Methylmercury (MeHg) is a ubiquitous environmental contaminant which bioaccumulates in marine biota. Fish constitute an important part of a balanced human diet contributing with health beneficial ...nutrients but may also contain contaminants such as MeHg. Interactions between the marine n-3 fatty acids eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) with MeHg-induced toxicity were investigated. Different toxic and metabolic responses were studied in Atlantic salmon kidney (ASK) cell line and the mammalian kidney-derived HEK293 cell line. Both cell lines were preincubated with DHA or EPA prior to MeHg-exposure, and cell toxicity was assessed differently in the cell lines by MeHg-uptake in cells (ASK and HEK293), proliferation (HEK293 and ASK), apoptosis (ASK), oxidation of the red-ox probe roGFP (HEK293), and regulation of selected toxicological and metabolic transcriptional markers (ASK). DHA was observed to decrease the uptake of MeHg in HEK293, but not in ASK cells. DHA also increased, while EPA decreased, MeHg-induced apoptosis in ASK. MeHg exposure induced changes in selected metabolic and known MeHg biomarkers in ASK cells. Both DHA and MeHg, but not EPA, oxidized roGFP in HEK293 cells. In conclusion, marine n-3 fatty acids may ameliorate MeHg toxicity, either by decreasing apoptosis (EPA) or by reducing MeHg uptake (DHA). However, DHA can also augment MeHg toxicity by increasing oxidative stress and apoptosis when combined with MeHg.
Increasing use of the chitin synthesis inhibitor diflubenzuron against the ectoparasitic salmon louse Lepeophtheirus salmonis in marine aquaculture has raised concerns over its environmental impacts. ...This study evaluated how diflubenzuron affects Atlantic cod Gadus morhua, a fish species often found near Atlantic salmon Salmo salar farms, focusing on uptake kinetics and hepatic transcriptional responses. Two experiments were conducted, one time-series trial in which the fish were given a daily dose (3 mg kg-1 fish) of diflubenzuron for 14 d followed by a 3 wk depuration period, and one dose-response trial with increasing concentrations (3, 10 and 50 mg kg-1 fish). The highest diflubenzuron concentrations were found in the liver at Day 15. No detectable levels of diflubenzuron were found in liver or muscle 3 wk after the end of the treatment. At the molecular level, small effects of diflubenzuron treatment on gene transcription were observed. In the time-series experiment, the strongest effects were seen at Day 8, with 2 transcripts being upregulated (bclx2 and cpt1a) and 8 transcripts being downregulated (gstp1, gstm1, gstt1, ugt1a, nat2, cat, p53 and slc16a9a). Five transcripts (cyp3a, cpt1a, ptgs2, elovl5 and mapk1) responded significantly to diflubenzuron exposure in the dose-response experiment. This study shows that diflubenzuron can be taken up by Atlantic cod, that it is rapidly cleared from the body and that when present this pharmaceutical causes only small effects on the expression of genes involved in detoxification pathways. Taken together, our data suggest that accumulated diflubenzuron at the levels studied would have a relatively small effect on wild Atlantic cod.
Cd, Cu and Zn concentrations were measured in ambient water as well as in gills, liver and kidney tissues of two natural populations of brown trout (
Salmo trutta) during a run-off episode in two ...rivers with different metal compositions due to mining pollution. Metallothionein (MT) was also measured in these tissues. The two rivers, Rugla (Cu contaminated) and Naustebekken (Cd and Zn contaminated), are located in two neighboring drainage basins separated by the topographic divide near the city of Røros in the County of Sør-Trøndelag, Norway. In Rugla, the Cu concentration increased from 15 μg/l at the low water level to 41 μg/l during the run-off episode. In Naustebekken, corresponding values for Cd were 90–170 ng/l and those for Zn were 49–91 μg/l. Gill concentrations of Cu and Cd/Zn MT in both populations of native trout clearly reflect the presence of these metals in the rivers during the run-off, in accordance with the hypothesis of protection caused by MT induction. When Rugla trout were transferred to Naustebekken and vice versa, both the amounts of MT itself and the Cu contents reflected the concentration of this metal in the new environment, indicating that MT induction also protects against acutely increased metal levels. The measured levels of MT in both native and transferred trout can account for all the Cd present in the tissues, but not for all of the Cu and Zn. The capacity of MT to regulate Cd and Cu in the trout populations in their natural habitat therefore seems clearly present. Our data also indicate that the MT I and II isoforms may bind metals selectively.
