E., Ruus, A., Jonsson, G., Sundt, R.C., Sanni, S., Utvik, T.I.R., _ J., Marigo mez, I., Feist, S.W., Johnsen, S., Nilssen, I., Pinturier, L., Balk, L., Barsiene, Børseth, J.F., 2008. Water column monitoring near oil installations in the North Sea 2001e2004. Mar. Pollut. Bull. 56, 414e429. http://dx.doi.org/10.1016/ j.marpolbul.2007.11.004. Isomaa, M., Kaitala, V., Laakso, J., 2013. Baltic cod (Gadus morhua callarias) recovery potential under different environment and fishery scenarios. Ecol. Modell. 266, 118e125. http://dx.doi.org/10.1016/j.ecolmodel.2013.06.015. Johansen, S.D., Coucheron, D.H., Andreassen, M., Karlsen, B.O., Furmanek, T., Jørgensen, T.E., Emblem, Å., Breines, R., Noreide, J.T., Moum, T., Nederbragt, A.J., Stenseth, N.C., Jacobsen, K.S., 2009. Large-scale sequence analyses of Atlantic cod. New Biotechnol. 25, 263e271. Jonsson, G., Taban, I.C., Jørgensen, K.B., Sundt, R.C., 2004. Quantitative determination of de-conjugated chrysene metabolites in fish bile by HPLC-fluorescence and GC-MS. Chemosphere 54, 1085e1097. http://dx.doi.org/10.1016/ j.chemosphere.2003.09.026.  Jensen, S., Hylland, K., Holth, T.F., Gunnlaugsdo €rundsdo ttir, H.O., ttir, H., Jo  El-Taliawy, H., Rige   ttir, A., t, F., Strand, J., Nyberg, E., Svavarsson, J., Olafsd o rsson, H.P., 2014. Pristine Arctic: background Bignert, A., Hoydal, K.S., Halldo mapping of PAHs, PAH metabolites and inorganic trace elements in the NorthAtlantic Arctic and sub-Arctic coastal environment. Sci. Total Environ. 493, 719e728. http://dx.doi.org/10.1016/j.scitotenv.2014.06.030. Karl, H., Kammann, U., Aust, M.-O., Manthey-Karl, M., Lüth, A., Kanisch, G., 2016. Large scale distributions of ditoxins, PCBs, heavy metals, PAH-metabolites and radionuclides in cod (Gadus morhua) from the North Atlantic and its adjacent seas. Chemosphere 149, 294e303. http://dx.doi.org/10.1016/ j.chemosphere.2016.01.052. Kim, K.-Y., Cho, T.S., Bang, I.-C., Nam, Y.K., 2009. Isolation and characterization of the apolipoprotein multigene family in Hemibarbus mylodon (Teleostei: Cypriniformes). Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 152, 38e46. http://dx.doi.org/10.1016/j.cbpb.2008.09.084. Kondo, H., Kawazoe, I., Nakaya, M., Kikuchi, K., Aida, K., Watabe, S., 2001. The novel sequence of major plasma apolipoproteins in the eel Anguilla japonica. Biochim. Biophys. Acta 1531, 132e142. PII: S1388-1981(01)00099-3. Kondo, H., Morinaga, K., Misaki, R., Nakaya, M., Watabe, S., 2005. Characterization of the pufferfish Takifugu rubripes apolipoprotein multigene family. Gene 346, 257e266. http://dx.doi.org/10.1016/j.gene.2004.11.015. Kvalheim, T.A.R., 2010. Mass Spectral Profiling and Multivariate Analysis for Detection of Biomarker Signatures (Doctoral Dissertation). University of Bergen. http://bora.uib.no/handle/1956/4451 (Accessed 06 September 2016). Larsen, B.K., Bjørnstad, A., Sundt, R.C., Taban, I.C., Pampanin, D.M., Andersen, O.K., 2006. Comparison of protein expression in plasma from nonylphenol and bisphenol A-exposed Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus) by use of SELDI-TOF. Aquat. Toxicol. 78S, S25eS33. http://dx.doi.org/ 10.1016/j.aquatox.2006.02.026. Li, C., Tan, X.F., Lim, T.K., Lin, Q., Gong, Z., 2016. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human. Sci. Rep. 6, 25329. http:// dx.doi.org/10.1038/srep24329. Lorentzen, M., Sydnes, M.O., Jørgensen, K.B., 2014. Enantioselective synthesis of (-)-(1R,2R)-1,2-dihydrochrysene-1,2-diol. Tetrahedron 70, 9041e9051. http:// dx.doi.org/10.1016/j.tet.2014.10.016.  