In vitro toxicity of glyphosate in Atlantic salmon evaluated with a 3D hepatocyte-kidney co-culture model

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Mixture toxicity of chlorpyrifos-methyl, pirimiphos-methyl, and nonylphenol in Atlantic salmon (Salmo salar) hepatocytes

Pesticide formulations typically contain adjuvants added to enhance the performance of the active ingredient. Adjuvants may modify the bioavailability and toxicity of pesticides. In this study, the aim was to examine to which degree nonylphenol (NP) may interfere with the toxicity of two organophosphorus pesticides found in aquafeeds, chlorpyrifos-methyl (CPM) and pirimiphos-methyl (PPM). Atlantic salmon liver cells were exposed to these compounds singly or in combinations for 48 h using 3D cell cultures. Cytotoxicity, gene expression (RT-qPCR), and lipidomics endpoints were used to assess toxicity. The dose-response assessment showed that NP was the most toxic compound at equimolar concentrations (100 μM). Shotgun lipidomics pointed to a general pattern of elevated levels of saturated 18:0 fatty acids and declined levels of 18:1 monounsaturated fatty acids by the combined treatment. All three compounds had a distinct effect on membrane phospholipids, in particular on phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Lipid species patterns predicted inhibited stearoyl CoA desaturase (SCD) activity and increased Δ6 desaturase (D6D) activity in co-treated cells. While all three compounds alone mitigated increased triacylglycerol (TAG) accumulation, combined treatment resulted in lower total TAG in the cells. Multivariate analysis with PLS regression showed significant combined effects for nine genes (d5d, d6d, scd, srebf2, vtg, esr1, cyp1, ugt1a, and cat) and four lipid species (FFA 22:5, LPC 18:0, TAG52:1-FA16:0, and TAG52:1-FA18:0). In summary, this study demonstrates that the adjuvant can be the main contributor to the toxicity of a mixture of two organophosphorus pesticides with relatively low toxicity in fish cells.publishedVersio

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.publishedVersio

Modifying effects of vitamin E on chlorpyrifos toxicity in Atlantic salmon

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Redox regulation in Atlantic cod (Gadus morhua) embryos developing under normal and heat-stressed conditions

With regard to predicted oceanic warming, we studied the effects of heat stress on the redox system during embryonic development of Atlantic cod (Gadus morhua), with emphasis on the glutathione balance, activities of key antioxidant enzymes, and their mRNA levels. The embryos were incubated at optimal temperature for development (6 °C) or slightly above the threshold temperature (10 °C). The regulation of all the redox-related parameters measured at optimum development was highly dynamic and complex, indicating the importance of both maternal and zygotic contributions to maintaining redox equilibrium. Development at 10 °C caused a significantly higher mortality at the blastula and early gastrula stages, indicating severe stress. Measures of the glutathione redox couple showed a significantly more reduced state in embryos at 10 °C compared to 6 °C at the post-gastrula stages. Mean normalized expression of nrf2, trxred, g6pd, gclc, nox1, CuZnsod, and mt in embryos kept at 10 °C revealed stage-specific significantly reduced mRNA levels. Activities of antioxidant enzymes changed both during ontogenesis and in response to temperature, but did not correlate with mRNA levels. As the embryos need a tightly regulated redox environment to coordinate between growth and differentiation, these findings suggest that the altered redox balance might participate in inducing phenotypic changes caused by elevated temperature.publishedVersio

Fluorescent Microplastic Uptake by Immune Cells of Atlantic Salmon (Salmo salar L.)

The ubiquitous presence of microplastics and their marine ecotoxicity are major public concerns. Microplastics are ingested accidentally by the marine fauna or are taken up indirectly through the food chain. These particles can accumulate in cells and tissues and affect the normal biological functions of organisms, including their defense mechanisms. There is limited information available about the response of immune cells to microplastics; the degree of uptake by the cells, the response of different organs or the impact of environmental concentrations of microplastic are matters that remain unclear. Moreover, very little is known about the toxicity of different polymer types. This study aimed to shed light on the physical impact of small microplastics (1–5 μm) on cells from Atlantic salmon. Immune cells from intestine, blood, and head kidney were exposed to green fluorescent polyethylene microplastic (PE-MP), yellow fluorescent polystyrene microplastic (PS-MP) and both. High (50 mg/L), medium (5 mg/L), and low (0.05 mg/L) concentrations were tested for 1, 24, 48, and 72 h to study cell mortality and microplastic uptake. Quantitative data of microplastic uptake by fish immune cells were obtained for the first time by imaging flow cytometry. Salmon immune cells showed a relatively low ability to phagocytose microplastics. Less than 6% of the cells ingested the particles after 48 h of exposure to high concentrations. Cells also phagocytosed microplastics at low concentrations although at low rates (<0.1%). PE-MPs was phagocytosed by higher percentage of cells compared to PS-MPs and the former bioaccumulated in time while the latter decreased over time. However, each cell generally phagocytosed more PS-MPs particles than PE-MPs. Cells from different tissues showed different responses to the microplastic polymers. In conclusion, this study shows that immune cells of Atlantic salmon can phagocytose microplastics, and the impact is dependent on the microplastic type. PE-MPs, the most abundant polymer in the oceans and a widely used plastic in salmon aquaculture, was more easily taken up than PS-MPs. Furthermore, the study demonstrates how imaging flow cytometry can be applied in microplastics research.publishedVersionUnit Licence Agreemen

