Environmentally realistic concentrations of chlorinated, brominated, and fluorinated persistent organic pollutants induce the unfolded protein response as a shared stress pathway in the liver of Atlantic cod (Gadus morhua).

In the North Sea and North Atlantic coastal areas, fish experience relatively high background levels of persistent organic pollutants. This study aimed to compare the mode of action of environmentally relevant concentrations of mixtures of halogenated compounds in Atlantic cod. Juvenile male cod with mean weight of 840 g were exposed by gavage to dietary mixtures of chlorinated (PCBs, DDT analogs, chlordane, lindane, and toxaphene), brominated (PBDEs), and fluorinated (PFOS) compounds for 4 weeks. One group received a combined mixture of all three compound groups. The results showed that the accumulated levels of chemicals in cod liver after 4 weeks of exposure reflected concentrations found in wild fish in this region. Pathway analysis revealed that the treatment effects by each of the three groups of chemicals (chlorinated, brominated, and fluorinated) converged on activation of the unfolded protein response (UPR). Upstream regulator analysis predicted that almost all the key transcription factors (XBP1, ERN1, ATF4, EIF2AK3, and NFE2L2) regulating the UPR were significantly activated. No additive effect was observed in cod co-treated with all three compound groups. In conclusion, the genome-wide transcriptomic study suggests that the UPR pathway is a sensitive common target of halogenated organic environmental pollutants in fish.

Spawning time in adult polar cod (Boreogadus saida) altered by crude oil exposure, independent of food availability.

Fish early life stages are well known for their sensitivity to crude oil exposure. However, the effect of crude oil exposure on adults and their gametes during their spawning period is not well studied. Polar cod, a key arctic fish, may be at risk for crude oil exposure during this potentially sensitive life stage. Additionally, this species experiences lower food availability during their spawning season, with unknown combined consequences. In the present study, wild-caught polar cod were exposed to decreasing levels of a water-soluble fraction (WSF) of crude oil or control conditions and fed either at a low or high feed ration to assess the combined effect of both stressors. Samples were taken during late gonadal development, during active spawning (spawning window), and in the post-spawning period. Histology analysis of gonads from fish sampled during the spawning window showed that oil-exposed polar cod were more likely to have spawned compared to controls. Oil-exposed females had 947 differentially regulated hepatic genes, and their eggs had a higher polycyclic aromatic hydrocarbon body burden compared to controls. Feed ration did not consistently affect polar cod's response to oil exposure for the endpoints measured, however, did alone result in decreases in some sperm motility parameters. These results suggest that polar cod's spawning period is a sensitive life event to crude oil exposure, while feed limitation may play a minor role for this supposedly capital breeder. The effects of adult exposure to crude oil on gamete quality and the next generation warrant further investigation.

Integrative omics-analysis of lipid metabolism regulation by peroxisome proliferator-activated receptor a and b agonists in male Atlantic cod.

Lipid metabolism is essential in maintaining energy homeostasis in multicellular organisms. In vertebrates, the peroxisome proliferator-activated receptors (PPARs, NR1C) regulate the expression of many genes involved in these processes. Atlantic cod (Gadus morhua) is an important fish species in the North Atlantic ecosystem and in human nutrition, with a highly fatty liver. Here we study the involvement of Atlantic cod Ppar a and b subtypes in systemic regulation of lipid metabolism using two model agonists after in vivo exposure. WY-14,643, a specific PPARA ligand in mammals, activated cod Ppara1 and Ppara2 in vitro. In vivo, WY-14,643 caused a shift in lipid transport both at transcriptional and translational level in cod. However, WY-14,643 induced fewer genes in the fatty acid beta-oxidation pathway compared to that observed in rodents. Although GW501516 serves as a specific PPARB/D ligand in mammals, this compound activated cod Ppara1 and Ppara2 as well as Pparb in vitro. In vivo, it further induced transcription of Ppar target genes and caused changes in lipid composition of liver and plasma. The integrative approach provide a foundation for understanding how Ppars are engaged in regulating lipid metabolism in Atlantic cod physiology. We have shown that WY-14,643 and GW501516 activate Atlantic cod Ppara and Pparb, affect genes in lipid metabolism pathways, and induce changes in the lipid composition in plasma and liver microsomal membranes. Particularly, the combined transcriptomic, proteomics and lipidomics analyses revealed that effects of WY-14,643 on lipid metabolism are similar to what is known in mammalian studies, suggesting conservation of Ppara functions in mediating lipid metabolic processes in fish. The alterations in the lipid profiles observed after Ppar agonist exposure suggest that other chemicals with similar Ppar receptor affinities may cause disturbances in the lipid regulation of fish. Model organism: Atlantic cod (Gadus morhua). LSID: urn:lsid:zoobank.org:act:389BE401-2718-4CF2-BBAE-2E13A97A5E7B. COL Identifier: 6K72F.