ttir, B., Jo nsdottir, H., Helgason, S., Bjo €rnsson, B., Jørgensen, T.Ø., Magnado € m, L., 1999a. Humoral immune parameters in Atlantic cod (Gadhus Pilstro morhua L.) I. The effects of environmental temperature. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 122, 173e180. PII: S0305-0491(98)10156-6.  ttir, B., Jo nsdottir, H., Helgason, S., Bjo €rnsson, B., Jørgensen, T.Ø., Magnado € m, L., 1999b. Humoral immune parameters in Atlantic cod (Gadhus Pilstro morhua L.) II. The effects of size and gender under different environmental conditions. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 122, 181e188. PII: S0305-0491(98)10157-8. Magnadottir, B., Gudmundsdottir, S., Gudmundsdottir, B.K., Helgason, S., 2009. Natural antibodies of cod (Gadus morhua L.): specificity, activity and affinity. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 154, 309e316. http:// dx.doi.org/10.1016/j.cbpb.2009.07.005. Magnadottir, B., 2014. The immune response of Atlantic cod, Gadus morhua L. Icel. Agric. Sci. 27, 41e61. Meier, S., Morton, H.C., Nyhammer, G., Grøsvik, B.E., Makhotin, V., Geffen, A., Boitsov, S., Kvestad, K.A., Bohne-Kjersem, A., Goksøyr, A., Folkvord, A., Kungsøyr, J., Svardal, A., 2010. Development of Atlantic cod (Gadus morhua) exposed to produced water during early life stages: effects on embryos, larvae, and juvenile fish. Mar. Environ. Res. 70, 383e394. http://dx.doi.org/10.1016/ j.marenvres.2010.08.002. Metcalf, V.J., Gemmel, N.J., 2005. Fatty acid transport in cartilaginous fish: absence of albumin and possible utilization of lipoproteins. Fish. Physiol. Biochem. 31, 55e64. http://dx.doi.org/10.1007/s10695-005-5124-y. Monsinjon, T., Knigge, T., 2007. Proteomic applications in ecotoxicology. Proteomics 7, 2997e3009. http://dx.doi.org/10.1002/pmic.200700101. Nilsen, M.M., Meier, S., Andersen, O.K., Hjelle, A., 2011a. SELDI-TOF MS analysis of

304

K. Skogland Enerstvedt et al. / Chemosphere 183 (2017) 294e304

alkylphenol exposed Atlantic cod with phenotypic variation in gonadosomatic index. Mar. Pollut. Bull. 62, 2507e2511. http://dx.doi.org/10.1016/ j.marpolbul.2011.08.006. Nilsen, M.M., Meier, S., Larsen, B.K., Andersen, O.K., Hjelle, A., 2011b. An estrogenresponsive plasma protein expression signature in Atlantic cod (Gadus morhua) revealed by SELDI-TOF MS. Ecotoxicol. Environ. Saf. 74, 2175e2181. http://dx.doi.org/10.1016/j.ecoenv.2011.07.036. Øverland, H.S., Petterseb, E.F., Rønneseth, A., Wergeland, H.I., 2010. Phagocytosis by B-cells and neutrophils in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.). Fish. Shellfish Immunol. 28, 193e204. http://dx.doi.org/ 10.1016/j.fsi.2009.10.021. Pampanin, D.M., Sydnes, M.O., 2013. Polycyclic aromatic hydrocarbons a constituent of petroleum: presence and influence in the aquatic environment. In: Kutcherov, V., Kolesnikov, A. (Eds.), Hydrocarbons. InTech, Rijeka, pp. 83e118. http://dx.doi.org/10.5772/48176. Pampanin, D.M., Larssen, E., Øysæd, K.B., Sundt, R.C., 2014. Study of the bile proteome of Atlantic cod (Gadus morhua): multi-biological markers of exposure to polycyclic aromatic hydrocarbons. Mar. Environ. Res. 101, 161e168. http:// dx.doi.org/10.1016/j.marenvres.2014.10.002. Pampanin, D.M., 2017. The presence of petrogenic PAHs in the aquatic environment, a focus on monitoring studies. In: Pampanin, D.M., Sydnes, M.O. (Eds.), Petrogenic Polycyclic Aromatic Hydrocarbons in the Aquatic Environment: