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.

PCB-126 spiked to polyethylene microplastic ingested by juvenile Atlantic cod (Gadus morhua) accumulates in liver and muscle tissues.

In the present study, polyethylene (PE) microplastics (150-300 µm) were added to Atlantic cod (Gadus morhua) feeds at 1 %, either in their present form (Virgin PE) or spiked with PCB-126 (Spiked PE). The feeds were given to juvenile cod for a 4-week period. The fish grew from 11 to 23 g with no significant difference between dietary treatments. Cod fed spiked PE showed a significantly higher concentration of PCB-126 in liver and muscle samples compared to control and fish ingesting virgin PE. In accordance with the accumulation of PCB-126 in the liver, the expression of hepatic cyp1a was higher in cod fed spiked PE. Notably, we observed that spiked PE, as well as virgin PE, have an effect on skin. Overall changes indicated a reduced skin barrier in fish fed a diet containing PE. Indicating that PE itself through interaction with gut tissue may influence skin health in fish.

West African e-waste-soil assessed with a battery of cell-based bioassays.

Soil samples randomly taken from major e-waste sites in West Africa (Nigeria, Benin and Ghana) were examined for an extensive range of organic contaminants. Cytotoxicity measurements and assessment of activation of xeno-sensing receptors from fish (Atlantic cod) were employed as a battery of in vitro biological assays to explore the quality and toxicity profile of West African e-waste soil. The concentrations of the measured contaminants of emerging concerns (CECs) and persistent organic pollutants (POPs) in the e-waste soil differs significantly from the reference soil with chemical profiles typically dominated by legacy polybrominated diphenyl ethers (PBDEs) (405.8 µgkg-1) and emerging organophosphate ester flame retardant tris (1-chloro-2-propyl) phosphate (TCPP) (404 µgkg-1), in addition to the short chain perfluorobutane sulfonate (PFBS) (275.3 µgkg-1) and perfluorobutanoate (PFBA) (16 µgkg-1). The study revealed that perfluorooctanoic acid (PFOA) occurred only in e-waste soil from Ghana and ranged from 2.6 to 5.0 µgkg-1. Overall, non-polar e-waste soil-derived extracts had a stronger effect on COS-7 cell viability than the polar extracts and elutriates. The highest receptor activation was observed with single polar and non-polar extracts from the Nigeria and Benin sites, indicating hotspots with Er-, PPARa- and Ahr-agonist activities. Thus, the results obtained with our battery of in vitro biological assays underscored these e-waste sites as remarkably polluted spots with complex toxicity profiles of great concern for human and environmental health.

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.

Xenobiotic metabolism and its physiological consequences in high-Antarctic Notothenioid fishes.

The Antarctic ecosystem is progressively exposed to anthropogenic contaminants, such as polycyclic aromatic hydrocarbons (PAHs). So far, it is largely unknown if PAHs leave a mark in the physiology of high-Antarctic fish. We approached this issue via two avenues: first, we examined the functional response of the aryl hydrocarbon receptor (Ahr), which is a molecular initiating event of many toxic effects of PAHs in biota. Chionodraco hamatus and Trematomus loennbergii served as representatives for high-Antarctic Notothenioids, and Atlantic cod, Gadus morhua as non-polar reference species. We sequenced and cloned the Ahr ligand binding domain (LBD) of the Notothenioids and deployed a GAL4-based luciferase reporter gene assay expressing the Ahr LBD. Benzo[a]pyrene (BaP), beta-naphthoflavone and chrysene were used as ligands for the reporter gene assay. Second, we investigated the energetic costs of Ahr activation in isolated liver cells of the Notothenioids during acute, non-cytotoxic BaP exposure. In the reporter assay, the Ahr LBD of Atlantic cod and the Antarctic Notothenioids were activated by the ligands tested herein. In the in vitro assays with isolated liver cells of high-Antarctic Notothenioids, BaP exposure had no effect on overall respiration, but caused shifts in the respiration dedicated to protein synthesis. Thus, our study demonstrated that high-Antarctic fish possess a functional Ahr that can be ligand-activated in a concentration-dependent manner by environmental contaminants. This is associated with altered cost for cellular protein synthesis. Future studies have to show if the toxicant-induced activation of the Ahr pathway may lead to altered organism performance of Antarctic fish. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00300-021-02992-4.