Intestinal permeability and gene expression after polyethylene and polyamide microplastic ingestion in Wistar rats.

Microplastic particles are ubiquitous in the environment. However, little is known about their uptake and effects in humans or mammalian model organisms. Here, we studied the effects of pristine polyamide (15-20 µm) and polyethylene (40-48 µm) particles after oral ingestion in rats. The animals received feed containing microplastic particles (0.1% polyamide or polyethylene, or a mixture of both polymers) or a control diet without microplastic particles, for 5 weeks. The permeability of the duodenum was investigated in an Ussing chamber, whereas gene expression and concentration of tight junction proteins were measured in gut tissue and plasma. Microplastic particles were quantified by pyrolysis-gas chromatography/mass spectrometry in rats' feces. Rats fed with microplastic particles had higher duodenal permeability. Expression of gene coding for the tight junction protein occludin (OCLN) was higher in PE treated animals compared to control or the PA group. No changes in the expression of the gene coding for zonula occludens protein 1 were detected. Occludin protein concentrations were below the limit of detection of the applied method in both gut and plasma. Zonula occludens protein 1 concentrations in the gut were significantly higher in groups exposed to PA and PE as compared to control, while zonula occludens protein 1 concentrations in plasma did not show significant changes. These results demonstrated that short-term exposure to a dose of 0.1% (w/w) microplastic particles in feed had limited effects on duodenal permeability, expression of pro-inflammatory protein genes and tight junction protein genes in the duodenum.

Transcriptome responses in copepods Calanus finmarchicus, Calanus glacialis and Calanus hyperboreus exposed to phenanthrene and benzo[a]pyrene.

Arctic and sub-arctic pelagic organisms can be exposed to effluents and spills from offshore petroleum-related activities and thus it is important to understand how they respond to crude oil related contaminants such as polycyclic aromatic hydrocarbons (PAHs). The copepod species Calanus finmarchicus, Calanus glacialis and Calanus hyperboreus represent key links in the arctic marine food web. We performed a transcriptome analysis of the three species exposed to phenanthrene (Phe) and benzo[a]pyrene (BaP) representing low and high molecular weight PAHs, respectively. Differential expression of several genes involved in many cellular pathways was observed after 72 h exposure to Phe (0.1 µM) and BaP (0.1 µM). In C. finmarchicus and C. glacialis, the exposure resulted in up-regulation of genes encoding enzymes in xenobiotic biotransformation, particularly the phase II cytosolic sulfonation system that include 3'-phosphoadenosine 5'-phosphosulfate synthase (PAPSS) and sulfotransferases (SULTs). The sulfonation pathway genes were more strongly induced by BaP than Phe in C. finmarchicus and C. glacialis but were not affected in C. hyperboreus. However, a larger number of genes and pathways were modulated in C. hyperboreus by the PAHs including genes encoding xenobiotic biotransformation and lipid metabolism enzymes, suggesting stronger responses in this species. The results suggest that the cytosolic sulfonation is a major phase II conjugation pathway for PAHs in C. finmarchicus and C. glacialis. Some of the biotransformation systems affected are known to be involved in metabolism of endogenous compounds such as ecdysteroids, which may suggest potential interference with physiological and developmental processes of the copepod species.

Agonistic and potentiating effects of perfluoroalkyl substances (PFAS) on the Atlantic cod (Gadus morhua) peroxisome proliferator-activated receptors (Ppars).