Analysis, Synthesis, Toxicity and Environmental Impact. Bentham Science Publishers. http://ebooks.benthamscience.com/future-books/D4/. Pampanin, D.M., Le Goff, J., Skogland, K., Marcucci, C.R., Øysæd, K.B., Lorentzen, M., Jørgensen, K.B., Sydnes, M.O., 2016. Biological effects of PAHs and their first metabolic products in in vivo exposed Atlantic cod (Gadus morhua). J. Toxicol. Environ. Health Part A 79, 633e646. http://dx.doi.org/10.1080/ 15287394.2016.1171993. rez-Casanova, J.C., Hamoutene, D., Samuelson, S., Burt, K., King, T.I., Lee, K., 2010. Pe The immune response of juvenile Atlantic cod (Gadus morhua L.) to chronic exposure to produced water. Mar. Environ. Res. 70, 26e34. http://dx.doi.org/ 10.1016/j.marenvres.2010.02.005. €m, L., Warr, G.W., Stro €mberg, S., 2005. Why is the antibody response of Pilstro Atlantic cod so poor? The search for genetic explanation. Fish. Sci. 71, 961e971. Qiao, Q., Manach, S.L., Huet, H., Duvernois-Berthet, E., Chaouch, S., Duval, C., ron, L., Lennon, S., Bolbach, G., Djediat, C., Sotton, B., Ponger, L., Marie, A., Mathe Bernard, C., Edery, M., Marie, B., 2016. An integrated omic analysis of hepatic alteration in medaka fish chronically exposed to cyanotoxins with possible mechanisms of reproductive toxicity. Environ. Pollut. 219, 119e131. http:// dx.doi.org/10.1016./j.envpol.2016.10.029. Qu, M., Huang, X., Zhang, X., Liu, Q., Ding, S., 2014. Cloning and expression analysis of apolipoprotein A-I (ApoA-I) in the Hong Kong grouper (Epinephelus akaara). Aquaculture 432, 85e96. http://dx.doi.org/10.1016/j.aquaculture.2014.04.023. Rajan, B., Fernandes, J.M.O., Caipang, C.M.A., Kiron, V., Rombout, J.H.W.M., Brinchmann, M.F., 2011. Proteome reference map of the skin mucus of Atlantic cod (Gadus morhua) revealing immune competent molecules. Fish. Shellfish Immunol. 31, 224e231. http://dx.doi.org/10.1016/j.fsi.2011.05.006. Reynaud, S., Deschaux, P., 2006. The effects of polycyclic aromatic hydrocarbons on the immune system of fish: a review. Aquat. Toxicol. 77, 229e238. http:// dx.doi.org/10.1016/j.aquatox.2005.10.018. Ruppert, J.L.M., Fortin, M.-J., Rose, G.A., Devillers, R., 2009. Atlantic cod (Gadus morhua) distribution response to environmental variability in the northern Gulf of St. Lawrence. Can. J. Fish. Aquat. Sci. 66, 909e918. http://dx.doi.org/10.1139/ F09-049. Sanchez, B.C., Ralston-Hooper, K., Sepúlveda, M.S., 2011. Review of recent proteomic applications in aquatic toxicology. Environ. Toxicol. Chem. 30, 274e282. http:// dx.doi.org/10.1002/etc.402. Sandnes, K., Lie, Ø., Waagbø, R., 1988. Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon. Salmo Salar. J. Fish. Biol. 32, 129e136. DOI: 0022-1112/88/010129. €rkblom, C., Jonsson, H., Godal, B.F., Liewenborg, B., Lyng, E., Sanni, S., Bjo

Pampanin, D.M., 2017. I: biomarker quantification in fish exposed to crude oil as input to species sensitivity distributions and threshold values for environmental monitoring. Mar. Environ. Res. 125, 10e24. http://dx.doi.org/10.1016/ j.marenvres.2016.12.002. Savaryn, J.P., Toby, T.K., Kelleher, N.L., 2016. A researcher's guide to mass spectrometry-based proteomics. Proteomics 0, 1e9. http://dx.doi.org/10.1002/ pmic.201600113. Smith, M.A.K., McKay, M.C., Lee, R.F., 1988. Catfish plasma lipoproteins: in vivo studies of apoprotein synthesis and catabolism. Comp. Biochem. Physiol. 246, 223e235. http://dx.doi.org/10.1002/jez.1402460302. Solem, S.T., Stenvik, J., 2006. Antibody repertoire development in teleosts - a review with emphasis on salmonids and Gadus morhua L. Dev. Comp. Immunol. 