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

A novel 3D Atlantic salmon co-culture model was developed using primary hepatocytes and kidney epithelial cells isolated from the same fish. Mono and co-cultures of primary hepatocytes and kidney epithelial cells were exposed for 48 h to glyphosate (5, 50 and 500 µM). For comparison, cells were also exposed to chlorpyrifos, benzo(a)pyrene and cadmium. Cell staining, cell viability assessments, RT-qPCR and global metabolomic profiling were used to examine the toxicological effects on liver and renal function and to compare responses in 3D and 2D cultures. The 3D hepatocyte cell culture was considered superior to the 2D culture due to the ATP binding cassette subfamily B member 1 (Abcb1) response and was thus used further in co-culture with kidney cells. Metabolomic analysis of co-cultured cells showed that glyphosate exposure (500 µM) altered lipid metabolism in both hepatocytes and kidney cells. Elevated levels of several types of PUFAs and long-chain fatty acids were observed in exposed hepatocytes, owing to increased uptake and phospholipid remodelling. Glyphosate suppressed the expression of estrogen receptor 1 (Esr1) and vitellogenin (Vtg) and altered histidine metabolism in exposed hepatocytes. Increased levels of cholesterol and downregulation of clusterin (Clu) suggest that glyphosate treatment affected membrane stability in Atlantic salmon kidney cells. This study demonstrates the usefulness of applying 3D co-culture models in risk assessment.

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.

Transcriptional effects of dietary chlorpyrifos­methyl exposure in Atlantic salmon (Salmo salar) brain and liver.

Elevated levels of chlorpyrifos­methyl have been detected in plant-based Atlantic salmon feeds. To evaluate the potential negative effects of long-term and continuous dietary exposure to chlorpyrifos­methyl in fish, we fed juvenile Atlantic salmon three concentrations (0.1, 1.0 and 8.0 mg/kg) of the pesticide for about two months. Brain and liver tissues were collected after 30 and 67 days of exposure. Homogenized brain tissue was examined for effects on acetylcholinesterase, and brain and liver tissue from fish exposed to 8.0 mg/kg were used for transcriptional analysis (RNA-seq). The results showed a transient accumulation of chlorpyrifos­methyl in the brain with lower levels after 67 days of exposure compared to after 30 days of exposure. In contrast, the liver showed a time-dependent accumulation pattern. No effect on acetylcholinesterase activity, the primary target of chlorpyrifos­methyl, was seen in the brain. However, after 30 days of exposure, 98 significantly differentially expressed genes (DEGs) were found in brain tissue and 239 DEGs in liver tissue. After 67 days of exposure, two and 258 DEGs were found in brain and liver tissue, respectively. Continuous dietary exposure of chlorpyrifos­methyl most profoundly affected mechanisms associated with protein degradation and lipid metabolism in both brain and liver. Specific for the brain, many of the significant DEGs encode proteins involved in neuron function. In conclusion, this study shows that chlorpyrifos­methyl affects the transcription of genes involved in neurological function in Atlantic salmon brain, even at exposure concentrations below the threshold for systemic toxicity as seen from brain acetylcholinesterase inhibition.

Sensitivity and toxic mode of action of dietary organic and inorganic selenium in Atlantic salmon (Salmo salar).