Toxicity mediated by per- and polyfluoroalkyl substances (PFAS), and especially perfluoroalkyl acids (PFAAs), has been linked to activation of peroxisome proliferator-activated receptors (Ppar) in many vertebrates. Here, we present the primary structures, phylogeny, and tissue-specific distributions of the Atlantic cod (Gadus morhua) gmPpara1, gmPpara2, gmPparb, and gmPparg, and demonstrate that the carboxylic acids PFHxA, PFOA, PFNA, as well as the sulfonic acid PFHxS, activate gmPpara1 in vitro, which was also supported by in silico analyses. Intriguingly, a binary mixture of PFOA and the non-activating PFOS produced a higher activation of gmPpara1 compared to PFOA alone, suggesting that PFOS has a potentiating effect on receptor activation. Supporting the experimental data, docking and molecular dynamics simulations of single and double-ligand complexes led to the identification of a putative allosteric binding site, which upon binding of PFOS stabilizes an active conformation of gmPpara1. Notably, binary exposures of gmPpara1, gmPpara2, and gmPparb to model-agonists and PFAAs produced similar potentiating effects. This study provides novel mechanistic insights into how PFAAs may modulate the Ppar signaling pathway by either binding the canonical ligand-binding pocket or by interacting with an allosteric binding site. Thus, individual PFAAs, or mixtures, could potentially modulate the Ppar-signaling pathway in Atlantic cod by interfering with at least one gmPpar subtype.

Single PFAS and PFAS mixtures affect nuclear receptor- and oxidative stress-related pathways in precision-cut liver slices of Atlantic cod (Gadus morhua).

The aim of the present study was to investigate effects of per- and polyfluoroalkyl substances (PFAS), both single compounds and a mixture of these, using precision-cut liver slices (PCLS) from Atlantic cod (Gadus morhua). PCLS were exposed for 48 h to perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA) and perfluorononanoate (PFNA) (10, 50 and 100 µM), and three mixtures of these at equimolar concentrations (10, 50 and 100 µM). Transcriptomic responses were assessed using RNA sequencing. Among exposures to single PFAS, PFOS produced the highest number of differentially expressed genes (DEGs) compared to PFOA and PFNA (86, 25 and 31 DEGs, respectively). Exposure to the PFAS mixtures resulted in a markedly higher number of DEGs (841). Clustering analysis revealed that the expression pattern of the PFAS mixtures were more similar to PFOS compared to PFOA and PFNA, suggesting that effects induced by the PFAS mixtures may largely be attributed to PFOS. Pathway analysis showed significant enrichment of pathways related to oxidative stress, cholesterol metabolism and nuclear receptors in PFOS-exposed PCLS. Fewer pathways were significantly enriched following PFOA and PFNA exposure alone. Significantly enriched pathways following mixture exposure included lipid biosynthesis, cancer-related pathways, nuclear receptor pathways and oxidative stress-related pathways such as ferroptosis. The expression of most of the genes within these pathways was increased following PFAS exposure. Analysis of non-additive effects in the 100 µM PFAS mixture highlighted genes involved in the antioxidant response and membrane transport, among others, and the majority of these genes had synergistic expression patterns in the mixture. Nevertheless, 90% of the DEGs following mixture exposure showed additive expression patterns, suggesting additivity to be the major mixture effect. In summary, PFAS exposure promoted effects on cellular processes involved in oxidative stress, nuclear receptor pathways and sterol metabolism in cod PCLS, with the strongest effects observed following PFAS mixture exposure.

Photo-enhanced toxicity of crude oil on early developmental stages of Atlantic cod (Gadus morhua).

Photo-enhanced toxicity of crude oil is produced by exposure to ultraviolet (UV) radiation. Atlantic cod (Gadus morhua) embryos were exposed to crude oil with and without UV radiation (290-400 nm) from 3 days post fertilization (dpf) until 6 dpf. Embryos from the co-exposure experiment were continually exposed to UV radiation until hatching at 11 dpf. Differences in body burden levels and cyp1a expression in cod embryos were observed between the exposure regimes. High doses of crude oil produced increased mortality in cod co-exposed embryos, as well as craniofacial malformations and heart deformities in larvae from both experiments. A higher number of differentially expressed genes (DEGs) and pathways were revealed in the co-exposure experiment, indicating a photo-enhanced effect of crude oil toxicity. Our results provide mechanistic insights into crude oil and photo-enhanced crude oil toxicity, suggesting that UV radiation increases the toxicity of crude oil in early life stages of Atlantic cod.