30, 57e76. http://dx.doi.org/10.1016/j.dci.2005.06.007. Song, Q., Chen, H., Li, Y., Zhou, H., Han, Q., Diao, X., 2016. Toxicological effects of benzo(a)pyrene, DDT and their mixture on the green mussel Perna viridis revealed by proteomic and metabolomic approaches. Chemosphere 144, 214e224. http://dx.doi.org/10.1016/j.chemosphere.2015.08.029. Star, B., Nederbragt, A.J., Jentoft, S., Grimholt, U., Malmstrom, M., Gregers, T.F., Rounge, T.B., Paulsen, J., Solbakken, M.H., Sharma, A., Wetten, O.F., Lanzen, A., Winer, R., Knight, J., Vogel, J.-H., Aken, B., Andersen, O., Lagesen, K., ToomingKlunderud, A., Edvardsen, R.B., Tina, K.G., Espelund, M., Nepal, C., Previti, C., Karlsen, B.O., Moum, T., Skage, M., Berg, P.R., Gjoen, T., Kuhl, H., Thorsen, J., Malde, K., Reinhardt, R., Du, L., Johansen, S.D., Searle, S., Lien, S., Nilsen, F., Jonassen, I., Omholt, S.W., Stenseth, N.C., Jakobsen, K.S., 2011. The genome sequence of Atlantic cod reveals a unique immune system. Nature 477, 207e210. http://dx.doi.org/10.1038/nature10342. Stensvik, J., Jørgensen, T.Ø., 2000. Immunoglobulin D (IgD) of Atlantic cod has a unique structure. Immunogenetics 51, 452e461. Vitale, F., 2008. Reproductive Aspects of Kategat Cod (Gadus morhua): Implications for Stock Assessment and Management (Doctoral Dissertation). University of Gothenburg. http://www.gu.se/digitalAssets/1175/1175309_vitale_phd_thesis. pdf (Accessed 02 January 2017). Wang, H.-S., Man, Y.-B., Wu, F.-Y., Zhao, Y.-G., Wong, C.K.C., Wong, M.-H., 2010. Oral bioaccessibility of polycyclic aromatic hydrocarbons (PAHs) through fish consumption, based on an in vitro digestion model. J. Agric. Food Chem. 58, 11517e11524. http://dx.doi.org/10.1021/jf102242m. Woo, S., Lee, A., Denis, V., Chen, C.A., Yum, S., 2014. Transcript response of soft coral (Scleronephthya gracillimum) on exposure to polycyclic aromatic hydrocarbons. Environ. Sci. Pollut. Res. 21, 901e910. http://dx.doi.org/10.1007/s11356-0131958-5. Xia, J.-H., Liu, J.-X., Zhou, L., Li, Z., Gui, J.-F., 2008. Apo-14 is required for digestive system organogenesis during fish embryogenesis and larval development. Int. J. Dev. Biol. 52, 1089e1098. http://dx.doi.org/10.1387/ijdb.072519jx. Yadetie, F., Bjørneklett, S., Garberg, H.K., Oveland, E., Berven, F., Goksøyr, A., Karlsen, O.A., 2016. Quantitative analyses of the hepatic proteome of methylmercury-exposed Atlantic cod (Gadus morhua) suggest oxidative stressmediated effects on cellular energy metabolism. BMC Genomics 17 (554), 1e15. http://dx.doi.org/10.1186/s12864-016-2864-2. Yaragina, N.A., 2010. Biological parameters of immature, ripening, and nonreproductive, mature northeast Arctic cod in 1984-2006. ICES J. Mar. Sci. 67, 2033e2041. http://dx.doi.org/10.1093/icesjms/fsq059. Zhang, Y., Fonslow, B.R., Shan, B., Baek, M.-C., Yates, J.R., 2013. Protein analysis by shotgun/bottom-up proteomics. Chem. Rev. 113, 2343e2394. http://dx.doi.org/ 10.1021/cr3003533. Zhou, Li, Wang, Y., Li, V., Ji, G.-D., Gui, J.F., 2005. Molecular cloning and expression pattern of 14 kDa apolipoprotein in orange-spotted grouper, Epinephelus coioides. Comp. Biochem. Physiol. 142, 432e437. http://dx.doi.org/10.1016/ j.cbpb.2005.09.007. Zybailov, B., Mosley, A.L., Sardiu, M.E., Coleman, M.K., Florens, L., Washburn, M.P., 2006. Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J. Proteom. Res. 5, 2339e2347. http://dx.doi.org/ 10.1021/pr060161n.