Depending on its chemical form, selenium (Se) is a trace element with a narrow range between requirement and toxicity for most vertebrates. Traditional endpoints of Se toxicity include reduced growth, feed intake, and oxidative stress, while more recent finding describe disturbance in fatty acid synthesis as underlying toxic mechanism. To investigate overall metabolic mode of toxic action, with emphasis on lipid metabolism, a wide scope metabolomics pathway profiling was performed on Atlantic salmon (Salmo salar) (572±7g) that were fed organic and inorganic Se fortified diets. Atlantic salmon were fed a low natural background organic Se diet (0.35mg Se kg-1, wet weight (WW)) fortified with inorganic sodium selenite or organic selenomethionine-yeast (SeMet-yeast) at two levels (∼1-2 or 15mgkg-1, WW), in triplicate for 3 months. Apparent adverse effects were assessed by growth, feed intake, oxidative stress as production of thiobarbituric acid-reactive substances (TBARS) and levels of tocopherols, as well as an overall metabolomic pathway assessment. Fish fed 15mgkg-1 selenite, but not 15mgkg-1 SeMet-yeast, showed reduced feed intake, reduced growth, increased liver TBARS and reduced liver tocopherol. Main metabolic pathways significantly affected by 15mgkg-1 selenite, and to a lesser extent 15mgkg-1 SeMet-yeast, were lipid catabolism, endocannabinoids synthesis, and oxidant/glutathione metabolism. Disturbance in lipid metabolism was reflected by depressed levels of free fatty acids, monoacylglycerols and diacylglycerols as well as endocannabinoids. Specific for selenite was the significant reduction of metabolites in the S-Adenosylmethionine (SAM) pathway, indicating a use of methyl donors that could be allied with excess Se excretion. Dietary Se levels to respectively 1.1 and 2.1mgkg-1 selenite and SeMet-yeast did not affect any of the above mentioned parameters. Apparent toxic mechanisms at higher Se levels (15mgkg-1) included oxidative stress and altered lipid metabolism for both inorganic and organic Se, with higher toxicity for inorganic Se.

Dietary histidine requirement to reduce the risk and severity of cataracts is higher than the requirement for growth in Atlantic salmon smolts, independently of the dietary lipid source.

The present study was carried out to investigate whether the dietary histidine requirement to reduce cataract development is higher than that for growth in Atlantic salmon smolts (Salmo salar L.) after seawater transfer and whether dietary vegetable oils contribute to cataractogenesis. Duplicate groups of salmon smolts were fed ten experimental diets with either fish oil (FO) or a vegetable oil (VO) mix replacing 70 % FO and histidine at five target levels (10, 12, 14, 16 and 18 g His/kg diet) for 13 weeks after seawater transfer. The VO diet-fed fish exhibited somewhat inferior growth and feed intakes compared with the FO diet-fed fish, irrespective of the dietary histidine concentration. Both cataract prevalence and severity were negatively correlated with the dietary histidine concentration, while lens N-acetyl-histidine (NAH) concentrations were positively correlated with it. The fatty acid profiles of muscle, heart and lens reflected that of the dietary oils to a descending degree and did not affect the observed cataract development. Muscle, heart and brain histidine concentrations reflected dietary histidine concentrations, while the corresponding tissue imidazole (anserine, carnosine and NAH) concentrations appeared to saturate differently with time. The expression level of liver histidase was not affected by the dietary histidine concentration, while the liver antioxidant response was affected in the VO diet-fed fish on a transcriptional level. The lowest severity of cataracts could be achieved by feeding 13·4 g His/kg feed, independently of the dietary lipid source. However, the present study also suggests that the dietary histidine requirement to minimise the risk of cataract development is 14·4 g His/kg feed.

Precision-cut liver slices of Atlantic cod (Gadus morhua): an in vitro system for studying the effects of environmental contaminants.

The Atlantic cod (Gadus morhua) is an economically important species commonly consumed by humans. The widespread distribution of cod in the North Atlantic Ocean makes it vulnerable to effluents from human activities, such as coastal industries and offshore petroleum exploration. It has been demonstrated that many effluents have adverse effects on cod reproduction and health, e.g. by disrupting endocrine signaling pathways. The liver, expressing important components of the biotransformation and the endocrine system, is one of the main target organs. Thus, reliable and reproducible in vitro systems of the liver are important for studying effects of environmental contaminants. The aim of this study was to investigate precision-cut liver slices (PCLS) as an alternative in vitro system for toxicological studies of the Atlantic cod liver. Slices of 8 mm in diameter and 250 µm thickness were prepared and cultivated from immature cod. Several analyses to measure the liver slice viability were performed: enzyme assays, histology, and morphometric analysis, all confirming cell viability for up to 72 h in culture. The liver slices were also exposed to two well-known model environmental contaminants, ß-naphthoflavone (BNF) and 17α-ethynylestradiol (EE2), representing established agonists for the aryl hydrocarbon receptor (AHR) and the estrogen receptor (ER), respectively. The results showed increased transcription of the target genes cytochrome P450 1A (CYP1A) and vitellogenin (VTG), both well-established biomarkers for exposure of fish to the selected compounds. In conclusion, PCLS is a promising in vitro system for toxicological studies of cod liver cells. The liver slices are viable in culture for several days and respond to environmental contaminants in a dose- and time-specific manner.