Transcriptome responses in polar cod (Boreogadus saida) liver slice culture exposed to benzo[a]pyrene and ethynylestradiol: insights into anti-estrogenic effects.

Polar cod (Boreogadus saida) is a key species in the arctic marine ecosystem vulnerable to effects of pollution, particularly from petroleum related activities. To facilitate studying the effects of those pollutants, we adapted a precision-cut liver slice culture protocol for this species. Using this system on board a research vessel, we studied gene expression in liver slice after exposure to the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP), ethynylestradiol (EE2), and their mixtures, to map their molecular targets and examine possible anti-estrogenic effects of BaP. The exposure experiments were performed with BaP alone (0.1, 1, and 10 µM) or in combination with low concentrations of EE2 (5 nM) to mimic physiological estradiol levels in early vitellogenic female fish. Transcriptome analysis (RNA-seq) was performed after 72 h exposure in culture to map the genes and cellular pathways affected. The results provide a view of global transcriptome responses to BaP and EE2, which resulted in enrichment of many pathways such as the aryl hydrocarbon (Ahr) and estrogen receptor pathways. In the mixture exposure, BaP resulted in anti-estrogenic effects, shown by attenuation of EE2 activated transcription of many estrogen target genes. The results from this ex vivo experiment suggest that pollutants that activate the Ahr pathway such as the PAH compound BaP can result in anti-estrogenic effects that may lead to endocrine disruption in polar cod.

Bioaccumulation of mercury and transcriptional responses in tusk (Brosme brosme), a deep-water fish from a Norwegian fjord.

High concentrations of mercury (Hg) have been documented in deep-water fish species from some Norwegian fjords. In this study, tusk (Brosme brosme) was sampled from four locations in the innermost parts of Sognefjorden in Western Norway. Total Hg and methylmercury (MeHg) levels were measured in liver tissue. To search for potential sublethal effects of Hg, we characterized the hepatic transcriptome in tusk with high and low levels of Hg bioaccumulation using global transcriptomics analysis (RNA-seq). The results showed that there was a significant correlation between fish weight and accumulated concentrations of MeHg but not total Hg. MeHg accounted for 30-40% of total Hg in liver of most of the fish, although at concentrations above 2-3 mg Hg/kg wet weight the percentage of MeHg dropped considerably. Transcriptome analysis resulted in hundreds of differentially expressed genes in the liver of tusk with high Hg levels. Functional enrichment analysis suggested that the top affected pathways are associated with protein folding, adipogenesis, notch signaling, and lipid metabolism (beta-oxidation and phospholipids). Based on transcriptional responses pointing to well-known effects of Hg compounds in fish, the study suggests that tusk in Sognefjorden could be negatively impacted by Hg bioaccumulation.

Application of quantitative transcriptomics in evaluating the ex vivo effects of per- and polyfluoroalkyl substances on Atlantic cod (Gadus morhua) ovarian physiology.