Chlorpyrifos-induced dysfunction of lipid metabolism is not restored by supplementation of polyunsaturated fatty acids EPA and ARA in Atlantic salmon liver cells

Exposure to contaminants can lead to accumulation of lipids in the liver. This study aimed to examine whether eicosapentaenoic acid (EPA) and arachidonic acid (ARA) supplementation can protect fish cells against the negative impact of chlorpyrifos (CPF). Atlantic salmon hepatocytes were exposed to either 100 μM CPF, 200 μM EPA, 200 μM ARA, or combinations of these for 48 h, and endpoints included lipid droplet formation, gene expression, and global metabolomic analysis. The results showed that polyunsaturated fatty acid (PUFA) supplementation modified the cell lipid composition, reduced uptake of CPF and increased the cellular number and size of lipid droplets. CPF exposure induced the transcription of ppara and fabp3, and reduced the levels of several PUFAs, and lead to accumulation of monoacylglycerols (MAGs) in the cells. Supplementation of EPA or ARA did not prevent CPF-induced accumulation of MAGs and only to a limited degree rescued the response on other lipids. CPF exposure further reduced energy metabolism, a response partly restored by PUFA supplementation. Reduced levels of glutathione indicated oxidative stress; an effect not ameliorated by the PUFAs. Altogether, this study shows that PUFA supplementation only modestly protects Atlantic salmon hepatocytes against the negative impact of CPF.publishedVersio

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.publishedVersio

Associations Between Behavioral Effects of Bisphenol A and DNA Methylation in Zebrafish Embryos

Endocrine-disrupting contaminants have been associated with aberrant changes in epigenetic pathways in animals. In this study, zebrafish embryos were exposed bisphenol A (BPA) to search for associations between behavior and epigenetic mechanisms in fish. For concentration-dependent responses, embryos were exposed to a range of BPA concentrations (0.1 nM to 30 μM). Embryos were analyzed for locomotor activity at 3-, 4-, and 5-days post fertilization (dpf) in response to changing light conditions. Based on concentration-dependent effects on behavior and gene expression, 10 μM BPA [from 24 to 96 hours post fertilization (hpf)] was used for a whole-genome bisulfite sequencing (WGBS) study searching for genome-wide impacts on DNA methylation. Over the examined concentration ranges, hyperactivity was demonstrated for exposures to 0.001 μM BPA in comparison to embryos exposed to lower or higher BPA concentrations. Transcriptional analysis showed significant effects at >0.01 μM BPA for two genes related to DNA methylation (dnmt1, cbs). BPA exposure did not significantly affect global DNA methylation, but 20,474 differentially methylated (DM) sites in 4,873 genes were identified by WGBS analysis. Most DM sites were identified within gene bodies. The genes with the most DM sites were all protocadherin 2 gamma subfamily genes, related to axon targeting, synaptic development and neuronal survival. KEGG pathways most significantly affected by BPA exposure were phosphatidylinositol signaling system, followed by VEGF and MAPK signaling pathways. This study shows that BPA can affect zebrafish embryo swimming activity at very low concentrations as well as affecting numerous methylated sites in genes which are overrepresented in functionally relevant metabolic pathways. In conclusion, altered methylation patterns of genes associated with nervous system development might lead to abnormal swimming activity.Associations Between Behavioral Effects of Bisphenol A and DNA Methylation in Zebrafish EmbryospublishedVersio

A transcriptomic analysis of diploid and triploid Atlantic salmon lenses with and without cataracts

To avoid negative environmental impacts of escapees and potential inter-breeding with wild populations, the Atlantic salmon farming industry has and continues to extensively test triploid fish that are sterile. However, they often show differences in performance, physiology, behavior and morphology compared to diploid fish, with increased prevalence of vertebral deformities and ocular cataracts as two of the most severe disorders. Here, we investigated the mechanisms behind the higher prevalence of cataracts in triploid salmon, by comparing the transcriptional patterns in lenses of diploid and triploid Atlantic salmon, with and without cataracts. We assembled and characterized the Atlantic salmon lens transcriptome and used RNA-seq to search for the molecular basis for cataract development in triploid fish. Transcriptional screening showed only modest differences in lens mRNA levels in diploid and triploid fish, with few uniquely expressed genes. In total, there were 165 differentially expressed genes (DEGs) between the cataractous diploid and triploid lens. Of these, most were expressed at lower levels in triploid fish. Differential expression was observed for genes encoding proteins with known function in the retina (phototransduction) and proteins associated with repair and compensation mechanisms. The results suggest a higher susceptibility to oxidative stress in triploid lenses, and that mechanisms connected to the ability to handle damaged proteins are differentially affected in cataractous lenses from diploid and triploid salmon.publishedVersionUnit Licence Agreemen