Effect of marine omega 3 fatty acids on methylmercury-induced toxicity in fish and mammalian cells in vitro.

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.

Differences in salinity tolerance and gene expression between two populations of Atlantic cod (Gadus morhua) in response to salinity stress.

Populations of marine fish, even from contrasting habitats, generally show low genetic differentiation at neutral genetic markers. Nevertheless, there is increasing evidence for differences in gene expression among populations that may be ascribed to adaptive divergence. Studying variation in salinity tolerance and gene expression among Atlantic cod (Gadus morhua) from two populations distributed across a steep salinity gradient, we observed high mortality (45% North Sea cod and 80% Baltic Sea cod) in a reciprocal common garden setup. Quantitative RT-PCR assays for expression of hsp70 and Na/K-ATPase α genes demonstrated significant differences in gene regulation within and between populations and treatment groups despite low sample sizes. Most interesting are the significant differences observed in expression of the Na/K-ATPase α gene in gill tissue between North Sea and Baltic cod. The findings strongly suggest that Atlantic cod are adapted to local saline conditions, despite relatively low levels of neutral genetic divergence between populations.

Susceptibility of Atlantic salmon lenses to hydrogen peroxide oxidation ex vivo after being fed diets with vegetable oil and methylmercury.

The development of cataract in farmed Atlantic salmon (Salmo salar L.) has been related to changes in feed composition resulting in sub-optimal lens nutrition. The present study was performed to investigate the ability of Atlantic salmon lenses to withstand oxidative stress ex vivo, with focus on the nutritional lipid history and exposure to methylmercury (MeHg) as a relevant dietary contaminant. Since dietary histidine has been shown to have a mitigating effect on the prevalence of cataract in farmed salmon, the antioxidative abilities of histidine and NAH, a major imidazole in the salmon lens, was also investigated ex vivo. Lenses from Atlantic salmon prefed diets based on either fish oil (FO) or vegetable oil (VO) as lipid source, with or without addition of 5 mg MeHgkg(-1) feed, were cultured for 96 h in normal medium (control), medium added 5 mM H(2)O(2) or in histidine enriched medium. Lipid class composition of the lenses was not affected by the dietary lipids; while VO fed fish had a decrease in lens n-3/n-6 fatty acid ratio due to minor but significant increase in the concentration of 18:2 n-6 and 20:4 n-6, and decrease in 20:5 n-3 fatty acids compared to FO fed fish. The lenses accumulated mercury in response to dietary levels, but neither the oxidative status nor any physiological responses were affected. The cultured lenses responded to H(2)O(2) exposure with loss of transparency, accumulation of auto-fluorescent compounds, volume increase and reduced glutathione concentration similarly and irrespective of the dietary history. Lenses extracted histidine from the media, and synthesised NAH during the culture period. The innate antioxidative defence system appeared to be influenced both by the dietary lipid history and histidine enrichment on a transcriptional level. Catalase and SPARC were expressed higher in lenses from FO fed fish, and glutaredoxin showed elevated expression levels in FO lenses cultured in histidine enriched medium, suggesting that histidine is related to the innate antioxidant defence in salmon lenses. Further, the concentration of NAH was significantly reduced in oxidatively stressed lenses. Based on the results from this study it is suggested that NAH has a novel role as antioxidant in the Atlantic salmon lens.

Effects of short-term starvation on ghrelin, GH-IGF system, and IGF-binding proteins in Atlantic salmon.