Because of their global consumption and persistence, per- and polyfluoroalkyl substances (PFASs), are ubiquitously distributed in the environment, as well as in wildlife and humans. In the present study, we have employed an ex vivo organ culture technique, based on the floating agarose method, of Atlantic cod ovarian tissue to investigate the effects of three different concentrations of PFOS, PFOA (1, 5 and 25 µM) and PFNA (0.5, 5 and 50 µM), used singly and in also in combination (1×, 20× and 100×). In the 1× exposure mixture, concentrations were decided based on their proportional levels (in molar equivalents) relative to PFOS, which is the most abundant PFAS in cod liver from a 2013 screening project. To investigate the detailed underlying mechanisms and biological processes, transcriptome sequencing was performed on exposed ovarian tissue. The number of differentially expressed genes (DEGs) having at least 0.75 log2-fold change was elevated in high, compared to low and medium concentration exposures. The highest PFNA, PFOA and PFOS concentrations, and the highest (100×) mixture exposure, showed 40, 68, 1295, and 802 DEGs, respectively. The latter two exposure groups shared a maximum of 438 DEGs. In addition, they both shared the majority of functionally enriched pathways belonging to biological processes such as cellular signaling, cell adhesion, lipid metabolism, immunological responses, cancer, reproduction and metabolism. Shortlisted DEGs that were specifically annotated to reproduction associated gene ontology (GO) terms were observed only in the highest PFOS and mixture exposure groups. These transcripts contributed to ovarian key events such as steroidogenesis (star, cyp19a1a), oocyte growth (amh), maturation (igfbp5b, tgfß2, tgfß3), and ovulation (pgr, mmp2). Contrary to other PFAS congeners, the highest PFOS concentration showed almost similar transcript expression patterns compared to the highest mixture exposure group. This indicates that PFOS is the active component of the mixture that significantly altered the normal functioning of female gonads, and possibly leading to serious reproductive consequences in teleosts.

ReCodLiver0.9: Overcoming Challenges in Genome-Scale Metabolic Reconstruction of a Non-model Species.

The availability of genome sequences, annotations, and knowledge of the biochemistry underlying metabolic transformations has led to the generation of metabolic network reconstructions for a wide range of organisms in bacteria, archaea, and eukaryotes. When modeled using mathematical representations, a reconstruction can simulate underlying genotype-phenotype relationships. Accordingly, genome-scale metabolic models (GEMs) can be used to predict the response of organisms to genetic and environmental variations. A bottom-up reconstruction procedure typically starts by generating a draft model from existing annotation data on a target organism. For model species, this part of the process can be straightforward, due to the abundant organism-specific biochemical data. However, the process becomes complicated for non-model less-annotated species. In this paper, we present a draft liver reconstruction, ReCodLiver0.9, of Atlantic cod (Gadus morhua), a non-model teleost fish, as a practicable guide for cases with comparably few resources. Although the reconstruction is considered a draft version, we show that it already has utility in elucidating metabolic response mechanisms to environmental toxicants by mapping gene expression data of exposure experiments to the resulting model.

Proteomics and lipidomics analyses reveal modulation of lipid metabolism by perfluoroalkyl substances in liver of Atlantic cod (Gadus morhua).

The aim of the present study was to investigate effects of defined mixtures of polycyclic aromatic hydrocarbons (PAHs) and perfluoroalkyl substances (PFASs), at low, environmentally relevant (1× = L), or high (20× = H) doses, on biological responses in Atlantic cod (Gadus morhua). To this end, farmed juvenile cod were exposed at day 0 and day 7 via intraperitoneal (i.p.) injections, in a two-week in vivo experiment. In total, there were 10 groups of fish (n = 21-22): two control groups, four separate exposure groups of PAH and PFAS mixtures (L, H), and four groups combining PAH and PFAS mixtures (L/L, H/L, L/H, H/H). Body burden analyses confirmed a dose-dependent accumulation of PFASs in cod liver and PAH metabolites in bile. The hepatosomatic index (HSI) was significantly reduced for three of the combined PAH/PFAS exposure groups (L-PAH/H-PFAS, H-PAH/L-PFAS, H-PAH/H-PFAS). Analysis of the hepatic proteome identified that pathways related to lipid degradation were significantly affected by PFAS exposure, including upregulation of enzymes in fatty acid degradation pathways, such as fatty acid ß-oxidation. The increased abundances of enzymes in lipid catabolic pathways paralleled with decreasing levels of triacylglycerols (TGs) in the H-PFAS exposure group, suggest that PFAS increase lipid catabolism in Atlantic cod. Markers of oxidative stress, including catalase and glutathione S-transferase activities were also induced by PFAS exposure. Only minor and non-significant differences between exposure groups and control were found for cyp1a and acox1 gene expressions, vitellogenin concentrations in plasma, Cyp1a protein synthesis and DNA fragmentation. In summary, our combined proteomics and lipidomics analyses indicate that PFAS may disrupt lipid homeostasis in Atlantic cod.