The present study was designed to evaluate if genetically modified (GM) maize (Bt maize, event MON810) compared with the near-isogenic non-modified (nGM) maize variety, added as a starch source at ...low or high inclusions, affected fish health of post-smolt Atlantic salmon, Salmo salar L. To evaluate the health impact, selected stress- and immune-response biomarkers were quantified at the gene transcript (mRNA) level, and some also at the protein level. The diets with low or high inclusions of GM maize, and its near-isogenic nGM parental line, were compared to a control diet containing GM-free suprex maize (reference diet) as the only starch source. Total superoxide dismutase (SOD) activity in liver and distal intestine was significantly higher in fish fed GM maize compared with fish fed nGM maize and with the reference diet group. Fish fed GM maize showed significantly lower catalase (CAT) activity in liver compared with fish fed nGM maize and to the reference diet group. In contrast, CAT activity in distal intestine was significantly higher for fish fed GM maize compared with fish fed reference diet. Protein level of heat shock protein 70 (HSP70) in liver was significantly higher in fish fed GM maize compared with fish fed the reference diet. No diet-related differences were found in normalized gene expression of SOD, CAT or HSP70 in liver or distal intestine. Normalized gene expression of interleukin-1 beta in spleen and head-kidney did not vary significantly between diet groups. Interestingly, fish fed high GM maize showed a significantly larger proportion of plasma granulocytes, a significantly larger sum of plasma granulocyte and monocyte proportions, but a significantly smaller proportion of plasma lymphocytes, compared with fish fed high nGM maize. In conclusion, Atlantic salmon fed GM maize showed some small changes in stress protein levels and activities, but none of these changes were comparable to the normalized gene expression levels analysed for these stress proteins. GM maize seemed to induce significant changes in white blood cell populations which are associated with an immune response.
A wide range of pollutants in the aquatic environment have the capacity to induce toxic effects expressed as cellular oxidative stress. In the current study, the potential of an
in vitro toxicity ...testing system was therefore investigated using rainbow trout (
Oncorhynchus mykiss) hepatocytes to assess different endpoints of oxidative stress. The pro-oxidants CuSO
4 and paraquat were used as models for comparison to a complex environmental sample. Results following 6, 24, 48 and 96
h exposure to different concentrations of these substances show cellular effects on intracellular ROS formation, glutathione levels and redox status, expression of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, γ-glutamyl-cysteine synthetase (GCS) and thioredoxin, as well as cytotoxicity parameters. The most consistent effects (maximum values within brackets), observed in dose and time parameters for both model compounds and environmental sample, were the depletion of total glutathione (9.4% of control), induced levels of oxidized glutathione (695% of control), and gene expression regulation depicted relative to the control gene beta-actin of GCS mRNA (239% of control) and catalase (29% of control). In conclusion, the responses on several antioxidant defence system parameters demonstrated the validity of the
in vitro toxicity testing system. Not only could multiple effects be detected at sub-lethal exposure concentrations, but these effects also gave valuable insight to the toxic mechanisms at the molecular level.
Atlantic salmon Salmo salar were fed fish feeds based on 100% replacement of fish oil (FO) with plant oil (PO) as compared to a 100% FO‐based diet. The transcript levels of eight genes’ encoding ...proteins involved in the cellular response to stressors metallothionein‐A isoform (MT‐A), cytochrome P450 1A (CYP1A), heat‐shock protein 70 (HSP70), catalase (CAT), glutathione S‐transferase (GST), glutathione reductase (GR), proliferating cell nuclear antigen (PCNA) and caspase 6A were quantified and compared to cell division (% PCNA‐positive cells) and apoptosis % terminal transferase mediated dUTP nick end labelling technique (TUNEL)‐positive cells in the mid intestinal (MI) section of the gastrointestinal tract. Five of these genes were also quantified in the pylorus caeca region (PR). Fish fed 100% PO‐based diets had significantly lower density of both PCNA and TUNEL‐positive cells in the MI section compared to fish fed a 100% FO‐based diet. The transcription levels of GST and caspase 6A were found to be significantly lower in the MI section of individuals fed a PO‐based diet compared to a FO‐based diet, analysed by a t‐test. In the PR, GR expression was significantly lower in the PO group compared to the FO group. The apoptosis markers PCNA and TUNEL were lower in the group fed a PO‐based diet. The results suggest that the transcription levels of three of the studied genes, GST, caspase 6A and GR, can be used as indicators of the MI response to feeding S. salar a PO‐based diet.