The effects of short-time fasting on appetite, growth, and nutrient were studied in Atlantic salmon (Salmo salar) smolts. Feed deprivation did change the energy metabolism with reduced plasma protein and muscle indispensible amino acid levels. Plasma levels of ghrelin were significantly higher in starved salmon compared with fed fish after 2 days, but no differences in circulating ghrelin were found between treatments after 14 days. Two mRNA sequences for ghrelin-1 and ghrelin-2, 430 and 533 bp long, respectively, were detected. In addition, the growth hormone secretagogues-receptor like receptor (GHSR-LR) 1a and 1b were identified. Ghrelin-1 but not ghrelin-2 mRNA levels were affected by starvation in the stomach. Lower ghrelin-1 mRNA levels were detected at day 2 in starved fish compared with fed fish. The mRNA levels of GHSR-LR1a were not affected by starvation. Fasting reduced the phenotypic growth and the transcription of insulin-like growth factor (IGF)-II together with IGF-IIR, but IGF-I mRNA were not regulated in fasted salmon after 14 days. Three IGF-binding proteins (IGFBP) at 23, 32, and 43 kDa were found in salmon, and circulating 23 kDa was significantly increased after 14 days of starvation compared with fed fish, indicating increased catabolism. The levels of IGFBP-1 mRNA were significantly higher in fed and starved fish after 14 days compared to those at the start of the experiment, but no significant difference was observed between the treatments. In conclusion, we have shown that circulating ghrelin and ghrelin-1 mRNA is related to changes in energy metabolism in Atlantic salmon.

Reducing persistent organic pollutants while maintaining long chain omega-3 fatty acid in farmed Atlantic salmon using decontaminated fish oils for an entire production cycle.

Oily fish are an important source of health promoting nutrients such as the very long chain marine omega-3 (VLC-n3) fatty acids and simultaneously a source of potentially hazardous contaminants. Fish oils that are used in fish feed are the main source for both contaminants and VLC-n3. Decontamination techniques have recently been developed to effectively remove persistent organic contaminants from fish oils. The aim of the present study was to assess the level of potentially hazardous contaminants and the health beneficial fatty acids in Atlantic salmon reared on novel decontaminated feeds. Atlantic salmon were fed for 18 months (an entire seawater production cycle) on diets based on decontaminated or non-treated (control) fish oils until market size (approximately 5 kg). The level of known notorious persistent organic pollutants (POPs, i.e. dioxins, dioxin-like polychlorinated biphenyls (DL-PCBs), non dioxin-like PCBs, poly brominated diphenyl ethers (PBDE), and organochlorine pesticides), as well as fatty acid composition were analysed in fish oils, the two diets, and Atlantic salmon fillet. The oil decontamination process was a two-step procedure using active carbon and short path distillation. The fillet levels of POPs in market size fish were reduced by 68-85% while the concentration of very long chain omega-3 fatty acids was reduced by 4-7%. No differences in biomarkers of dioxin-like component exposures, such as hepatic gene expression of CYP1A or AhR2B, CYP1A protein expression and 7-ethoxyresorufin O-deethylase (EROD) activity, were observed between salmon raised on normal or decontaminated feeds, thus indicating that the difference in POPs levels were of no biological significance to the fish. Atlantic salmon reared on decontaminated feeds had sum polychlorinated dibenzodioxins/furans (PCDD/Fs) and DL-PCB concentrations that were comparable with terrestrial food products such as beef, while the level of marine omega-3 fatty acids remained as high as for commercially farmed Atlantic salmon.

Transcriptional effects on glutathione S-transferases in first feeding Atlantic cod (Gadus morhua) larvae exposed to crude oil.

Polycyclic aromatic hydrocarbons (PAHs) and other oil compounds are known to induce stress and impact health of marine organisms. Water-soluble fractions of oil contain components known to induce glutathione S-transferases (GSTs), one of the major classes of phase II detoxifying enzymes present in essentially all eukaryotic organisms. In this study, the transcriptional responses of six GSTs (GST pi, GST mu, GST omega, GST theta, GSY zeta and GST kappa) were examined in early larvae of Atlantic cod Gadus morhua exposed to five concentrations of dispersed oil (containing oil droplets and water-soluble fraction) and water-soluble fractions (WSF) of oil. When Atlantic cod larvae were exposed to WSF (containing 1.31+/-0.31microg summation PAH/L for 4 days), expression of GSTM3 and GSTO1 was significantly increased, whereas no differences in GST expression were observed in larvae exposed to a corresponding 50% lower amount of dispersed oil (containing 0.36+/-0.10 microg summation PAH/L for 4 days). The study suggest that although the oil clearly had severe negative effects on the larvae (i.e. concentration-dependent lethality and growth reduction), only minor effects on GST transcription could be observed using RNA obtained from pooled whole-larvae homogenates. This result indicates that the expression of these important detoxification enzymes is only moderately inducible at such an early developmental stage either reflecting low tolerance of cod larvae to dispersed oil or alternatively that using whole-larvae homogenates may have masked tissue-specific mRNA induction.