Quantitative transcriptomics, and lipidomics in evaluating ovarian developmental effects in Atlantic cod (Gadus morhua) caged at a capped marine waste disposal site.

In the present study, a previously capped waste disposal site at Kollevåg (Norway) was selected to study the effects of contaminant leakage on biomarkers associated with Atlantic cod (Gadus morhua) reproductive endocrinology and development. Immature cod were caged for 6 weeks at 3 locations, selected to achieve a spatial gradient of contamination, and compared to a reference station. Quantitative transcriptomic, and lipidomic analysis was used to evaluate the effects of the potential complex contaminant mixture on ovarian developmental and endocrine physiology. The number of expressed transcripts, with 0.75 log2-fold differential expression or more, varied among stations and paralleled the severity of contamination. Particularly, significant bioaccumulation of ∑PCB-7, ∑DDTs and ∑PBDEs were observed at station 1, compared to the other station, including the reference station. Respectively 1416, 698 and 719 differentially expressed genes (DEGs), were observed at stations 1, 2 and 3, compared to the reference station, with transcripts belonging to steroid hormone synthesis pathway being significantly upregulation. Transcription factors such as esr2 and ahr2 were increased at all three stations, with highest fold-change at Station 1. MetaCore pathway maps identified affected pathways that are involved in ovarian physiology, where some unique pathways were significantly affected at each station. For the lipidomics, sphingolipid metabolism was particularly affected at station 1, and these effects paralleled the high contaminant burden at this station. Overall, our findings showed a novel and direct association between contaminant burden and ovarian toxicological and endocrine physiological responses in cod caged at the capped Kollevåg waste disposal site.

Molecular and Functional Properties of the Atlantic Cod (Gadus morhua) Aryl Hydrocarbon Receptors Ahr1a and Ahr2a.

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that mediates the toxicity of halogenated and polycyclic aromatic hydrocarbons in vertebrates. Atlantic cod (Gadus morhua) has recently emerged as a model organism in environmental toxicology studies, and increased knowledge of Ahr-mediated responses to xenobiotics is imperative. Genome mining and phylogenetic analyses revealed two Ahr-encoding genes in the Atlantic cod genome, gmahr1a and gmahr2a. In vitro binding assays showed that both gmAhr proteins bind to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but stronger binding to gmAhr1a was observed. Transactivation studies with a reporter gene assay revealed that gmAhr1a is one order of magnitude more sensitive to TCDD than gmAhr2a, but the maximal responses of the receptors were similar. Other well-known Ahr agonists, such as ß-naphthoflavone (BNF), 3,3',4,4',5-pentachlorobiphenyl (PCB126), and 6-formylindolo[3,2-b]carbazole (FICZ), also activated the gmAhr proteins, but gmAhr1a was, in general, the more sensitive receptor and produced the highest efficacies. The induction of cyp1a in exposed precision-cut cod liver slices confirmed the activation of the Ahr signaling pathway ex vivo. In conclusion, the differences in transcriptional activation by gmAhr's with various agonists, the distinct binding properties with TCDD and BNF, and the distinct tissue-specific expression profiles indicate different functional specializations of the Atlantic cod Ahr's.

Effects of Agricultural Pesticides in Aquafeeds on Wild Fish Feeding on Leftover Pellets Near Fish Farms.

Screening has revealed that modern-day feeds used in Atlantic salmon aquaculture might contain trace amounts of agricultural pesticides. To reach slaughter size, salmon are produced in open net pens in the sea. Uneaten feed pellets and undigested feces deposited beneath the net pens represent a source of contamination for marine organisms. To examine the impacts of long-term and continuous dietary exposure to an organophosphorus pesticide found in Atlantic salmon feed, we fed juvenile Atlantic cod (Gadus morhua), an abundant species around North Atlantic fish farms, three concentrations (0.5, 4.2, and 23.2 mg/kg) of chlorpyrifos-methyl (CPM) for 30 days. Endpoints included liver and bile bioaccumulation, liver transcriptomics and metabolomics, as well as plasma cholinesterase activity, cortisol, liver 7-ethoxyresor-ufin-O-deethylase activity, and hypoxia tolerance. The results show that Atlantic cod can accumulate relatively high levels of CPM in liver after continuous exposure, which is then metabolized and excreted via the bile. All three exposure concentrations lead to significant inhibition of plasma cholinesterase activity, the primary target of CPM. Transcriptomics profiling pointed to effects on cholesterol and steroid biosynthesis. Metabolite profiling revealed that CPM induced responses reflecting detoxification by glutathione-S-transferase, inhibition of monoacylglycerol lipase, potential inhibition of carboxylesterase, and increased demand for ATP, followed by secondary inflammatory responses. A gradual hypoxia challenge test showed that all groups of exposed fish were less tolerant to low oxygen saturation than the controls. In conclusion, this study suggests that wild fish continuously feeding on leftover pellets near fish farms over time may be vulnerable to organophosphorus pesticides.