Atlantic salmon smolts were exposed to three doses of the fungicide azoxystrobin for 4 days, and physiological blood parameters and transcriptional effects in liver and muscle were evaluated in ...search for potential negative effects. Azoxystrobin exposure mediated up-regulation of catalase, MAPK1 and IGFBP1 in liver tissue. Catalase, transferrin, IGFBP1 and TNFR were up-regulated and CYP1A down-regulated in muscle tissue. Blood parameters glucose, hematocrit, pCO
2, HCO
3 and pH grouped together with transcripts levels of MnSOD, MAPK1, IGFBP1, MAP3K7 and GPx4 in liver of fish exposed to the highest azoxystrobin concentration (352
μg
L
−1) using principal component analysis (PCA). In muscle, the blood parameters glucose, hematocrit, pCO
2, HCO
3 and pH grouped together with transcript levels of heme oxygenase, thioredoxin, MnSOD, TNFR and MMP9. These results suggest that the fungicide azoxystrobin affects mitochondrial respiration and mechanisms controlling cell growth and proliferation in fish and may have negative effects on juvenile Atlantic salmon.
Atlantic cod were fed five different diets consisting of a plant protein mixture replacing fishmeal in a regression design up to total replacement. Growth was high and maintained equal up to 75% ...plant protein inclusion, while a total plant protein diet resulted in significantly reduced appetite, feed conversion, and growth. To reveal if the transcription of stress responsive genes or any metabolic pathways were affected by plant proteins, a microarray consisting of 744 EST library-clones from cod was used on liver samples after the fish doubled its weight. Liver mRNA-expression of insulin-like growth factor II (IGF-II), insulin-like growth factor receptors I and II (IGF-IR, IGF-IIR), genes coding for proteolytic enzymes; cathepsinD (CatD), cathepsinF (CatF), calpain 2, and seven proteins involved in protein-biosynthesis and energy-turnover, were studied by means of quantitative real-time PCR. Transcriptional levels of IGF-IIR and CatD were reduced in the plant fed fish. With few exceptions, dietary inclusion of plant protein did not affect the expression of genes related to cellular stress, protein-biosynthesis or energy turnover when the fish was fed up to 75% plant protein inclusion. The data on down-regulation of the IGF receptors and CatD coincided with the reduced growth in the high plant inclusions, indicating that IGF and CatD responses were closely related to protein turnover.
The pesticide chlorpyrifos-methyl (CLP-m) has been identified in plant ingredients intended for aquaculture feed production. To investigate systemic effects of CLP-m with emphasis on lipid ...metabolism, post-smolt Atlantic salmon were fed in duplicate (n=2) either diets with no CLP-m (Control) or CLP-m at different concentrations (0.1, 1.0 or 8.0mgkg−1) for a total of 67days (Low, Medium, High). Fish in all groups almost doubled their weight during the feeding trial from 262±26g (mean±SD) to 465±64g (overall mean), with no significant effects on any growth parameters. There was a significant dose-dependent inhibition of plasma cholinesterase activity (BuChE) after 67days. The CLP-m biotransformation metabolite, TCP was detected in liver and bile, with low levels of the parent compound in the organs. Spleen somatic index decreased significantly with increasing dietary CLP-m intake. Hematocrit (%) decreased linearly with increasing dietary exposure to CLP-m after 30days of exposure, but this decrease was less at 67days of exposure. A significantly reduced content of arachidonic acid (ARA 20:4n−6), accompanied by a significantly increased content of the saturated fatty acid, palmitic acid (PA 16:0), was observed in liver phospholipids (PLs) with increasing dietary content of CLP-m. Major effects were seen on the PL classes in liver which showed a significantly decreased absolute content, possibly indicating inhibition of PL remodeling pathways or other membrane perturbation effects from CLP-m exposure. In conclusion, this study shows that the pesticide CLP-m is a relatively potent toxicant in Atlantic salmon, especially affecting liver PLs and ARA metabolism.
•Dietary CLP-m inhibits plasma cholinesterase activity and affects the immune system in Atlantic salmon.•Dietary CLP-m reduces the content of arachidonic acid (ARA) in liver phospholipids (PLs).•Dietary CLP-m has membrane perturbation effects in Atlantic salmon.
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