Oxidative stress responses in rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to pro-oxidants and a complex environmental sample.

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 96h 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, gamma-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.

Effects of combined gamma-irradiation and metal (Al+Cd) exposures in Atlantic salmon (Salmo salar L.).

These experiments were designed to investigate transcriptional effects in Atlantic salmon (Salmo salar) after exposure in vivo to ionizing gamma radiation combined with subtoxic levels of aluminum (Al) and cadmium (Cd). Juvenile fish (35 g) in freshwater with or without Al and Cd (255 microg Al/L + 6 microg Cd/L) were exposed to a 75 mGy dose of gamma-irradiation, and induced responses were compared to those of controls. The transcriptional levels of eight genes encoding proteins known to respond to stress in fish were quantified in liver of fish exposed for 5 h to gamma radiation, to Al and Cd or to the combination of Al, Cd and gamma radiation. The studied genes were caspase 3B, caspase 6A, caspase 7, p53 (apoptosis), glutathione reductase (GR), phospholipid hydroperoxide glutathione peroxidase (GSH-Px), (oxidative stress), metallothionein (MT-A) (metal stress) and ubiquitin (Ubi) (protein degradation). The results showed that gamma-irradiation alone induced significant upregulation of caspase 6A, GR, GSH-Px, MT-A and Ubi compared to the control group, while 5 h exposure to Al+Cd alone did not induce any of the studied genes compared to the control. No significant upregulation of the series of investigated genes could be observed in fish exposed to gamma-irradiation in combination with Al+Cl. In conclusion, the results suggest that the presence of Al+Cd in the water counteracted the gamma-irradiation effect by modifying the transcription of genes encoding proteins involved in the defense mechanisms against free radicals in the cells.

The partial pressure of oxygen affects biomarkers of oxidative stress in cultured rainbow trout (Oncorhynchus mykiss) hepatocytes.

Oxidative stress, the imbalance between production of reactive oxygen species and the cellular detoxification of these reactive compounds, is believed to be involved in the pathology of various diseases. Several biomarkers for oxidative stress have been proposed to serve as tools in toxicological and ecotoxicological research. Not only may exposure to various pro-oxidants create conditions of cellular oxidative stress, but hyperoxic conditions may also increase the production of reactive oxygen species. The objective of the current study was to determine the extent to which differences in oxygen partial pressure would affect biomarkers of oxidative stress in a primary culture of hepatocytes from rainbow trout (Oncorhynchus mykiss). Membrane integrity, metabolic activity, levels of total and oxidized glutathione (tGSH/GSSG) was determined, as well as mRNA expression levels of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), gamma-glutamyl-cystein synthetase (GCS) and thioredoxin (TRX). The results show that different biomarkers of oxidative stress are affected when the cell culture is exposed to atmospheric oxygen, and that changes such as increased GSSG content and induction of GSSG-R and GSH-Px can be reduced by culturing the cells under lower oxygen tension. Oxygen tension may thus influence results of in vitro based cell research and is particularly important when assessing parameters in the antioxidant defence system. Further research is needed to establish the magnitude of this effect in different cellular systems.

Lysine intake affects gene expression of anabolic hormones in Atlantic salmon, Salmo salar.