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.

Substituted Two- to Five-Ring Polycyclic Aromatic Compounds Are Potent Agonists of Atlantic Cod (Gadus morhua) Aryl Hydrocarbon Receptors Ahr1a and Ahr2a.

Polycyclic aromatic hydrocarbons (PAHs) are among the most toxic and bioavailable components found in petroleum and represent a high risk to aquatic organisms. The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other planar aromatic hydrocarbons, including certain PAHs. Ahr acts as a xenosensor and modulates the transcription of biotransformation genes in vertebrates, such as cytochrome P450 1A (cyp1a). Atlantic cod (Gadus morhua) possesses two Ahr proteins, Ahr1a and Ahr2a, which diverge in their primary structure, tissue-specific expression, ligand affinities, and transactivation profiles. Here, a luciferase reporter gene assay was used to assess the sensitivity of the Atlantic cod Ahrs to 31 polycyclic aromatic compounds (PACs), including two- to five-ring native PAHs, a sulfur-containing heterocyclic PAC, as well as several methylated, methoxylated, and hydroxylated congeners. Notably, most parent compounds, including naphthalene, phenanthrene, and partly, chrysene, did not act as agonists for the Ahrs, while hydroxylated and/or alkylated versions of these PAHs were potent agonists. Importantly, the greater potencies of substituted PAH derivatives and their ubiquitous occurrence in nature emphasize that more knowledge on the toxicity of these environmentally and toxicologically relevant compounds is imperative.

Toxicity assessment of urban marine sediments from Western Norway using a battery of stress-activated receptors and cell-based bioassays from fish.

A luciferase reporter gene-based bioassay battery consisting of stress-activated receptors from fish, complemented with traditional fish cell-based bioassays, were used to assess the toxicity of marine sediment samples from the Byfjorden area around the city of Bergen (Norway). The reporter assays covered a wide range of cellular signalling and metabolic pathways, representing different molecular initiating events in the adverse outcome pathway framework. Cytotoxicity, generation of reactive oxygen-species, and induction of 7-ethoxyresorufin-O-deethylase activity were analysed using fish liver and gill cell lines. Chemical analyses of the sediment extracts revealed complex contamination profiles, especially at the innermost stations, which contained a wide array of persistent organic pollutants, polycyclic aromatic hydrocarbons, and metals. Sediment extracts from these sites were more potent in activating the stress-activated receptors than the other extracts, reflecting their toxicant profiles. Importantly, receptor- and cell-based bioassays complemented the chemical analyses and provided important data for future environmental risk assessments of urban marine sediments.

Polycyclic aromatic hydrocarbons modulate the activity of Atlantic cod (Gadus morhua) vitamin D receptor paralogs in vitro.

Vitamin D receptor (VDR) mediates the biological function of the steroid hormone calcitriol, which is the metabolically active version of vitamin D. Calcitriol is important for a wide array of physiological functions, including calcium and phosphate homeostasis. In contrast to mammals, which harbor one VDR encoding gene, teleosts possess two orthologous vdr genes encoding Vdr alpha (Vdra) and Vdr beta (Vdrb). Genome mining identified the vdra and vdrb paralogs in the Atlantic cod (Gadus morhua) genome, which were further characterized regarding their phylogeny, tissue-specific expression, and transactivational properties induced by calcitriol. In addition, a selected set of polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene, fluorene, pyrene, chrysene, benzo[a]pyrene (BaP), and 7-methylbenzo[a]pyrene, were assessed for their ability to modulate the transcriptional activity of gmVdra and gmVdrb in vitro. Both gmVdra and gmVdrb were activated by calcitriol with similar potencies, but gmVdra produced significantly higher maximal fold activation. Notably, none of the tested PAHs showed agonistic properties towards the Atlantic cod Vdrs. However, binary exposures of calcitriol together with phenanthrene, fluorene, or pyrene, antagonized the activation of gmVdra, while chrysene and BaP significantly potentiated the calcitriol-mediated activity of both receptors. Homology modeling, solvent mapping, and docking analyses complemented the experimental data, and revealed a putative secondary binding site in addition to the canonical ligand-binding pocket (LBP). Calcitriol was predicted to interact with both binding sites, whereas PAHs docked primarily to the LBP. Importantly, our in vitro data suggest that PAHs can interact with the paralogous gmVdrs and interfere with their transcriptional activities, and thus potentially modulate the vitamin D signaling pathway and contribute to adverse effects of crude oil and PAH exposures on cardiac development and bone deformities in fish.