Modulation of Neuro-Dopamine Homeostasis in Juvenile Female Atlantic Cod ( Gadus morhua) Exposed to Polycyclic Aromatic Hydrocarbons and Perfluoroalkyl Substances.

The dopaminergic effect of PAH and PFAS mixtures, prepared according to environmentally relevant concentrations, has been studied in juvenile female Atlantic cod ( Gadus morhua). Benzo[a]pyrene, dibenzothiophene, fluorene, naphthalene, phenanthrene, and pyrene were used to prepare a PAH mixture, while PFNA, PFOA, PFOS, and PFTrA were used to prepare a PFAS mixture. Cod were injected intraperitoneally twice, with either a low (1×) or high (20×) dose of each compound mixture or their combinations. After 2 weeks of exposure, levels of plasma 17ß-estradiol (E2) were significantly elevated in high PAH/high PFAS treated group. Brain dopamine/metabolite ratios (DOPAC/dopamine and HVA+DOPAC/dopamine) changed with E2 plasma levels, except for high PAH/low PFAS and low PAH/high PFAS treated groups. On the transcript levels, th mRNA inversely correlated with dopamine/metabolite ratios and gnrh2 mRNA levels. Respective decreases and increases of drd1 and drd2a after exposure to the high PAH dose were observed. Specifically, high PFAS exposure decreased both drds, leading to high plasma E2 concentrations. Other studied end points suggest that these compounds, at different doses and combinations, have different toxicity threshold and modes of action. These effects indicate potential alterations in the feedback signaling processes within the dopaminergic pathway by these contaminant mixtures.

Contaminant accumulation and biological responses in Atlantic cod (Gadus morhua) caged at a capped waste disposal site in Kollevåg, Western Norway.

The aim of this study was to assess whether fish in Kollevåg, a sheltered bay on the western coast of Norway, previously utilized as a waste disposal site, could be affected by environmental contaminants leaking from the waste. Farmed, juvenile Atlantic cod (Gadus morhua) were caged for six weeks at three different locations in Kollevåg bay and at one reference location. Sediments and cod samples (bile and liver) were analyzed for polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), brominated flame retardants (BFRs), per-and polyfluoroalkyl substances (PFASs) and polycyclic aromatic hydrocarbon (PAH) metabolites, revealing a contamination gradient at the four stations. Furthermore, hepatosomatic index (HSI) and Fulton's condition factor (CF) were significantly lower in cod caged closest to the disposal site. Levels and activities of biomarker proteins, such as vitellogenin (Vtg), metallothionein (Mt), and biotransformation and oxidative stress enzymes, including cytochrome P450 1a and 3a (Cyp1a, Cyp3a), glutathione s-transferase (Gst) and catalase (Cat), were quantified in blood plasma and liver tissue. Hepatic Cat and Gst activities were significantly reduced in cod caged at the innermost stations in Kollevåg, indicating modulation of oxidative stress responses. However, these results contrasted with reduced hepatic lipid peroxidation. Significant increases in transcript levels were observed for genes involved in lipid metabolism (fasn and acly) in cod liver, while transcript levels of ovarian steroidogenic enzyme genes such as p450scc, cyp19, 3ß-hsd and 20ß-hsd showed significant station-dependent increases. Cyp1a and Vtg protein levels were however not significantly altered in cod caged in Kollevåg. Plasma levels of estradiol (E2) and testosterone (T) were determined by enzyme immunoassay (EIA) and showed elevated E2 levels, but only at the innermost station. We conclude that the bay of Kollevåg did not fullfill adequate environmental condition based on environmental quality standards (EQSs) for chemicals in coastal waters. Following a six weeks caging period, environmental contaminants accumulated in cod tissues and effects were observed on biomarker responses, especially those involved in reproductive processes in cod ovary.