Nutritional factors influence regulation of the growth hormone (GH) and the insulin-like growth factor (IGF) system in fish, but so far there are no published studies describing how single indispensable amino acids influence these systems. Therefore, the present study aimed to test whether lysine (Lys) intake at low (LL=2.85 g/16 gN), medium (ML=4.91 g/16 gN) and high levels (HL=9.19 g/16 gN) affected the expression of genes related to the GH-IGF system (i.e. GH receptor, GH-R, IGF-I, IGF-II, IGF binding protein 1, IGFBP-1, IGF-I receptor IGF-IR) in Atlantic salmon during seawater growth phase. Salmon fed the HL diet significantly up-regulated hepatic IGF-I mRNA level by a factor of 2.2 as compared to those with medium Lys intake. In addition a significant up-regulation of 2.7-fold in muscle IGF-II mRNA was present. Low Lys intake decreased the nitrogen deposition and muscle protein accretion in fish and significantly down-regulated hepatic IGFBP-1 as well as muscle GH-R and IGF-II, as compared to those fed the ML diet. mRNA of IGF-IR on the other hand was not affected by Lys intake. High Lys intake resulted in a 7-fold up-regulation of muscle IGF-II mRNA level as compared to low Lys intake, and thus might be an important local anabolic regulator in fast muscle tissue. The single indispensable amino acid Lys indeed affected signalling through the genes of IGF-I, IGFBP-1 in hepatic tissue and GH-R, IGF-II in fast muscle in Atlantic salmon. Concomitantly the higher Lys intake increased nitrogen deposition to a certain level.

Evaluation of stress- and immune-response biomarkers in Atlantic salmon, Salmo salar L., fed different levels of genetically modified maize (Bt maize), compared with its near-isogenic parental line and a commercial suprex maize.

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.

Effects of hypo- and hyperoxia on transcription levels of five stress genes and the glutathione system in liver of Atlantic cod Gadus morhua.

The transcript levels of three genes coding for antioxidants, Cu/Zn superoxide dismutase (SOD), catalase and phospholipid hydroperoxide glutathione peroxidase (GSH-Px), and those of two stress proteins, metallothionein (MT) and CYP1A, were examined with real-time quantitative (q) RT-PCR in hepatic tissue of Atlantic cod exposed to 46% (hypoxia), 76% (normoxia) and 145% (hyperoxia) O(2) saturation (tank outlet). To evaluate the oxidative stress state, the levels of total glutathione (tGSH), reduced glutathione (GSH) and oxidized glutathione (GSSG) and subsequently the oxidative stress index (OSI), were determined in the same tissue samples. The transcript level of GSH-Px was significantly upregulated in fish exposed to hyperoxia, and significantly downregulated in fish exposed to hypoxia, compared to the normoxia group. Significant downregulation was also found for SOD and CYP1A transcriptional levels in fish exposed to hypoxia. The transcript levels of catalase and MT did not change in liver of cod exposed to suboptimal oxygen levels. No significant differences were seen between the groups for tGSH, GSH, GSSG or OSI. Prolonged exposure to unfavourable oxygen saturation levels did not alter the OSI, indicating that the antioxidant glutathione system is maintained at an unchanged level in liver of the examined cod.

Antioxidative stress proteins and their gene expression in brown trout (Salmo trutta) from three rivers with different heavy metal levels.

Three populations of brown trout (Salmo trutta) exposed to different metal levels in their natural environments, were studied with respect to antioxidants metallothionein (MT), superoxide dismutase (SOD) and catalase (CAT) as well as for corresponding mRNA levels. In addition, mRNA levels were studied for glutathione peroxidase (GPx) and glutathione reductase (GR). The Cd/Zn-exposed trout (Naustebekken River) had higher accumulated levels of Cd, Cu and Zn in gills, and higher levels of MT (both protein and mRNA) in liver and kidney as well as in gills compared to the Cu-exposed trout (Rugla River) and trout from an uncontaminated reference river (Stribekken River). Less MT found in the Cu-exposed trout may increase susceptibility to oxidative stress, but no higher levels of antioxidant mRNAs were found in gills of these trouts. The data indicated that chronic exposures of brown trout to Cd, Zn and/or Cu did not involve maintenance of high activities of SOD and CAT enzymes in gills, although SOD mRNA levels were higher in the Cd/Zn-exposed trout. In livers, mRNA levels of SOD, CAT and GPx were higher in the metal-exposed trout, but in the case of GR this was only seen in kidneys of Cd/Zn-exposed trout. However, both metal-exposed groups had higher activities of SOD enzyme in liver compared to the unexposed reference trout, and CAT activity was found to be higher in kidneys of Cu-exposed trout. The Cu-exposed trout did not seem to rely on MT production to avoid Cu toxicity in gills, but rather by keeping the Cu uptake at a low level. A coordinated expression of different stress genes may also be important in chronic metal exposure. It may be concluded that the observed metal effects relies on acclimation rather than on genetic adaptation in the metal exposed populations.