The chemical defensome of five model teleost fish.

How an organism copes with chemicals is largely determined by the genes and proteins that collectively function to defend against, detoxify and eliminate chemical stressors. This integrative network includes receptors and transcription factors, biotransformation enzymes, transporters, antioxidants, and metal- and heat-responsive genes, and is collectively known as the chemical defensome. Teleost fish is the largest group of vertebrate species and can provide valuable insights into the evolution and functional diversity of defensome genes. We have previously shown that the xenosensing pregnane x receptor (pxr, nr1i2) is lost in many teleost species, including Atlantic cod (Gadus morhua) and three-spined stickleback (Gasterosteus aculeatus), but it is not known if compensatory mechanisms or signaling pathways have evolved in its absence. In this study, we compared the genes comprising the chemical defensome of five fish species that span the teleosteii evolutionary branch often used as model species in toxicological studies and environmental monitoring programs: zebrafish (Danio rerio), medaka (Oryzias latipes), Atlantic killifish (Fundulus heteroclitus), Atlantic cod, and three-spined stickleback. Genome mining revealed evolved differences in the number and composition of defensome genes that can have implication for how these species sense and respond to environmental pollutants, but we did not observe any candidates of compensatory mechanisms or pathways in cod and stickleback in the absence of pxr. The results indicate that knowledge regarding the diversity and function of the defensome will be important for toxicological testing and risk assessment studies.

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.

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.

Expression and localization of the aryl hydrocarbon receptors and cytochrome P450 1A during early development of Atlantic cod (Gadus morhua).

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that mediates the toxicity of dioxins and dioxin-like compounds (DLCs) in vertebrates. Two clades of the Ahr family exist in teleosts (Ahr1 and Ahr2), and it has been demonstrated that Ahr2 is the main protein involved in mediating the toxicity of dioxins and DLCs in most teleost species. Recently, we characterized the Atlantic cod (Gadus morhua) Ahr1a and Ahr2a receptors. To further explore a possible subfunction partitioning of Ahr1a and Ahr2a in Atlantic cod we have mapped the expression and localization of ahr1a and ahr2a in early developmental stages. Atlantic cod embryos were continuously exposed in a passive-dosing exposure system to the Ahr agonist, benzo[a]pyrene (B[a]P), from five days post fertilization (dpf) until three days post hatching (dph). Expression of ahr1a, ahr2a, and the Ahr-target genes, cyp1a and ahrrb, was assessed in embryos (8 dpf and 10 dpf) and larvae (3 dph) with quantitative real-time PCR analyses (qPCR), while in situ hybridization was used to assess the localization of expression of ahr1a, ahr2a and cyp1a. Quantitative measurements showed an increased cyp1a expression in B[a]P-exposed samples at all sampling points, and for ahr2a at 10 dpf, confirming the activation of the Ahr-signalling pathway. Furthermore, B[a]P strongly induced ahr2a and cyp1a expression in the cardiovascular system and skin, respectively, of embryos and larvae. Induced expression of both ahr2a and cyp1a was also revealed in the liver of B[a]P-exposed larvae. Our results suggest that Ahr2a is the major subtype involved in mediating responses to B[a]P in early developmental stages of Atlantic cod, which involves transcriptional regulation of biotransformation genes, such as cyp1a. The focused expression of ahr1a in the eye of embryos and larvae, and the presence of ahr2a transcripts in the jaws and fin nodes, further indicate evolved specialized roles of the two Ahrs in ontogenesis.

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.