RNA-Seq analysis of transcriptome responses in Atlantic cod (Gadus morhua) precision-cut liver slices exposed to benzo[a]pyrene and 17α-ethynylestradiol.

Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) that activate the aryl hydrocarbon receptor (Ahr) pathway, and endocrine disruptors acting through the estrogen receptor pathway are among environmental pollutants of major concern. In this work, we exposed Atlantic cod (Gadus morhua) precision-cut liver slices (PCLS) to BaP (10 nM and 1000 nM), ethynylestradiol (EE2) (10 nM and 1000 nM), and equimolar mixtures of BaP and EE2 (10 nM and 1000 nM) for 48 h, and performed RNA-Seq based transcriptome mapping followed by systematic bioinformatics analyses. Our gene expression analysis showed that several genes were differentially expressed in response to BaP and EE2 treatments in PCLS. Strong up-regulation of genes coding for the cytochrome P450 1a (Cyp1a) enzyme and the Ahr repressor (Ahrrb) was observed in BaP treated PCLS. EE2 treatment of liver slices strongly up-regulated genes coding for precursors of vitellogenin (Vtg) and eggshell zona pellucida (Zp) proteins. As expected, pathway enrichment and network analysis showed that the Ahr and estrogen receptor pathways are among the top affected by BaP and EE2 treatments, respectively. Interestingly, two genes coding for fibroblast growth factor 3 (Fgf3) and fibroblast growth factor 4 (Fgf4) were up-regulated by EE2 in this study. To our knowledge, the fgf3 and fgf4 genes have not previously been described in relation to estrogen signaling in fish liver, and these results suggest the modulation of the FGF signaling pathway by estrogens in fish. The signature expression profiles of top differentially expressed genes in response to the single compound (BaP or EE2) treatment were generally maintained in the expression responses to the equimolar binary mixtures. However, in the mixture-treated groups, BaP appeared to have anti-estrogenic effects as observed by lower number of differentially expressed putative EE2 responsive genes. Our in-depth quantitative analysis of changes in liver transcriptome in response to BaP and EE2, using PCLS tissue culture provides further mechanistic insights into effects of the compounds. Moreover, the analyses demonstrate the usefulness of PCLS in cod for omics experiments.

Quantitative proteomics analysis reveals perturbation of lipid metabolic pathways in the liver of Atlantic cod (Gadus morhua) treated with PCB 153.

PCB 153 is one of the most abundant PCB congeners detected in biological samples. It is a persistent compound that is still present in the environment despite the ban on production and use of PCBs in the late 1970s. It has strong tendencies to bioaccumulate and biomagnify in biota, and studies have suggested that it is an endocrine and metabolic disruptor. In order to study mechanisms of toxicity, we exposed Atlantic cod (Gadus morhua) to various doses of PCB 153 (0, 0.5, 2 and 8mg/kg body weight) for two weeks and examined the effects on expression of liver proteins using label-free quantitative proteomics. Label-free liquid chromatography-mass spectrometry analysis of the liver proteome resulted in the quantification of 1272 proteins, of which 78 proteins were differentially regulated in the PCB 153-treated dose groups compared to the control group. Functional enrichment analysis showed that pathways significantly affected are related to lipid metabolism, cytoskeletal remodeling, cell cycle and cell adhesion. Importantly, the main effects appear to be on lipid metabolism, with up-regulation of enzymes in the de novo fatty acid synthesis pathway, consistent with previous transcriptomics results. Increased plasma triglyceride levels were also observed in the PCB 153 treated fish, in agreement with the induction of the lipogenic genes and proteins. The results suggest that PCB 153 perturbs lipid metabolism in the Atlantic cod liver. Elevated levels of lipogenic enzymes and plasma triglycerides further suggest increased synthesis of fatty acids and triglycerides.